doi:10.1006/cyto.1999.0661, available online at http://www.idealibrary.

com on

TRANSCRIPTIONAL ACTIVATION OF
INDOLEAMINE DIOXYGENASE BY
INTERLEUKIN 1 AND TUMOR NECROSIS
FACTOR
IN INTERFERON-TREATED
EPITHELIAL CELLS
Tricia A. Babcock and Joseph M. Carlin

Interferon (IFN)--induced indoleamine 2,3-dioxygenase (IDO) activity is enhanced synergisti-

cally by interleukin (IL-)1, tumor necrosis factor-
(TNF-
) and LPS in IFN-treated macro-
phages by increasing IDO mRNA concentration. These studies demonstrate that IFN-treated
HeLa cells also exhibit dose-dependent enhancement of IDO induction by TNF-
and IL-1, with
maximal effects at concentrations of 5 ng/ml and 3 ng/ml, respectively. Furthermore, with
sub-optimal IFN concentrations, cells treated with maximally effective concentrations of TNF-

or IL-1
required 3–5 times less IFN to induce the same level of IDO activity as that observed
with IFN alone. To detect changes in transcriptional activation of the IDO gene, HeLa cells
were transfected with a plasmid containing the IDO 5 regulatory region upstream of a green
fluorescent protein (GFP) reporter gene. In transfected cells, IFN induced both IDO activity and
GFP that was detected by flow cytometry. When cell-sorted, transfected cells were stimulated
with IFN in combination with TNF-
or IL-1 but not LPS, increased GFP was detected in
comparison to transfected cells treated with IFN alone. Furthermore, increases in GFP
expression correlated with IDO enzymatic activity, indicating that combinations of IFN with
IL-1 or TNF-
increase the transcriptional activity of the IDO promoter region.
 2000 Academic Press

Although intracellular pathogens are sheltered
from humoral immune responses within the host cell,
they remain susceptible to cytokine-mediated immune
responses. In particular, IFN-
has been implicated
as one of the principal immune effector molecules
that induces several antimicrobial mechanisms effective
at inhibiting the growth of intracellular patho-
gens. Among these mechanisms are the release of
reactive oxygen intermediates,1 the generation of
nitrogen intermediates by nitric oxide synthase,2,3 and
production of indoleamine 2,3-dioxygenase (IDO).4
IDO is a heme-containing enzyme that catalyses
the oxidative cleavage of the indole ring of the essential
amino acid -tryptophan to N-formylkynurenine;5 its
From the Department of Microbiology, Miami University, Oxford,
Ohio 45056, USA
Correspondence to: Joseph M. Carlin, Miami University,
Department of Microbiology, Oxford, Ohio 45056, USA; E-mail:
CarlinJM@MUOhio.edu
Received 21 July 1999; received in revised form 22 November 1999;
accepted for publication 29 December 1999
 2000 Academic Press
1043–4666/00/060588+07 $35.00/0
KEY WORDS: indoleamine dioxygenase/interferon/interleukin 1/
tumour necrosis factor/tryptophan
588
induction rapidly depletes both intracellular and extra-
cellular tryptophan.6 Thus, IDO restricts the growth of
tryptophan auxotrophs by limiting the availability of
an amino acid required for protein synthesis. Among
the pathogens known to be sensitive to IDO activity
are Chlamydia trachomatis, C. psittaci, C. pneumoniae
and Toxoplasma gondii.4,7–10 Several lines of evidence
suggest that IDO-mediated inhibition of these patho-
gens is due to tryptophan starvation. Pathogen repli-
cation can be reversed by the addition of excess
tryptophan to the medium,8 and in a mutant cell line
defective in IFN-
-induced IDO activity, IFN-
fails
to suppress the growth of these pathogens.11 Further-
more, expression of IDO activity in a cell line
transfected with a metallothionein-regulated IDO
cDNA inhibits pathogen growth independent of other
IFN-induced proteins.12
Regulation of IDO expression can be influenced
by several cytokines and immunomodulating agents,
including TNF-, IL-1 and lipopolysaccharide (LPS).
Each can enhance the amount of IDO activity induced
in IFN-
-treated macrophages and the myelomono-
cytic THP-1 cell line.13–16 Furthermore, this increase in
IDO activity enhances the antimicrobial effect of
CYTOKINE, Vol. 12, No. 6 (June), 2000: pp 588–594

