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. Author manuscript; available in PMC: 2007 Jan 1.
Published in final edited form as: J Interferon Cytokine Res. 2006 Jan;26(1):53–62. doi: 10.1089/jir.2006.26.53

Upregulation of IFN-γ Receptor Expression by Proinflammatory Cytokines Influences IDO Activation in Epithelial Cells

KARI ANN SHIREY 1, JOO-YONG JUNG 1, GREGORY S MAEDER 1, JOSEPH M CARLIN 1
PMCID: PMC1550344  NIHMSID: NIHMS10695  PMID: 16426148

Abstract

Interferon-γ (IFN-γ) induces the enzyme indoleamine dioxygenase (IDO) in a variety of human cell types. Furthermore, tumor necrosis factor-α (TNF-α) and interleukin-1 (IL-1) synergistically increase IFN-induced IDO activity. Inasmuch as cytokines can upregulate cytokine receptor expression, one mechanism of cytokine synergy may be at the level of receptor expression. To test the hypothesis that this mechanism of IDO regulation is active in epithelial cells, HeLa cells were treated with IFN-γ, TNF-β, or IL-1 α to determine optimal cytokine concentrations and time for maximal cytokine receptor expression. Flow cytometric analysis with antibodies to receptors for IFN-γ, TNF-α, or IL-1 β indicated that each cytokine upregulated expression of the other cytokine receptors by 4 h, with maximal expression observed between 16 and 20 h after cytokine treatment. Furthermore, increases in IFN-γ receptors (IFNGR) induced by IL-1 β were found to be dependent on NF-κB transactivation. To determine if increases in IFNGR expression alone contributes to synergistic IDO induction, cells were stimulated with IL-1 β to upregulate receptor expression, and the NF-κB concentration was allowed to return to basal levels. When treated with IFN-γ, enhanced Stat1 signaling and IDO induction were still observed, indicating that increased cytokine receptor expression contributes to synergistic increases in IDO activity.

INTRODUCTION

Interferon-γ (IFN-γ) induces a variety of antimicrobial effector mechanisms, including the activation of mononuclear phagocytes to release reactive oxygen intermediates,1 the activation of inducible nitric oxide synthase (iNOS), which catalyzes the production of various antimicrobial reactive nitrogen intermediates,2,3 the induction of intracellular iron deficiency by a process that involves the downregulation of transferring receptors and iron transport,4 and the induction of indoleamine 2,3-dioxygenase (IDO). IDO catalyzes the decyclization of l-tryptophan into N-formylkynurenine,5 thereby limiting the availability of this essential amino acid and, consequently, inhibiting the growth of tryptophan auxotrophs. IFN-γ-induced IDO activity is effective in restricting the growth of the intracellular pathogens Chlamydia and Toxoplasma, as well as group B Streptococcus.6-8 Furthermore, replenishment of tryptophan to infected cells early in infection can restore microbial growth,9 suggesting that decyclization of l-tryptophan is critical to the ability of IFN-γ to inhibit the replication of some microbial pathogens.

The interactions between IFN-γ and other cytokines are important in regulating antimicrobial mechanisms; combinations can be either antagonistic or synergistic in their modulation of responses.10,11 Several inflammatory agents, such as interleukin-1 (IL-1), tumor necrosis factor-α (TNF-α), and lipopolysaccharide (LPS), enhance IFN-γ-induced IDO activity, resulting in increased inhibition of tryptophan auxotrophs in both epithelial and myeloid cells.12,13 Increased transcriptional activation of the IDO gene has been observed in cells treated with combinations of IFN-γ and inflammatory cytokines,14 although the molecular mechanism for this synergy has not been fully determined. Some of the synergy is due to an increase in specific transcription factors, notably Stat1α, IFN regulatory factor-1 (IRF-1), and NF-κB (C.M. Robinson et al., unpublished observations).15,16 The IDO gene contains multiple binding elements for Stat1 and IRF-1.17,18 Although combined treatment with IFN-γ and TNF-α causes an immediate increase in Stat1 κ transactivation,15,16 activation of IRF-1 requires binding of both Stat1 and NF-κB to their respective binding elements within the IRF-1 regulatory region.19,20 Furthermore, the increase in nuclear NF-κB in response to TNF-α is essential to the increase in IRF-1 activation. When NF-κB transactivation is inhibited by interference with proteasomal degradation of I κB,21,22 IRF-1 upregulation and subsequent IDO upregulation are blocked (C. M. Robinson et al., unpublished observations). The mechanism for synergy, however, may also include an increase in cytokine receptor expression.23 Cytokines, such as IL-1 and TNF-α, may stimulate an increase in expression of the IFN-γ receptor (IFNGR), resulting in an enhanced ability of the cells to respond to lower concentrations of IFN-γ. Although such changes in IFNGR expression have been well characterized in mononuclear cells,23 cross-regulation of cytokine receptors in nonimmunocompetent epithelial cells is less clear.

The objectives of this study were twofold: to assess whether cytokine receptor expression in epithelial cells is cross-regulated and to correlate changes in cytokine receptor expression with IDO synergy. The results of this study indicate that the cytokines tested are able to cross-regulate cytokine receptor expression, resulting in increased signaling by IFN-γ and enhanced activation of the IDO gene.

