Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
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Originally published online as doi:10.1189/jlb.0802420 on May 22, 2003

Published online before print May 22, 2003
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(Journal of Leukocyte Biology. 2003;74:287-294.)
© 2003 by Society for Leukocyte Biology

Pathways for the regulation of interferon-{gamma}-inducible genes by iron in human monocytic cells

Horst Oexle*, Arthur Kaser*, Johannes Möst*, Rosa Bellmann-Weiler*, Ernst R. Werner{dagger}, Gabriele Werner-Felmayer{dagger} and Günter Weiss*

* Department of Internal Medicine, University Hospital Innsbruck, Austria; and
{dagger} Institute for Medical Chemistry and Biochemistry, University of Innsbruck, Austria

Correspondence: Günter Weiss, M.D., Department of Internal Medicine, University Hospital, Anichstr. 35, A-6020 Innsbruck, Austria. E-mail: guenter.weiss{at}uibk.ac.at


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To elucidate iron-regulated interferon-{gamma} (IFN-{gamma}) effector functions, we investigated three IFN-{gamma}-inducible genes [intercellular adhesion molecule-1 (ICAM-1), human leukocyte antigen (HLA)-DR, guanosine 5'-triphosphate-cyclohydrolase I (GTP-CH)] in primary human monocytes and the cell line THP-1. IFN-{gamma} increased the surface expression of ICAM-1 and HLA-DR and stimulated GTP-CH activity. Addition of iron before cytokine stimulation resulted in a dose-dependent reduction of these pathways, and iron restriction by desferrioxamine (DFO) enhanced ICAM-1, HLA-DR, and GTP-CH expression. Iron neither affected IFN-{gamma} binding to its receptor nor IFN-{gamma} receptor surface expression. IFN-{gamma}-inducible mRNA expression of ICAM-1, HLA-DR, and GTP-CH was reduced by iron and increased by DFO by a transcriptional mechanism. Moreover, ICAM-1 and to a lesser extent, GTP-CH and HLA-DR mRNA expression were regulated post-transcriptionally, as iron pretreatment resulted in shortening the mRNA half-life compared with cells treated with IFN-{gamma} alone. Thus, iron perturbations regulate IFN-{gamma} effector pathways by transcriptional and post-transcriptional mechanisms, indicating that iron rather interferes with IFN-{gamma} signal-transduction processes.

Key Words: monocytes/macrophages • gene regulation • cytokines • adhesion molecules


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Iron is an essential metal for immune surveillance via its stimulatory role on lymphocyte proliferation and its central function in mitochondrial respiration and by catalyzing the formation of toxic radicals by the Haber-Weiss reaction for host defense [1 ]. In addition, the metal also down-regulates cellular immune effector mechanisms by inhibiting the activity of interferon-{gamma} (IFN-{gamma}) toward macrophages [2 ]. This is of major importance for immune surveillance, as IFN-{gamma} is a central cytokine for the regulation of antimicrobical effector mechanisms of macrophages. This T helper cell type 1-derived cytokine promotes antigen presentation via stimulation of major histocompatibility complex (MHC) classes I and II expression [3 ], modulates leukocyte-endothelium interaction, stimulates the synthesis of proinflammatory cytokines by monocytes such as interleukin (IL)-1, tumor necrosis factor {alpha} (TNF-{alpha}), and IL-6, and in synergism with the latter, induces the formation of toxic oxygen radicals and nitric oxide (NO) for host defense [4 ]. A regulatory effect of iron on IFN-{gamma} activity was first observed in human monocytic cells, where iron down-regulated the IFN-{gamma}-inducible formation of neopterin and in parallel, the degradation of tryptophan to kynurenin [5 ]. Moreover, in murine macrophages, iron inhibited the expression of inducible NO synthase (iNOS) by a transcriptional mechanism involving the deactivation of the transcription factor nuclear factor (NF)-IL-6 [6 ]. The relevance of these observations was substantiated by experiments, which demonstrated that macrophages that have been loaded with iron lose their ability to kill intracellular pathogens such as Legionella pneumophila, Ehrlichia chaffeensis, or Listeria monocytogenes by IFN-{gamma}-mediated pathways [7 8 9 ].