IFN-
in a dose-dependent manner (Currier et al.,
unpublished data).10,17,18 Although the effect of these
inflammatory molecules has been demonstrated pri-
marily in cells of a macrophage lineage, there is some
evidence that TNF- might also enhance IDO induc-
tion in epithelial cells. The TNF-enhanced anti-
chlamydial effect of IFN-
can be significantly reduced
by the addition of excess tryptophan to the medium,9,19
suggesting the involvement of IDO in restricting
intracellular pathogen growth.
Although the synergistic increase in IDO activity
has also been correlated with increased IDO mRNA
expression following stimulation with these immuno-
modulating agents in IFN-treated macrophages,14,15
the molecular mechanism by which these cytokines and
immunomodulating agent are able to potentiate IDO
activity has not been identified. The objectives of this
study were two-fold: to characterize and quantify the
effects of IDO-enhancing agents in epithelial cells, and
to assess whether enhancement of IDO activity and
mRNA expression was due to increased transcriptional
activity of the IDO promoter. To accomplish these
objectives, a human cervical epithelial cell line was
transfected with a green fluorescent protein reporter
linked to the 5 regulatory region of the IDO gene.
RESULTS
Synergistic enhancement of IDO activity
Baseline IDO activity in HeLa cells was estab-
lished by stimulation with increasing concentrations of
IFN-
from 1 ng/ml to 30 ng/ml for 48 h at which time
IDO activity was determined. IFN-
concentrations of
c1 ng/ml were unable to induce IDO activity, yet
higher concentrations of IFN-
induced a dose-
dependent increase in IDO activity (Fig. 1). TNF-
(5 ng/ml) and IL-1 (3 ng/ml) alone did not induce IDO
activity in HeLa cells. However, when each was added
to IFN-treated cells, significantly greater IDO activity
was induced at IFN-
concentrations d3 ng/ml.
TNF- induced seven-fold (P<0.02) and three-fold
(P<0.001) increases in IDO activity at IFN-
concen-
trations of 3 and 10 ng/ml, respectively, while IL-1
induced a three-fold (P<0.02) increase in IDO activity
with IFN-
at 10 ng/ml. Although TNF- and IL-1
had similar effects on IDO activity in IFN-treated cells,
LPS was unable to enhance IDO activity.
To characterize dose-dependent effects of
immunomodulating agents on IFN-
induced IDO
activity, HeLa cells were cultured with systematically
varied concentrations of TNF- (0.005–50 ng/ml), IL-1
(0.0003–3 ng/ml) and LPS (0.01-10 ng/ml), with and
without IFN-
at 10 ng/ml. This concentration of
IFN-
was chosen because it induced significant IDO
activity (P<0.007) when used as a single agent, yet this
Activation of IDO in epithelial cells / 589
80
70
Specific catabolism (%)
60
50
40
30
20
10
0
0 1 3 10 30
[IFN] (ng/ml)
Figure 1. Effect of IFN concentration on enhancement of IDO
activity.
Triplicate samples of HeLa cells were treated with increasing con-
centrations of IFN-
(1-30 ng/ml) alone ( ) or combined with
TNF- (5 ng/ml) ( ), IL-1 (3 ng/ml) ( ) or LPS (10 ng/ml) ( ) for
48 h before assessment of IDO activity. IDO activity is represented
as percentage specific catabolismSE of three separate experiments.
activity could be significantly enhanced by cytokine
combinations. By itself, TNF- was unable to induce
IDO activity at any concentration [Fig. 2(A)]. How-
ever, in the presence of IFN-
, a dose-dependent
increase in IDO activity was observed with a maximum
four-fold increase in IDO activity induced at a TNF-
concentration of 5 ng/ml. Similar results were obtained
in cells treated with increasing concentration of IL-1.
While IL-1 alone was unable to induce IDO activity,
when used in combination with IFN-
it induced a
synergistic increase in IDO activity in a dose-
dependent manner, with a maximum two-fold increase
in activity at 3 ng/ml [Fig. 2(B)]. Unlike THP-1 and
human macrophages, HeLa cells were not responsive
to LPS; no synergistic increase in IDO activity
was observed even when IFN-
was combined with
10 ng/ml of LPS [Fig. 2(C)].
Transcriptional Activation of the IDO Promoter
Previous studies using RT-PCR have correlated
the synergistic increase in IDO activity with increased
IDO mRNA expression in macrophages and the
THP-1 monocytic cell line.14,15 To determine if
increased IDO mRNA expression was due to transcrip-
tional activation of the IDO gene, HeLa cells were
transfected with the pEGFP/IDOp reporter construct,
which contained the enhanced GFP gene downstream
of the IDO promoter. To demonstrate that GFP
expression was under the control of the IDO promoter,
cells were cultured with and without IFN-
(10 ng/ml).
After 48 h incubation, cells were harvested with
EDTA/trypsin and resuspended in PBS for analysis by
flow cytometry (Fig. 3A). Cells activated with IFN-