MATERIALS AND METHODS

Cytokines and immunoreagents

Human recombinant TNF-α (specific activity 2 × 107 U/mg, <0.1 ng LPS/mg) and IL-1 β (specific activity 1 × 107 U/mg, <0.1 ng LPS/mg) were purchased from Peprotech (Rocky Hill, NJ). IFN-γ (specific activity 108 U/mg, <0.4 ng LPS/mg) was provided by Biogen Corp. (Cambridge, MA). Rabbit polyclonal antihuman IFNGR alpha chain (IFNGR-1) and beta chain (IFNGR-2) were purchased from Research Diagnostics Inc. (Flanders, NJ). Goat polyclonal antihuman TNF-α receptor I (TNFR) and fluorokine biotinylated IL-1 β and TNFα kits were purchased from R&D Systems (Minneapolis, MN). Rabbit polyclonal antihuman IL-1 receptor I (IL-1RI) and receptor II (IL-1RII) were purchased from Rockland Immunochemicals (Gilbertsville, PA). Mouse monoclonal antihuman NF-κB p50 IgG and goat polyclonal anti-pStat1 (Tyr701) IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Fluorescein isothiocyanate (FITC)-conjugated sheep antimouse IgG F(ab′)2, phycoerythrin (PE)-conjugated goat antirabbit IgG F(ab′)2, FITC-conjugated rabbit antigoat IgG F(ab′)2, rabbit IgG, goat IgG, and mouse IgG were purchased from Sigma Chemical Co. (St. Louis, MO). PE-conjugated donkey antigoat IgG F(ab′)2 was purchased from Jackson ImmunoResearch (West Grove, PA). Photoprobe was purchased from Vector Laboratories (Burlingame, CA), and FITC-conjugated streptavidin was purchased from Southern Biotechnology (Birmingham, AL). The 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) stain was purchased from Pierce Biotechnology (Rockford, IL). Proteasome inhibitor I (PSI) was purchased from Calbiochem (San Diego, CA).

Cell cultivation

HeLa 229 cells, obtained from American Type Culture Collection (Rockville, MD), were cultivated in minimal essential medium (MEM) supplemented with 10% fetal bovine serum (FBS) (v/v), 10 μg/ml gentamicin sulfate, and 100 γg/ml streptomycin sulfate (complete medium). Cells were maintained at 106 cells/ml at 37°C in 5% CO2 in air. HeLa cells transfected with pGASinsIDO/EGFP-C1, encoding the enhanced green fluorescent protein (GFP) under regulation of the IDO promoter, were cultivated in complete medium containing G418 (100 μg/ml) for continual selection of transfected cells.14,16

Cytokine dose-response and time course

To determine the effect of cytokine concentration on receptor expression, HeLa 229 cells (106 cells/ml) were treated with increasing concentrations of IL-1 β (1-100 ng/ml), TNF-α (2-200 ng/ml), IFN-γ (1-100 ng/ml), or with complete medium alone for 24 h at 37°C in 5% CO2. The medium was replaced with 0.2% EDTA in phosphate-buffered saline (PBS) (w/v), and the cells were collected by gentle scraping with rubber policemen. After the cells were fixed in 1% p-formaldehyde for 20 min, they were washed and incubated with antireceptor antibodies for 45 min, FITC-conjugated or PE-conjugated secondary antibodies for 45 min, and analyzed for receptor expression using a FACScan flow cytometer (Becton Dickinson, San Jose, CA). CellQuest software (Becton Dickinson) was used for analysis of the data.

To assess changes in receptor expression over time, HeLa cells (106 cells/ml) were treated with IL-1 β (100 ng/ml), TNF-α (20 ng/ml), IFN-γ (10 ng/ml), or complete medium alone for up to 48 h. These concentrations were found to generate maximal increases in cytokine receptor expression. The cells were collected at various times, stained with antireceptor antibodies as described, and analyzed by flow cytometry. Fold increase in receptor expression was calculated by comparing the mean fluorescence intensity of cytokine-treated cells with the mean fluorescence intensity (MFI) of cells cultured in complete medium alone at each time point.

Biotinylation of IFN-γ

IFN-γ was diluted in 10 mM Tris, pH 8.0, 1 mM EDTA (TE) to a final concentration of 100 ng/ml. Biotin photoprobe was added to IFN-γ at a 20:1 biotin/IFN-γ ratio and UV photocoupled for 30 min at 2 cm distance from the UV lamp (30 W) according to manufacturer’s instructions.

Cytokine binding assay

To quantify changes in the capacity of cells to bind cytokine after upregulation, HeLa cells (106 cells/ml) were treated with IL-1 β (100 ng/ml), IFN-γ (10 ng/ml), or complete medium alone for 20 h in triplicate to allow for maximum receptor expression. Parallel sets of cells were collected by scraping and then incubated with biotinylated-IFN-γ, IL-1β, or TNF-α or with antireceptor antibodies for 1 h on ice. FITC-conjugated streptavidin was added to the cells receiving biotinylated cytokines at a streptavidin/biotinylated cytokine ratio of 10:1, and PE-conjugated antibodies were added to cells receiving antireceptor antibodies. An additional 1 h of incubation on ice followed to allow the streptavidin and the secondary antibodies to bind to biotin and primary antibodies, respectively. The cells were washed with PBS and analyzed by flow cytometry.