However, the underlying mechanism by which iron regulates IFN-{gamma} activity in human monocytic cells remained elusive so far. To investigate this and to see whether iron may also affect IFN-{gamma} function not only in cell lines (THP-1) but also in peripheral blood mononuclear cells (PBMC), the current study was undertaken. We examined the metabolic effects of iron on three IFN-{gamma}-inducible genes.

The first one, intercellular adhesion molecule-1 (ICAM-1), is an important surface protein expressed on a wide range of peripheral blood leukocytes. It serves as a ligand to leukocyte function-associated antigen 1 (CD11a/CD18) or Mac-1 (CD11b/CD18) and has an important role in the initiation and progress of important immunological processes such as leukocyte migration, T cell-mediated killing, but also T and B cell responses [10 ]. PBMC express very low amounts of ICAM-1, but this surface marker can be strongly and rapidly induced after stimulation with cytokines such as IFN-{gamma} [11 ,12 ]. This enables leukocytes to adhere to postcapillary venules and to migrate to the place of inflammation.

The second IFN-{gamma}-inducible protein we investigated was human leukocyte antigen (HLA)-DR, a MHC class II protein, which is expressed on the surface of monocytes, dendritic cells, and B cells. MHC class II proteins are involved in antigen presentation, MHC restriction, and T cell activation [13 ]. This surface protein is induced by IFN-{gamma}, similar to ICAM-1, but the exact regulatory mechanisms and signal-transduction factors for this regulatory pathway have not yet been fully identified [3 ,14 ].

Finally, we investigated guanosine 5'-triphosphate-cyclohydrolase I (GTP-CH; EC. 3.5.4.16), which is induced by IFN-{gamma} and cleaves GTP to D-erythro-7,8-dihydroneopterin triphosphate. This intermediate is further metabolized to 5,6,7,8-tetrahydrobiopterin. In humans and especially in monocytes/macrophages, this pathway also yields substantial amounts of 7,8-dihydroneopterin and neopterin [15 ]. Although neopterin is able to modulate radical formation [16 ], an exact biological role of neopterin has not been elucidated so far. Nevertheless, determination of neopterin in serum has turned out to be a clinically reliable marker for monitoring patients with activated cellular immunity [17 ].


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture
THP-1 cells, a human myelomonocytic cell line, which is widely used to study monocyte/macrophage biology in a cell culture system [18 ], were used for this study. Cells were cultured in RPMI 1640, supplemented with 10% (v/v) heat-inactivated fetal calf serum (FCS), 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (all obtained from Life Technologies, Vienna, Austria) at 37°C in humidified air containing 5% CO2. Cells were treated with 100–500 U/ml recombinant human (rh)IFN-{gamma} (Life Technologies) and/or ferric iron chloride and desferrioxamine (DFO; both obtained from Sigma, Munich, Germany) or were left untreated. Treatment with the indicated concentrations of IFN-{gamma}, iron, or DFO did not result in reduced viability of cells in comparison with untreated controls as checked by trypan blue exclusion (data not shown).

Isolation of monocytes
PBMC were isolated from the blood of healthy adult volunteers (buffy coats from blood donors were kindly provided by the local blood transfusion unit) by density gradient centrifugation with Ficoll-Paque (Pharmacia, Uppsala, Sweden). The cells were washed twice in phosphate-buffered saline (PBS) and were resuspended in medium RPMI 1640 (HyClone, Cramlington, UK) supplemented with 2 mM glutamine (PAA Laboratories, Linz, Austria) and 10% FCS (PAA Laboratories). After incubation on hydrophilic Petri dishes (Petriperm, Bachofer, Reutlingen, Germany) for 1 h at 37°C (2x107 cells/ml), the nonadherent cells were removed by repeated, vigorous washings. The resulting cell population contained at least 90% monocytes, as shown by morphology and phenotype fluorescein-activated cell sorter (FACS) analysis. For flow cytometric analysis, we cultured 2 x 106 PBMC in six-well dishes in 2 ml medium together with 50 µM FeCl3 or 100 µM DFO in the absence or presence of 250 U/ml IFN-{gamma} (kind gift from Jan Brzoska, Bioferon, Laupheim, Germany).