590 / Babcock and Carlin
A B
70
60
Specific catabolism (%)
50
40
30
20
10
0
0 0.05 0.5
TNF (ng/ml)
Figure 2. Enhancement of IFN-induced IDO activity by TNF
, IL-1 or LPS.
Triplicate samples of HeLa cells were treated with increasing concentrations of TNF- (A) IL-1 (B) or LPS (C) alone ( ) or in combination with
IFN-
(10 ng/ml) ( ) for 48 h before assessment of IDO activity. IDO activity is represented as percentage specific catabolismSE of three
separate experiments.
had a 13-fold increase in mean fluorescence intensity
(MFI=77) over background fluorescence observed in
unstimulated cells (MFI=6), illustrating that GFP
expression was under the control of the IDO promoter.
To determine if synergistic enhancement of
IDO activity was associated with increased transcrip-
tional activation, pEGFP/IDOp-transfected cells
(2105 cells/ml) were cultured with and without
IFN-
(10 ng/ml) in the presence of the TNF-
(5 ng/ml), IL-1 (3 ng/ml), LPS (10 ng/ml) or medium
alone. After 48 h incubation, cells were harvested and
resuspended in PBS for flow cytometric analysis
(Fig. 3B). Cells cultured with medium or immuno-
modulating agents alone showed no increase in MFI
(Fig. 4A). IFN-
-treated cells had a 12-fold increase in
MFI over medium alone. Furthermore, cells cultured
with IFN-
in combination with TNF- or IL-1
showed a significant two-fold increase (P<0.001) in
MFI above that of IFN-
alone. LPS in combination
with IFN-
had no effect on MFI. Transfected cells
were tested for IDO activity in parallel and showed a
similar increase in IDO activity to that of untransfected
cells. While TNF-, IL-1, and LPS induced no IDO
activity when used alone, both TNF- and IL-1
induced two-fold increases in IDO activity in
IFN-treated cells (Fig. 4B).
To correlate IDO activity with GFP expression,
duplicate sets of enriched pEGFP/IDOp-transfected
cells were cultivated with or without IFN-
(10 ng/ml)
in the presence of increasing concentrations of TNF-
(0.005–50 ng/ml). After 48 h incubation, one set of
stimulated cells was tested for IDO activity while the
other set was analysed for GFP expression. A dose-
dependent increase in IDO activity was observed in the
transfected HeLa cells similar to that observed in
untransfected cells (Fig. 5). This dose-dependent
increase in IDO activity correlated with MFI that was
quantified in parallel cultures at all but the highest
CYTOKINE, Vol. 12, No. 6 (June, 2000: 588–594)
C
5 50 0 0.003 0.03 0.3 3 0 0.01 0.1 1 10
IL-1 (ng/ml) LPS (ng/ml)
TNF- concentration (r2 =0.92 and 0.82 for TNF-
concentrations <50 ng/ml and c50 ng/ml, respect-
ively). When IFN-
-stimulated cells were treated with
TNF- at 50 ng/ml, MFI continued to increase, but
IDO activity consistently decreased.
DISCUSSION
While previous studies on the regulation of expres-
sion of the IDO gene in response to combined treat-
ment with IFNs and pro-inflammatory cytokines were
performed in cells of a macrophage lineage,13–16 some
studies suggested that TNF- enhanced the anti-
microbial effects of IFN-
in an epithelial-like cell line
(HEp-2) by a tryptophan-dependent mechanism.9,19
Although increased IDO activity was measured in only
one of these studies,19 in both, administration of excess
tryptophan was able to reverse the enhanced anti-
microbial effect of combined IFN-
and TNF- treat-
ment, indicating that synergistic enhancement of IDO
activity could be demonstrated in non-macrophage cell
lines.
To extend these observations and more fully
characterize the enhancing effect of pro-inflammatory
cytokines on IFN-
-induced IDO activity, the HeLa
human epithelioid cervical carcinoma cell line was
chosen for study. Similar to what has been observed in
macrophages (Currier et al., unpublished data),13
TNF- and IL-1 synergistically increase IDO activity
in IFN-
-treated HeLa cells. Furthermore, the concen-
trations of IL-1 and TNF- that maximally enhanced
IDO activity in macrophages and HeLa cells were
identical. However, there are some differences in IDO
induction in macrophages and HeLa cells. While
IFN-
at 1 ng/ml alone induced no IDO activity in
macrophages, maximum IDO activity was induced
when it was used in combination with IL-1 or TNF-.
 Activation of IDO in epithelial cells / 591