Nuclei isolation and transcription factor translocation

To determine the amount of NF-κB translocated into the nucleus upon IL-1 β stimulation, the amount of NF-κB in isolated nuclei was quantified using a protocol published on the NCI ETIB Flow Cytometry Core Laboratory website. HeLa cells (106 cells/ml) were treated with IL-1 β (100 ng/ml) or complete medium for 0, 1, 12, or 24 h before harvesting. The cells were incubated on ice in cold PBS for 1 h and collected by gentle scraping with rubber policemen before being washed with PBS by centrifugation. Nuclei were extracted by suspending the cell pellets in cold nuclei extraction buffer (320 mM sucrose, 5 mM MgCl2, 10 mM HEPES, 1% Triton X-100) and incubating on ice for 10 min. The nuclei were pelleted by centrifugation and washed with nuclei wash buffer (320 mM sucrose, 5 mM MgCl2, 10 mM HEPES). Nuclei yield was determined by microscopic examination using DAPI stain. The extracted nuclei were incubated with a 1:50 diluted anti-NF-κB p50 PE-conjugated primary antibody in nuclear labeling buffer (320 mM sucrose, 5 mM MgCl2, 10 mM HEPES, 1% bovine serum albumin [BSA], 0.1% sodium azide) overnight. The nuclei were washed with labeling buffer and analyzed for the presence of NF-κB p50 by flow cytometry.

To determine the amount of phosphorylated Stat1 (pStat1) found in the nucleus after induction with IFN-γ, HeLa cells (106 cells/ml) were treated with IL-1 β (100 ng/ml) or complete medium for 16 h. The cells were washed and incubated an additional 8 h in complete medium and then treated with IFN-γ (20 ng/ml) for 4 h before the nuclei were harvested in the same manner as described. The extracted nuclei were incubated with a 1:100 diluted anti-pStat1 primary antibody in nuclear labeling buffer overnight. The nuclei were washed with labeling buffer and incubated with a 1:500 diluted secondary antibody for 2 h. The nuclei were washed again in labeling buffer and analyzed for the presence of pStat1 by flow cytometry.

IDO quantitation

To assess the effect of cytokine stimulation by IL-1 β or TNF-α on IDO induction, HeLa cells (105 cells/ml) were cultivated with IL-1 β (100 ng/ml) or complete medium alone and incubated for 16 h. After 16 h, the cells were washed with PBS and incubated a further 8 h in complete medium. At the end of this 8 h, IFN-γ was added in increasing concentrations (up to 100 ng/ml) and incubated for an additional 24 h. For determination of IDO activity, medium was replaced with 0.4 ml Hanks balanced salt solution (HBSS) containing [5-3H]tryptophan (1μCi/ml, specific activity 20 Ci/mmol) (MP Biomedicals, Irvine, CA) and 25μM tryptophan carrier. After an additional 4 h incubation, supernatants were collected and analyzed for tryptophan metabolites by reversed phase HPLC.24 Briefly, aliquots (50μl) were injected into aμBondapak C18 column (Millipore, Bedford, MA) and eluted with 1 mM KH2PO4 buffer, pH 4.0, containing 10% MeOH, at a flow rate of 1.6 ml/min. Radioactivity in tryptophan and metabolite fractions was quantified by flowthrough scintillation spectroscopy using an HPLC (Isco, Lincoln, NE) equipped with a radioisotope detector (Radiomatic instruments, Tampa, FL). The percentage of specific tryptophan catabolism was calculated by the following equation:

Percentage specific catabolism=cpmmetabolites−cpmbackgroundcpmtryptophan×100

where cpmmetabolites corresponds to the counts per minute (cpm) present in metabolite fractions, cpmbackground represents cpm resulting from nonspecific breakdown of tryptophan to metabolites, and cpmtryptophan equals the total cpm from all fractions. All statistical analyses were performed with Microsoft Excel X for Mac software (Redmond, WA) using two-tailed t-tests for independent samples.

NF-κB inhibition by proteasome inhibitor

NF-κB may be important in cytokine-induced receptor increase as well as in IDO transcriptional activation. To characterize the effect of receptor upregulation on IDO transcriptional activation, HeLa cells stably transfected with pGASinsIDO/EGFP-C1 were seeded into 24-well plates (105 cells/ml). Cells were treated with IL-1 β (100 ng/ml) for 8 h. The medium was then replaced with fresh medium for 16 h to permit nuclear NF-κB to return to basal levels. Finally, the cells were treated with IFNγ (20 ng/ml) for 6 h to induce IDO reporter activity. In some experiments, PSI (0-3μM) was added along with IL-1 β during the first 8 h incubation to inhibit NF-κB transactivation and subsequent receptor upregulation. In other experiments, PSI (0-3μM) was added along with IFN-γ during the final 6 h incubation to assess the effect of NF-κB transactivation on IFN-γ-induced IDO reporter activity. After the final incubation, the cells were harvested, and GFP expression was analyzed by flow cytometry.