Cytofluorometric analysis of IFN-{gamma} binding to its receptor
We stimulated 1 x 106 U937 cells with the indicated agents. Then the cells were extensively washed in PBS/2% FCS, resuspended in 250 µg/ml h immunoglobulin G (IgG)/PBS/2% FCS, and incubated for 20 min at 4°C. After pelleting, cells were incubated with or without 1 x 104 IU/mL rhIFN-{gamma} for 30 min at 4°C [IFN-{gamma} receptor (IFN-{gamma}R) binding at this presumably saturating concentration of rhIFN-{gamma} was too faint to allow a further titration]. After washing in PBS/2% FCS, a biotinylated anti-IFN-{gamma} monoclonal antibody (mAb; 10 µg/mL; Becton Dickinson, Heidelberg, Germany) was added for 30 min at 4°C. Following another wash, a 1:25 dilution of streptavidin-phycoerythrin (PE; from Becton Dickinson) in PBS/2% FCS was incubated for 30 min at 4°C. Cells were immediately analyzed on a FACSCalibur, and data analysis was performed with Cellquest software (Becton Dickinson).

Cytofluorometric analysis of IFN-{gamma}R expression, ICAM-1, and HLA-DR expression
Fluorescein isothiocyanate-labeled anti-CD54 (purchased from Immunotech, Marseille, France), anti-HLA-DR (Dako, Glostrup, Denmark), and anti-IgG1{kappa} (Sigma) as an isotype control were used. Immunofluorescence was performed according to standard procedures [12 ]. For determination of IFN-{gamma}, receptor expression cells were incubated with 10 µg/ml biotinylated anti-IFN-{gamma}R mAb (Becton Dickinson) or biotinylated istotype-matched control. Briefly, 1 x 106 U937 cells stimulated with indicated agents were washed twice in PBS/2% FCS, resuspended in 250 µg/ml hIgG/PBS/2% FCS, and incubated for 20 min at 4°C. After washing in PBS/2% FCS, a 1:25 dilution of streptavidin-PE (Becton Dickinson) in PBS/2% FCS was incubated for 30 min at 4°C, and cells were analyzed as described above. Values are given as specific mean fluorescence intensities; i.e., fluorescence of isotype-matched control antibodies is subtracted.

Determination of GTP-CH activity
Enzyme activity was assessed by a method modified from Viveros et al. [19 ]. Sephadex G-25 cell extract eluates were prepared in 0.1 M Tris/HCl, pH 7.8, 0.3 M KCl, and 10% glycerol. The eluate was incubated with 4 mM GTP (Serva, Munich, Germany) at 37°C for 90 min in the dark, and then, the thereby-formed dihydroneopterin triphosphate was oxidized to neopterin triphosphate using 0.1 M HCl/0.01 M I2 (final concentrations). After centrifugation at 10,000 g, excess iodine was destroyed with 0.1 M ascorbic acid, and then, the pH was adjusted to 7.6 with 1 M NaOH. Neopterin triphosphate was then cleaved to neopterin by incubation with 16 U/ml alkaline phosphatase (Serva) for 1 h at 37°C. Neopterin was finally quantified after acidification of 100 µl of the sample with 10 µl 5 M H3PO4 by high-pressure liquid chromatography (HPLC) determination. The samples were then extracted using AASP-SCX cartridges (Analytichem, Harbor City, CA), and neopterin was eluted onto a reverse-phase HPLC column (Lichrosorb Rp-18, Merck, Darmstadt, Germany) and quantified by fluorescence determination at 353 nm excitation and 438 nm emission [20 ]. The detection limit for neopterin was 1 nM.