80
A

70

60

50

40

30

20
Relative cell number

10

0
B

70

60

50

40

30

20

10

0
100 101 102 103 104
Fluorescence
Figure 3. GFP expression is under the control of the IDO promoter.

HeLa cells were transfected with the pEGFP/IDOp reporter vector, cultured with medium alone, IFN-
(10 ng/ml), or IFN-
combined with
TNF- (5 ng/ml) for 48 h, and analysed for GFP expression by flow cytometry. Representative histogram overlays of unstimulated (– – – –) and
IFN-
-stimulated cells ( ) (A) and cells stimulated with IFN-
( ) or combined IFN-
and TNF- ( ) (B) are presented.

This differs from HeLa cells, in which at least 3 ng/ml
of IFN-
was required for enhancement of IDO
activity by TNF- and IL-1, and 30 ng/ml of IFN-
in
combination with TNF- or IL-1 was required for
maximum IDO induction. Furthermore, HeLa cells did
not respond to LPS, even at 100 times the concen-
tration used to maximally enhance IDO activity in
IFN-
-treated macrophages. This was not surprising,
since macrophages are known for their responsiveness
to sub-nanogram concentrations of LPS.20
Initial work in characterizing the molecular mech-
anism involved in synergistic cytokine interactions
resulting in increased IDO enzyme activity evaluated the
relative changes in mRNA occurring in stimulated hu-
man macrophages and THP-1 cells by RT-PCR. When
these cells were stimulated with IFN-
in combination
with IL-1 or LPS, each generated a dose-dependent
increase in IDO mRNA expression, and this increase
correlated with the increase in IDO activity.14,15
Inasmuch as IFN-
alone transcriptionally activates of
592 / Babcock and Carlin
160
140
120
Fluorescence (MFI)
100
80
60
40
20
0 [TNF] (ng/ml)
50
Specific catabolism (%)
40
30
20
10
0
Medium TNF IL-1 LPS
Figure 4. Comparison of GFP expression and IDO activity.
Triplicate samples of HeLa cells were cultured with IFN-
(10 ng/ml)
(hatched bars) or medium alone (open bars). Cells also received
TNF- (5 ng/ml), IL-1 (3 ng/ml) or LPS (10 ng/ml). After 48 h
incubation, one set of samples were analysed for GFP by flow
cytometry (A) and another set of samples were analysed for IDO
activity (B). The results from one of several similar experiments
(meanSD) are presented.
the IDO gene,21 these results suggest that the increase
in IDO activity in response to combined treatments in
these cells was at least in part due to transcriptional
activation of the IDO gene.
Although much of the work on synergistic
cytokine induction of IDO activity has been in macro-
phage cultures, these cells do not support the growth of
Chlamydia trachomatis biovars relevant to trachoma
and genital chlamydial infections. Since HeLa cells
were found to respond similarly to macrophages with
respect to enhancement of IDO activity by IL-1 and
TNF-, and since HeLa cells are a standard for
cultivation of Chlamydia trachomatis and investigation