To assess the role of NF-κB transactivation in receptor increase, HeLa 229 cells were cultivated in medium alone or with IL-1 β (100 ng/ml) for 8 h. Cultures were simultaneously treated with increasing concentrations (0-3μM) of PSI for 8 h. Concentrations of PSI within this range have been shown to maintain the nuclear NF-κB concentration at steady-state levels in TNF-α-treated cells (C.M. Robinson et al., unpublished observations). The cells were harvested, and receptor expression was analyzed by antireceptor antibodies and flow cytometry.

Quantification of IFNGR mRNA by RT-PCR

Total RNA was extracted from HeLa 229 cells treated with complete medium, IL-1 β (100 ng/ml), or IL-1 β in combination with PSI I (3μM) for 8 h with TRI-reagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer’s instructions. Extracted total RNA was used as a template for the synthesis of cDNA by reverse transcription, using an MMLV-RT kit from Invitrogen (Carlsbad, CA), according to the manufacturer’s protocol. Oligonucleotide primers used for PCR amplification were synthesized by Integrated DNA Technologies, Inc. (Coralville, IA). Each specific primer was synthesized based on the NCBI sequence database and was chosen to flank an intron so that amplified DNA could be differentiated from genomic DNA. The primer sequences were as follows: IFNGR-1 forward primer: 5 ′-TTGGGAGTACCAGATCATGCCACA-3′; IFNGR-1 reverse primer: 5 ′-GCTGAAACACAGTACTGAGAATTC-3′;β-actin forward primer: 5 ′-GCACCACACCTTCTACAATGAG-3′;β-actin reverse primer: 5 ′-ATAGCACAGCCTGGATAGCAAC-3 ′.

The sizes of cDNA amplified by PCR are 501 bp and 166 bp for IFNGR-1 and β-actin, respectively.

cDNA was amplified by PCR as described previously15 in a 100-μl reaction mixture containing 50 mM KCl, 10 mM Tris, pH 9.0, 0.1% Triton X-100, 2 mM MgCl2, 0.4 mM dNTPs, 25 μCi ofα32P-dATP (10 mCi/ml, specific activity 800 Ci/mmol) (Perkin-Elmer, Boston, MA), 2.5 U Taq DNA polymerase (Eppendorf, Hamburg, Germany), and 0.2μM forward and reverse primers of IFNGR-1 orβ-actin. PCR amplification was performed in a Perkin-Elmer thermal cycler according to the following protocol: denaturation at 94°C for 1 min, annealing at 55°C for 2 min, and extension at 72°C for 2 min for 26 cycles, followed by a final 5 min extension at 72°C. PCR products (20μl) were separated electrophoretically on 8% polyacrylamide. Images were detected by scanning with a STORM 860 phosphorimager (Molecular Dynamics, Amersham Biosciences, Piscataway, NJ). Band intensities were analyzed using ImageQuant 5.2 software (Molecular Dynamics), and radioactivity in the IFNGR-1-specific band was normalized to that of theβ-actin band. The fold induction of mRNA expression was calculated with Microsoft Excel 2004 software.

Statistics

All statistical analyses were performed with Microsoft Excel 2004 software using two-tailed t-tests for independent samples.

RESULTS

IL-1β, TNF-α, and IFN-γ increase cytokine receptor expression in HeLa 229 cells

To determine if cytokine treatment affected cytokine receptor expression in epithelial cells, changes in receptor expression on HeLa 229 cells were detected using antireceptor antibodies that bind to specific cytokine receptor components and analyzed by flow cytometry. A range of IL-1 β and TNF-α concentrations was tested to determine the effects these cytokines had on IFNGR expression (Fig. 1). Significant increases (p < 0.001) in MFI of IFNGR-1 expression were observed at all concentrations of IL-1 β tested (Fig. 1A), with a 3.6-fold increase at 100 ng/ml. IL-1 β also significantly increased IFNGR-1 expression (p < 0.001) at concentrations ≥10 ng/ml, with a 2.3- fold increase at 100 ng/ml. Small but significant increases in expression were seen with TNF-α at all tested concentrations (Fig. 1B) of both IFNGR-1 (p < 0.03 at 2 ng/ml, p < 0.001 at 2-200 ng/ml) and IFNGR-2 (p < 0.002 at 2 ng/ml, p < 0.001 at 2-200 ng/ml), with a 1.3-fold increase in both chains at 200 ng/ml. To determine the optimal exposure time, the increase in IFNGR expression induced by IL-1 β and TNF-α was measured over a 48-h period. Increases in IFNGR expression were observed by 4 h posttreatment with either IL-1 β or TNF-α, with maximum expression occurring by 20 h posttreatment (Fig. 2). IL-1 β increased IFNGR-1 and IFNGR-2 expression by 2.5-fold and 2.7-fold, respectively. TNF-α stimulated a maximum 1.3- fold increase in IFNGR-1 expression and 1.6-fold increase in IFNGR-2 expression at 20 h posttreatment.

FIG. 1.