of Chlamydia-host cell interactions, they were used to
determine if the increase in IDO activity observed was
due to transcriptional activation of the IDO gene.
Following transfection of HeLa cells with the pEGFP/
IDOp reporter vector, IFN-inducible transfectants
were selected by fluorescence-activated cell sorting and
maintained in culture with G418. While cells treated
with TNF- or IL-1 alone showed no increase in MFI,
CYTOKINE, Vol. 12, No. 6 (June, 2000: 588–594)
220 35
200
30
Specific catabolism (%) ( )
180
160 25
Fluorescence ( )
140
20
120
100 15
80
10
60
40
5
20
0 0
0 0.005 0.05 0.5 5 50
Figure 5. TNF-
concentration-dependent increases in GFP
expression and IDO activity.
Duplicate sets of transfected HeLa cells were cultured in triplicate
with IFN-
(10 ng/ml) or medium alone, in the presence of increasing
concentrations of TNF-. After 48 h incubation, one set of cells were
analysed for IDO activity and the other set was analysed for GFP by
flow cytometry. The results from one of several experiments
(meanSD) are presented.
cells cultured with IFN-
in combination with TNF-
or IL-1 had increased MFI above that induced by
IFN-
alone. Furthermore, this increase in MFI was
associated with the increase in IDO activity that was
observed in the transfected cells. In conjunction with
previous studies which have shown that TNF- and
IL-1 increase both IDO mRNA and IDO enzymatic
activity in IFN-treated cells,13–16 the fact that GFP
under the regulation of the IDO 5 flanking region
possesses similar responsiveness suggests that TNF-
and IL-1 increase transcriptional activity of the IDO
gene promoter in IFN-
-treated cells.
This correlation was extended by quantifying
dose-dependent increases in MFI and IDO activity in
the response to increasing concentrations of TNF- in
IFN-
-stimulated HeLa cells. Although MFI increased
in IFN-induced cells in proportion to the IDO activity,
at 50 ng/ml of TNF-, IDO activity in the cells
decreased while the MFI continued to rise. It may be
that this amount of TNF- was toxic to HeLa
cells, resulting in decreased tryptophan transport and
decyclization. However, GFP that has been already
transcribed and translated before the cells were
damaged would remain active, resulting in the
increased fluorescence at the highest TNF- concen-
tration tested.
The mechanism by which TNF- and IL-1
enhance the transcriptional activity of the IDO gene
remains unknown. The IDO gene has two interferon-
stimulated response elements (ISRE) and a gamma
activation sequence (GAS) element that are required
for the induction of IDO activity by IFN-
.22,23 There
may be other regulatory elements present in the IDO
promoter that could be involved in potentiating IDO

activity by TNF- and IL-1. Alternatively, these
proinflammatory cytokines might upregulate IFN-

receptor expression,24 resulting in greater sensitivity to
sub-optimal concentrations of IFN-
.
MATERIALS AND METHODS
Cytokines and Reagents
Recombinant IFN-
(specific activity=1.6107 U/mg
protein, <0.4 ng LPS/mg) was provided by Biogen
(Cambridge, MA, USA). Recombinant human TNF- [50%
effective dose (ED50, the concentration of cytokine required
for 50% maximal response)=0.02–0.05 ng/ml] was purchased
from R&D Systems (Minneapolis, MN, USA). Recombinant
human IL-1- (specific activity=3108 U/mg protein,
<0.5 ng LPS/mg) was generously provided by Peter
Lomedico of Hoffman-LaRoche (Nutley, NJ, USA).
Dulbecco’s minimal essential eagle medium (DMEM) and
G418 sulfate were obtained from Mediatech (Herndon,
VA, USA). Streptomycin sulfate, gentamicin sulfate, Hanks’
balanced salt solution (HBSS), LPS (Escherichia coli 026:B6)
and phosphate buffered saline (PBS) were purchased from
Sigma Chemical Company (St. Louis, MO, USA). Charac-
terized fetal bovine serum (FBS) was purchased from
HyClone (Logan, UT, USA). Radiolabeled [5-3H]-
tryptophan (specific activity=20 Ci/mmol) was obtained
from New England Nuclear (Boston, MA, USA). Taq DNA
polymerase and kanamycin monophosphate were purchased
from Fisher Scientific (Pittsburgh, PA, USA). Restriction
enzymes EcoR1 and BamH1 were purchased from Promega
(Madison, WI, USA) and the T4 DNA ligase was obtained
from New England Biolabs (Beverly, MA, USA). The
enhanced GFP expression vector (pEGFP-1), which contains
neomycin and kanamycin resistance markers, was purchased
from Clontech (Palo Alto, CA, USA).
Cell Culture
The HeLa human cervical epithelial cell line, obtained
from American Type Culture Collection (Rockville, MD,
USA), was routinely maintained in DMEM supplemented
with 10% FBS (v/v), 100 mg/ml of gentamicin, and 100 g/ml
of streptomycin (complete medium). Cells were maintained
at a cell density between 3105 and 106 cells/ml at 37C in
5% CO2.
Induction of IDO activity
IDO activity in HeLa cells was determined in a manner
similar to that previously described.25 Cells (105 cells/ml of
complete medium) were cultivated in 24-well tissue culture
plates, at a final volume of 1 ml. To determine the effects of
immunomodulating agents on IFN-induced IDO activity in
HeLa cells, IFN concentration was held constant, while the
amount of immunomodulatory agents was varied. Wells
received IFN-
(10 ng/ml) or medium alone, with increasing
concentrations of LPS (0.01–10 ng/ml), TNF- (0.005–50 ng/
ml), IL-1 (0.003–3 ng/ml), or medium. To determine the
effect of immunomodulating agents on IFN responsiveness,
the concentration of immunomodulatory agents was held
constant, while the amount of IFN was varied. Cells were
Activation of IDO in epithelial cells / 593
treated with increasing concentrations of IFN-
(1–30 ng/ml)
alone or in combination with TNF (5 ng/ml), IL-1 (3 ng/ml)
or LPS (10 ng/ml). Plates were then incubated for 48 h at
37C, at which time the IDO activity was determined.
Determination of IDO activity
After incubation, the medium in each well was replaced
with 0.4 ml HBSS containing [5-3H]tryptophan (1 Ci/ml)
and a 25 M unlabeled tryptophan carrier.8 Some wells
received radiolabeled medium without cells to determine the
amount of background tryptophan decyclization that
occurred. Cultures were incubated another 4 h and then
supernatants were collected and frozen at
20C until
further analysis by reverse-phase high pressure liquid
chromatography (HPLC) to determine the conversion of
tryptophan to its decyclized metabolites, L-N-
formylkynurenine and kynurenine.26 These metabolites were
quantified by flow-through scintillation spectroscopy using
an automated HPLC (Isco, Lincoln, NE, USA) equipped
with a radioisotope detector (Radiomatic Instruments,
Tampa, FL, USA). Enzymatic activity was expressed as a
percentage of the specific tryptophan catabolism, calculated
using the following equation:
percentage specific catabolism=
cpmmetabolities
cpmbackground
100,
cpmtryptophan
where cpmmetabolites corresponds to the cpm present in the
metabolite fractions, cpmbackground represents cpm resulting