FIG. 1

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Upregulation of cytokine receptor expression. HeLa 229 cells were treated in triplicate increasing concentrations of IFN-γ, IL-1β, TNF-α, or complete medium alone for 24 h. The relative amount of cytokine receptor expression was assessed by incubating cells with antibodies to specific receptor components followed by incubation with FITC-conjugated or PE-conjugated secondary antibodies and analyzing by flow cytometry. (A) Effect of IL-1 β on IFNGR expression. (B) Effect of TNF on IFNGR expression. (C) Effect of IFN-γ on TNFR expression. (D) Effect of IL-1 β on TNFR expression. (E) Effect of TNF-α on IL-1R expression. (F) Effect of IFN-γ on IL-1R expression. The results from one of three similar experiments (MFI\m=+-SD) are presented.

FIG. 2.

FIG. 2

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Upregulation of cytokine receptor expression over time. HeLa 229 cells were treated in triplicate with IFN-γ (10 ng/ml), IL-1 β (100 ng/ml), TNF-α (20 ng/ml), or complete medium alone for up to 48 h. Cytokine receptor expression (IFNGR-1, IFNGR-2, IL-1RI, IL-1RII, and TNFR) was assessed at several points by incubating cells with antibodies to specific receptor components, followed by incubation with FITC-conjugated or PE-conjugated secondary antibodies and analyzing by flow cytometry. The fold increase in cytokine receptor expression was calculated by comparing the mean fluorescence intensity of cytokine-stimulated cells relative to that of unstimulated cells. One of three experiments with similar results (fold increase in MFI ± SD) is presented.

To assess whether IL-1R expression was regulated similarly, IFN-γ and TNF-γ were tested to determine their effects on the expression of IL-1RI and IL-1RII (Fig. 1E, F), the signal transducing receptor and the decoy receptor of IL-1, respectively. Little variation in IL-1RI or IL-1RII expression was seen when treated with 1 ng/ml IFN-γ or 2 ng/ml TNF-α. However, significant 1.5-1.7-fold increases (p < 0.001) were seen in the expression of both receptors when treated with 10-100 ng/ml IFN-γ or 20-200 ng/ml TNF-α. A study of change in expression over a 48-h period (Fig. 2) revealed results similar to those obtained with IFNGR. IFN-γ induced a maximal increases in IL-1RI and IL-1RII by 16 h (2.2-fold and 2.4-fold increases, respectively). Maximum IL-1RI and IL-1RII expression in response to TNF-α> also occurred at 16 h posttreatment, with increases of 1.8-fold and 1.4 fold, respectively.

The effect of increasing concentrations of IFN-γ and IL-1 β on TNFR expression was also examined (Fig. 1C,D). Although minimal increases in TNFR expression were observed with either IFN-γ or IL-1 β at 1 ng/ml, a significant increase (p 0.001) was observed in cells treated with ≥10 ng/ml of either cytokine. The time of maximum TNFR expression was similar to that observed with IFNGR and IL-1R, with maximum expression observed 16 h posttreatment (Fig. 2).

Cytokine binding assay

Although increased binding of antibodies to cytokine receptor subunits suggests that receptor expression was upregulated, this approach does not indicate whether this increase correlates with an increased capacity to bind cytokine. To confirm that increased binding of antireceptor antibodies was due to increased receptor expression, an alternative technique of detecting changes in cytokine receptor expression was employed. HeLa 229 cells were stimulated with cytokines (IL-1 β for IFNGR, IFN-γ for IL-1R and TNFR) or with complete medium alone for 20 h to permit maximum receptor expression. Cytokine binding was assessed by first incubating the cells with biotinylated IFN-γ, IL-1β, or TNF-α, followed by FITC-conjugated streptavidin to bind to the biotin. Binding of biotinylated cytokines was compared to binding of antireceptor antibodies in parallel cultures (Fig. 3). Fold increases in binding were 2.0 and 2.0 for anti-IFNGR-1 and biotinylated IFN-γ, 2.3 and 1.9 in for anti-IL-1RI and biotinylated IL-1, and 1.4 and 1.5 in for anti-TNFR and biotinylated TNF-α, respectively, relative to binding measured in unstimulated cells. In each case, flow cytometry revealed that increases in cytokine binding were similar to increases in binding of antireceptor antibodies. This indicates that for each cytokine tested, the increased binding of antireceptor antibody in stimulated cells was due to an increase in receptors capable of binding their corresponding cytokine.

FIG. 3.

FIG. 3

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Upregulation of cytokine binding to stimulated cells. Parallel sets of HeLa 229 cells were cultivated with IL-1 β (100 ng/ml) to stimulate IFNGR expression, IFN-γ (10 ng/ml) to stimulate IL-1R and TNFR expression, or complete medium for 20 h. One set was then incubated with biotinylated-IFN-γ, IL-1β, or TNF-α for 1 h, followed by FITC-conjugated streptavidin (open bars). The other set was stained with the corresponding antireceptor antibody and PE-conjugated secondary antibody (hatched bars). The MFI of each was determined using flow cytometry. The results from one of several experiments with similar results (MFI ± SD) are shown.