from nonspecific breakdown of tryptophan to metabolites,
and cpmtryptophan equals the cpm from tryptophan prior to
metabolic activity. Statistical significance was determined by
t-test (two-tailed) for independent samples.
Generation of the pEGFP/IDOp Reporter Vector
The 1245 bp promoter region of the human IDO gene21
was graciously provided by Dr Milton Taylor (Indiana
University, Bloomington, IN, USA) within the pINDOCAT
vector. From this plasmid, the promoter region was amplified
by PCR as previously described,22 ligated into the EcoR1/
BamH1 sites of the EGFP-1 vector, and selected in JM109
competent cells. To confirm insertion of the promoter into
the 4.2 kb plasmid, restriction digestion and PCR amplifi-
cation of the promoter from the pEGFP/IDOp vector was
performed.
Transfection and selection of IFN-responsive
transfectants
The pEGFP/IDOp construct was purified on a Qiagen
maxi column (Qiagen Inc., Santa Clarita, CA, USA), and
transfected into HeLa cells using an Effectene transfection kit
(Qiagen). At 24 h after transfection, cells containing pEGFP/
IDOp were cultivated in complete medium supplemented
G418 sulfate (1.5 mg/ml) to maintain the plasmid within the
cultured cells. Following induction of GFP expression by
IFN-
, transfectants were selected based on fluorescent inten-
sity using a Coulter EPICS 753 Flow Cytometric Sorter.
Flow cytometric analysis of IFN-
induced cells allowed for
quantitation of the GFP-expressing population.
594 / Babcock and Carlin
Assessment of promoter activity in transfectants
Transfected HeLa cells (2105 cells/ml) were plated in
24-well plates and cultured with or without IFN-
(10 ng/ml)
in the presence of the TNF- (5 ng/ml), IL-1 (3 ng/ml), LPS
(10 ng/ml) or medium alone. After 48 h incubation, half the
cultures were analysed for IDO activity as previously
described. The other half of the cultures were harvested with
EDTA/trypsin and resuspended in 400 l of PBS for flow
cytometric analysis using a FACScan flow cytometer (Becton
Dickinson, San Jose, CA, USA). Dead cells and debris were
excluded from analysis based on forward angle and side
scatter light gating. At least 10 000 gated events were col-
lected for analysis of each sample. Excitation of the enhanced
GFP was achieved using an argon laser tuned to 488 nm.
Green fluorescence was recorded in the FL1 emission channel
containing a 530/30 nm filter. CELLQuest software (Becton
Dickinson) was used for analysis of the data. Mean fluor-
escence intensity was calculated using the linear value for
each event.
Acknowledgements
This work was supported by Public Health Service
Grant No. AI/OD39727 from the National Institute of
Allergy and Infectious Diseases (JMC) and a Grant-in
Aid from Sigma Xi (TAB). We thank Sondra
Karipides for synthesizing the oligonucleotide primers
used in this study, and Dr. George Babcock at the
University of Cincinnati Medical Center for the use of
and assistance with the cell sorter.
REFERENCES
1. Nathan CF, Murray HW, Wiebe ME, Rubin BY (1983)
Identification of interferon-
as the lymphokine that activated
human macrophage oxidative metabolism and antimicrobial activity.
J Exp Med 158:670–689.
2. Adams LB, Hibbs Jr. JB, Taintor RR, Krahenbuhl JL
(1990) Microbiostatic effects of murine-activated macrophages for
Toxoplasma gondii: role of synthesis of inorganic nitrogen oxides
from L-arginine. J Immunol 144:2725–2729.
3. Green SJ, Meltzer MS, Hibbs Jr. JB, Nacy CA (1990)
Activated macrophages destroy intracellular Leishmania major
amastigotes by an -arginine-dependent killing mechanism.
J Immunol 144:278–283.
4. Pfefferkorn ER (1982) Interferon-
blocks the growth of
Toxoplasma gondii in human fibroblasts by inducing host cells to
degrade tryptophan. Proc Natl Acad Sci USA 81:908–912.
5. Shimizu T, Nomiyama S, Hirata F, Hayaishi O (1978)
Indoleamine 2,3-dioxygenase. Purification and some properties.
J Biol Chem 253:4700–4706.
6. Pfefferkorn ER, Rebhun S, Eckel M (1986) Characteriz-
ation of an indoleamine 2,3-dioxygenase induced by gamma-
interferon in cultured human fibroblasts. J Interferon Res 6:
267–279.