IL-1-stimulated HeLa cells increase IDO activity

Because treatment with IL-1 β caused greater increases in IFNGR expression than did treatment with TNF-α, IL-1 β was chosen for further study with IDO. In a previous study of synergy between IFN-γ and TNF-α or IL-1 in upregulation of IDO activity in epithelial cells, cells were treated simultaneously with cytokines.14 If upregulation of IFNGR by IL-1 β is a mechanism of IDO synergy, however, it may be possible to separate the upregulation of IFNGR from transcriptional activation of the IDO gene temporally. When cells were stimulated with complete medium containing IL-1 β (100 ng/ml) for 16 h and then cultivated in complete medium alone, IFNGR expression remained stably elevated, a 2.1-2.3-fold greater MFI, for at least 48 h after removal of IL-1 from the system (data not shown). Therefore, if increased receptor expression influences IDO induction, sequential treatment of cells—first with IL-1 β to upregulate receptors, followed by IFN-γ to induce the activity— should cause enhanced IDO activity. To test this hypothesis, HeLa cells were incubated sequentially with or without IL-1 β for 16 h and then complete medium alone for 8 h prior to IFN-γ treatment. After 24 h incubation with IFN-γ, IDO activity was assessed by HPLC. As shown in Figure 4, cells treated first with IL-1 β before induction of IDO had increased enzyme activity compared with cells induced with only IFN-γ. A 3-fold increase in IDO induction was observed when cells were treated with IL-1 β prior to treatment with 3 ng/ml IFN-g=g.

FIG. 4.

FIG. 4

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Enhancement of IFN-γ-induced IDO activity in IL-1 β-stimulated cells. HeLa 229 cells were sequentially treated in triplicate with IL-1 β (100 ng/ml) or complete medium alone for 16 h, followed by an additional 8 h of treatment with complete medium before IFN-γ treatment for 48 h. IDO activity was then assessed by reverse phase HPLC. One of three experiments with similar results (mean percentage specific catabolism ± SD) is shown.

Effect of NF-κB on receptor expression and IDO transcriptional activation

Although the increase in IDO activity following sequential cytokine treatment suggested that changes in IFNGR expression can influence the amount of IDO activity induced, the possibility existed that NF-κB transactivated in response to IL-1 β stimulation directly enhanced IDO transcriptional activation. NF-κB, upregulated in response to TNF-α, has been shown to bind upstream of IRF-1 and participate in its transcription.20,25 Furthermore, inhibition of proteasomal degradation of I κB to prevent NF-κB transactivation21,22 blocks the ability of TNF-α to enhance IFN-γ-induced IDO activity (C.M. Robinson et al., unpublished observations). To distinguish the effect of IL-1 β on IFNGR expression from its effect on IRF-1 activation, a PSI was used to maintain nuclear NF-κB at steady-state levels. HeLa cells stably transfected with the pGASinsIDO/EGFP-C1 reporter were activated with IL-1 β for 8 h, followed by cultivation in complete medium for 16 h. IDO was then induced for 6 h with IFN-γ. Surprisingly, when PSI was included in the culture medium along with IL-1β, no synergistic enhancement of IDO reporter activity was observed; cells receiving both IL-1 β and IFN-γ exhibited IDO reporter activity comparable to cells receiving only IFN-γ (Fig. 5, top). When PSI was included in the culture medium only during the exposure to IFN-γ, however, there was no effect on synergistic induction of IDO reporter activity (Fig. 5, bottom), indicating that NF-κB activation during the exposure to IL-1 β was critical to the development of synergy. To determine if NF-κB transactivation could influence the upregulation if IFNGR, HeLa cells were treated with IL-1 β and PSI (0, 1, or 3μM) for 8 h before receptor expression was analyzed. A dose-dependent decrease in receptor expression was observed in cells treated with IL-1 β and PSI (Fig. 6A), suggesting that NF-κB transactivation is required for upregulation of IFNGR expression. Furthermore, RT-PCR revealed that whereas IL-1 β treatment increased expression of IFNGR-1 mRNA 2-fold at 8 h posttreatment, simultaneous treatment with PSI blocked this effect (Fig. 6B). Thus, the increase in IFNGR mRNA expression induced by IL-1 β also appears to be dependent on NF-κB transactivation.

FIG. 5.

FIG. 5

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Effect of PSI on IDO regulation. (Top) HeLa cells stably transfected with pGASinsIDO/EGFP-C1 were treated with IL-1 β (100 ng/ml) with or without PSI (0-3μM) for 8 h, followed by a 16-h incubation with medium alone. IDO was induced for 6 h with IFN-γ (20 ng/ml). The cells were harvested and analyzed for GFP expression by flow cytometry. The MFI ± SD from one of several similar experiments is represented. (Bottom) HeLa cells stably transfected with pGAS insIDO/EGFP-C1 were treated with medium alone or IL-1 β (100 ng/ml) for 16 h, followed by treatment with medium alone for 8 h. The cells were treated with IFN-γ (20 ng/ml) for 6 h in the presence or absence of PSI (0-3μM). Cells were harvested and analyzed for GFP expression by flow cytometry. The MFI ±SD from one of several experiments with similar results is represented.

FIG. 6.