7. Rapoza PA, Tahija SG, Carlin JM, Miller SL, Padilla ML,
Byrne GI (1991) Effect of interferon on a primary conjunctival
epithelial cell model of trachoma. Invest Ophthalmol Vis Sci
32:2919–2923.
CYTOKINE, Vol. 12, No. 6 (June, 2000: 588–594)
8. Byrne GI, Lehmann LK, Landry GJ (1986) Induction of
tryptophan catabolism is the mechanism for gamma-interferon-
mediated inhibition of intracellular Chlamydia psittaci replication in
T24 cells. Infect Immun 53:347–351.
9. Summersgill JT, Sahney NN, Gaydos CA, Ramirez JA
(1995) Inhibition of Chlamydia pneumoniae growth in HEp-2 cells
pre-treated with gamma interferon and tumor necrosis factor alpha.
Infect Immun 63:2801–2803.
10. Schmitz JL, Carlin JM, Borden EC, Byrne GI (1989)
Beta-interferon inhibits Toxoplasma gondii growth in human
monocyte-derived macrophages. Infect Immun 57:3254–3256.
11. Thomas SM, Garrity LF, Brandt CR, Schobert CS, Feng
GS, Taylor MW, Carlin JM, Byrne GI (1993) IFN-
-mediated
antimicrobial response: indoleamine 2,3-dioxygenase-deficient
mutant host cells no longer inhibit intracellular Chlamydia spp. or
Toxoplasma growth. J Immunol 150:5529–5534.
12. Gupta SL, Carlin JM, Pyati P, Dai W, Pfefferkorn ER,
Murphy MJ Jr (1994) Antiparasitic and antiproliferative effects
of indoleamine 2,3-dioxygenase enzyme expression in human
fibroblasts. Infect Immun 62:2277–2284.
13. Hissong BD, Byrne GI, Padilla ML, Carlin JM (1995)
Upregulation of indoleamine 2,3-dioxygenase in human macrophage
cultures by lipopolysaccharide, muramyl tripeptide and
interleukin-1. Cell Immunol 160:264–269.
14. Hu B, Hissong BD, Carlin JM (1995) Interleukin-1
enhances indoleamine 2,3- dioxygenase activity by increasing specific
mRNA expression in human mononuclear phagocytes. J Interferon
Cyto Res 15:617–624.
15. Hissong BD, Carlin JM (1997) Potentiation of interferon-
induced IDO mRNA in human mononuclear phagocytes by
lipopolysaccharide and interleukin-1. J Interferon Cyto Res 17:
387–393.
16. Werner-Felmayer G, Werner ER, Fuchs D, Hausen A,
Reibnegger G, Wachter H (1990) Neopterin formation and
tryptophan degradation by a human myelomonocytic cell line
(THP-1) upon cytokine treatment. Cancer Res 50:2863–2867.
17. Carlin JM, Weller JB (1995) Potentiation of interferon-
mediated inhibition of Chlamydia infection by interleukin-1 in
human macrophage cultures. Infect Immun 63:1870–1875.
18. Carlin JM, Borden EC, Byrne GI (1989) Interferon-induced
indoleamine 2,3-dioxygenase activity inhibits Chlamydia psittaci
replication in human macrophages. J Interferon Res 9:329–337.
19. Shemer-Avni Y, Wallach D, Sarov I (1989) Reversion of the
antichlamydial effect of tumor necrosis factor by tryptophan and
antibodies to beta interferon. Infect Immun 57:3484–3490.
20. Kornbluth RS, Edgington TS (1986) Tumor necrosis factor
production by human monocytes is a regulated event: induction of
TNF--mediated cellular cytotoxicity by endotoxin. J Immunol
137:2585–2591.
21. Dai W, Gupta SL (1990) Regulation of indoleamine 2,3
dioxygenase gene expression in human fibroblasts by interferon-
.
J Biol Chem 265:19871–19877.
22. Konan KV, Taylor MW (1996) Importance of the two
interferon-stimulated response element (ISRE) sequences in the
regulation of the human indoleamine 2,3- dioxygenase gene. J Biol
Chem 271:19140–19145.
23. Chon SY, Hassanain HH, Pine R, Gupta SL (1995)
Involvement of two regulatory elements in interferon-
-regulated
expression of human indoleamine 2,3- dioxygenase gene. J Interferon
Cyto Res 15:517–521.
24. Krakauer T, Oppenheim JJ (1993) IL-1 and tumor necrosis
factor-alpha each up-regulate both the expression of IFN-gamma
receptors and enhance IFN-gamma-induced HLA-DR expression on
human monocytes and a human monocytic cell line (THP-1).
J Immunol 150:1205–1211.
25. Carlin JM, Borden EC, Sondel PM, Byrne GI (1989)
Interferon-induced indoleamine 2,3-dioxygenase activity in human
mononuclear phagocytes. J Leuk Biol 45:29–34.
26. Yong S, Lau S (1979) Rapid separation of tryptophan,
kynurenine and indoles using reversed phase high performance liquid
chromatography. J Chromatogr 175:343–347.