FIG. 6

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Effect of PSI on IFNGR expression. (A) HeLa cells were treated with medium alone or IL-1 β (100 ng/ml) in the presence or absence of PSI (0-3μM) for 8 h. The cells were harvested, and IFNGR expression was determined by antireceptor antibody and flow cytometry. The results from one of several experiments are presented (MFI ± SD). (B) HeLa cells were cultivated in medium alone or treated with IL-1 β (100 ng/ml) alone or in combination with PSI (3μM) for 8 h. At that time, total mRNA was isolated, subjected to RT-PCR, and phosphorimaged. Data for IFNGR-1 mRNA expression were normalized to those of β-actin mRNA (internal standard), and fold induction was calculated by comparing with values obtained from cells cultivated in medium alone. The results from one of several experiments with similar results are presented.

Because NF-κB is important both in upregulation of IFNGR and in synergistic transcriptional activation of IRF-1, both of which may enhance IDO activity, and because inhibition of NF-κB transactivation blocks activation of the IDO promoter, it was necessary to determine if the increase in nuclear NF-κB in response to IL-1 κ stimulation remained elevated long enough to influence the response to IFN-β. HeLa cells were stimulated with IL-1 β or complete medium alone. At various times after treatment, the amount of the p50 subunit of NF-κB translocated into the nucleus was quantified by flow cytometry. A >2-fold increase in p50 translocation was observed after 1 h of stimulation, although after 12 h of IL-1 β exposure, nuclear p50 had returned to basal levels (Fig. 7). Similar results were obtained with the p65 subunit, indicating that elevated NF-κB was not available to participate in synergistic induction of IDO transcriptional activation when IL-1 β and IFN-γ were administered sequentially.

FIG. 7.

FIG. 7

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Effect of IL-1 β on NF-κB nuclear translocation and IFNGR expression over time. HeLa cells were cultivated with IL-1 β (100 ng/ml) or complete medium alone for up to 24 h. Nuclei were isolated as described and stained with anti-p50 NF-κB subunit antibody and analyzed by flow cytometry. The results from one of several experiments with similar results (fold increase in MFI ± SD) are shown.

Effect of IL-1 β on IFNGR signal transduction

The increase in IFNGR expression correlated with the increase in IDO activity in cells treated sequentially with IL-1 β and IFN-γ. Inasmuch as NF-κB was shown to have returned to basal levels before IFN-\g=g exposure, the increase in IDO activity is most likely due to an increase in IFN-induced signaling mediated by these receptors. To confirm that the increase in IFNGR contributes to an increase in receptor-specific signal transduction, HeLa cells were incubated with IL-1 β or complete medium for 16 h before being washed and incubated an additional 8 h in complete medium alone. The cells were then stimulated with IFN-γ for 4 h before the nuclei were harvested and analyzed for the amount of phosphorylated Stat1 present in the nucleus. A 1.7-fold increase (p < 0.001) in nuclear Stat1 phosphorylation was observed in the cells treated first with IL-1β, then with IFN-γ, over that detected in cells receiving only IFN-γ (Fig. 8). Thus, an increase in the number of expressed IFNGR contributes to an increase in IFN-induced Stat1 signaling, resulting in a synergistic increase in IDO activity.

FIG. 8.

FIG. 8

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Effect of IL-1 β on Stat1 phosphorylation in IFN-γ-stimulated cells. HeLa 229 cells were sequentially treated in replicates of six with IL-1 β (100 ng/ml) or complete medium alone for 16 h, followed by an additional 8 h of treatment with complete medium before IFN-γ treatment for 4 h. Nuclei were isolated as described, stained with anti-pStat1, and analyzed by flow cytometry. The results from one of several experiments with similar results (fold increase in MFI ± SD) are shown.

DISCUSSION

Synergy among cytokines and the defense mechanisms they initiate is a hallmark of immune system regulation and response amplification; this synergy is important in the response to a variety of pathogens. iNOS and IDO are important antimicrobial defense mechanisms that demonstrate regulation by cytokine synergy. With iNOS, the combination of TNF-α with IFN-γ synergistically inhibits replication of microbial pathogens through enhanced production of nitrogen oxides,2 whereas IFN-γ in combination with LPS induces NOS to restrict the growth of Chlamydia trachomatis in mouse fibroblasts.26 Furthermore, the antimicrobial activity of iNOS has been shown to be synergistic with that of reactive oxygen species (ROS) to induce double-stranded DNA (dsDNA) breakage.27,28 With IDO, various inflammatory agents, including LPS, TNF-α, and IL-1β, enhance IFN-induced IDO activity that can restrict the growth of tryptophan auxotrophs by depleting intracellular tryptophan. Both TNF-α and IL-1 α have been shown to increase IDO mRNA expression, resulting in increased IDO promoter activity.13,14,29

There are several mechanisms by which cytokine synergy can occur. These include modulation of receptor expression for one cytokine by another, regulation of cytokine affinity for its cognate receptor, and changes in gene expression by either altered transcription, mRNA stability, or translation.30,31 Several reports have shown the capacity of cytokines to increase receptor expression on immunologically competent cells, such as monocytes and macrophages.32-34 Similarly, IFNGR has been reported to be regulated by IL-1, IL-6, and TNF-α in highly specialized murine oligodendrocytes.35 This study shows that IL-1β, TNF-α, and IFN-γ also have the ability to increase expression of receptors for IFN-γ, IL-1, and TNF-α in epithelial cells, suggesting that cytokine receptor regulation may be more universal in both immunocompetent and nonimmunologic cells.36 Furthermore, cytokine receptor upregulation in immunologically competent cells would render them more sensitive to stimuli, facilitating amplification of immunologic responses. Upregulation of cytokine receptors in nonimmunologic epithelial cells, normally the first cells to encounter pathogens, would facilitate induction of intracellular defense mechanisms.

To test whether the proinflammatory cytokines upregulated IFNGR, antireceptor antibodies were used to measure changes in IFNGR expression in HeLa cells. Both IL-1 β and TNF-α increased IFNGR expression, with a maximum occurring 16-20 h posttreatment. This is consistent with reports by Krakauer and Oppenheim,23 in which they demonstrated by radiometric techniques that the THP-1 monocytic cell line responded to IL-1 and TNF-α with increased IFNGR expression and affinity. Use of flow cytometry in this study precluded the ability to quantify absolute receptor number and affinity. Nevertheless, flow cytometric techniques were able to identify relative increases in both IFNGR-1 and IFNGR-2 and increased binding of IFN-γ to cytokine-stimulated cells. These results were confirmed using RT-PCR, in which IL-1 β> treatment generated a 2-fold increase in IFNGR-1 expression. In addition, results obtained with biotinylated IFN-γ support the hypothesis that the increase in binding of anti-IFNGR antibodies reflects an increase in receptors capable of binding cytokine. This increase in the number of receptors expressed on the surface was also shown to increase the amount of nuclear phosphorylated Stat1 resulting from IFN treatment. IFNGR expression was increased on stimulation by cytokines in the HEp-2 epithelial cell line (data not shown), indicating that the increase in receptor expression is not a phenomenon unique to HeLa cells.

Similar increases were seen in IL-1RI/RII expression when treated with either IFN-γ or TNF-α, both of which gave maximum expression by 16-20 h posttreatment. There also was increased binding of biotinylated IL-1β, which is consistent with increased expression of receptors capable of binding cytokine. Although increases in TNFR when stimulated with IFN-γ or IL-1 β were not as profound as those observed with IFNGR and IL-1RI/RII, maximum expression still occurred around 16-20 h posttreatment. The reason for this smaller increase is not known, but IL-1 is known to downregulate TNFR expression in monocytes and macrophages.37 Thus, each cytokine tested was able to cross-regulate expression of the other cytokines.

Previous studies have shown that IL-1 β and TNF-α synergistically enhance IDO induction in IFN-γ-treated cells13,14,29 and that synergistic increases in Stat1 and IRF-1 mediate the increase.15,16 Furthermore, elevation of nuclear NF-κB in response to TNF-α was required for synergistic transcriptional activation of the IRF-1 gene (C.M. Robinson et al., unpublished observations). However, IL-1-dependent increases in IFNGR expression also appear to be dependent on NF-κB transactivation. Although IL-1 β increased IFNGR mRNA and cell surface expression, PSI effectively blocked this increase by inhibiting proteasomal degradation of I κB. Previous experiments used simultaneous exposure to the cytokines to induce change, and NF-κB-dependent increases in IFNGR expression were occurring at the same time as NF-κB-dependent increases in IRF-1 activation. Distinguishing the influence of IFNGR expression from synergy at the level of IDO transcriptional activation required an experimental design in which each could be studied independently. In this study, sequential application of IL-1 β followed by IFN-γ also induced similar increases in IDO activity. This is consistent with a mechanism by which the proinflammatory cytokines upregulate expression of IFNGR, permitting a greater response to IFN-γ stimulation. Furthermore, when PSI was used to prevent transactivation of NF-κB, it was effective in inhibiting the synergistic increase in IDO promoter activity only when it was added along with IL-1 β. When PSI was added after IFNGR expression had been upregulated, it had no effect. However, if NF-κB that was transactivated during IL-1 stimulation remained elevated, it could continue to promote synergistic activation of IRF-1. The observation that elevated nuclear NF-κB returned to basal levels before IDO could be induced argues against this hypothesis. Furthermore, the relatively brief elevation in NF-κB is consistent with other findings showing NF-κB translocation by 30 min after induction and decrease to basal levels after 6 h.38,39 Thus, upregulation of IFNGR in the absence of other synergistic mechanisms is an effective mechanism of synergistic IDO activation; as long after IL-1 was removed and nuclear NF-κB had returned to basal levels, the expression of IFNGR remained elevated. This does not suggest that NF-κB-mediated enhancement of IRF-1 with subsequent enhancement of IDO transcriptional activation is unimportant. Rather, this suggests that the increase in IFNGR also contributes significantly to the mechanism of synergy.

Overall, this study extends the observation that increases in IFNGR expression in response to proinflammatory cytokines occur in nonimmunologic cells as well as in immunocompetent cells. Furthermore, this upregulation is driven by the transcription factor NF-κB, which also plays a role in transcriptional activation of IRF-1, a transcription factor required in IFN-induced IDO induction.

ACKNOWLEDGMENTS

This work was supported by Public Health Service grant No. AI45836 from the National Institute of Allergy and Infectious Diseases (J.M.C.).

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