Northern blot analysis
Total RNA was isolated from cells by using RNAclean (Dipro Diagnostic Products, Vienna, Austria). RNA (10 µg) was size-fractionated on a denaturing 1% formaldehyde/ 3-N-morpholinopropanesulfonic acid agarose gel exactly as described in ref. [21 ]. After blotting onto a Duralon UV® membrane (Stratagene, La Jolla, CA) and cross-linking, the membranes were hybridized with radiolabeled DNA probes for GTP-CH (600 bp EcoRI fragment in TA cloning vector), ICAM-1 (1.8 kb XbaI fragment in pCDM8 vector), HLA-DR (1.3 kb BamHI fragment of HLA-DR-B1 in pcDV1-pL2 vector), and glyceraldehyde 3-phosphate-dehydrogenase (GAPDH; 1.4 kb PstI fragment of rat GAPDH in pBR322 vector) or ß-actin (1.9 kb HindIII fragment of chicken ß-actin in pBR322 vector) as housekeeping genes. Specific RNA bands were imaged by autoradiography for 24–72 h using Amersham HyperfilmTMMP (Amersham Life Science, Buckinghamshire, UK).

RNA stability
RNA half-life was estimated in cells stimulated with the appropriate additives as described above for 24 h, which were then treated with actinomycin D (final concentration, 5 µg/ml) for 1.5, 3, 4.5, and 6 h before RNA isolation for Northern blotting. All Northern blots were scanned and densitometrically evaluated using the Gel Doc system from Bio-Rad Laboratories (Hercules, CA).

Nuclear runoff assays [22 ]
We stimulated 2.5 x 107 cells with 500 U/ml rhIFN-{gamma} alone or in the presence/absence of 100 µM ferric iron chloride or 200 µM DFO for 4 h. After washing, nuclei were prepared following lysis of cells with 2 ml detergent buffer [10 mM Tris, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.5% Nonidet P-40 (NP-40)]. Nuclei were then resuspended in 100 µl glycerol storage buffer (50 mM Tris, pH 8.0, 5 mM MgCl2, 0.5 mM dithiothreitol, 40% glycerol) and stored in liquid nitrogen until use. Plasmids were linearized and then spotted onto a Duralon UV® membrane using a Slot blot chamber (Hoefer Pharmacia Biotech, San Francisco, CA). After cross-linking, membranes were prehybridized for 4 h at 65°C, and then radiolabeled nuclear transcripts were added for 24 h. Labeling of transcripts was performed using isolated nuclei in glycerol storage buffer, 150 µl assay buffer (25 mM Tris-HCl, pH 8.0, 12.5 mM MgCl2, 325 mM KCl), 1.25 mM adenosine 5'-triphosphate/cytidine 5'-triphosphate/GTP, and 120 µCi [{alpha}-32P]-uridine 5'-triphosphate, which were incubated for 1 h at 37°C. Then, labeled transcripts were recovered following digestion of nuclear DNA with 200 U RNase-free DNase (Pharmacia) and of nuclear proteins with proteinase K (Sigma). Radiolabeled RNA was then isolated with RNA-phenol-chloroform, precipitated with isopropanol, and washed with 75% ethanol before resuspending the RNA in 100 µl sterile 10 mM Tris-EDTA, pH 7.0.

Western blot analysis
After stimulation, cells were washed in PBS and lysed with radio immunoprecipitation assay buffer [NaCl 150 mM, 50 mM Tris-HCl, adjusted to pH 8.0, NP-40 1%, deoxycholate 0.5%, sodium dodecyl sulfate (SDS) 0.1%, phenylmethylsulfonyl fluoride 100 µM, leupeptin 10 µg/ml] for 20 min. After centrifugation at 13,000 g for 5 min, the supernatant was used as cell extract. The protein separation of 10 µg protein was performed with SDS-polyacrylamide gel electrophoresis and blotted on a polyvinylidene difluoride membrane (Bio-Rad Laboratories). Blocking was done with Tris-buffered saline containing 5% nonfat dry milk and 0.05% Tween-20 for 30 min. Phosphorylated signal transducer and activator of transcription (STAT)1{alpha} was detected by a polyclonal rabbit antiphospho-STAT1 antibody (Upstate Biotechnology, Lake Placid, NY) by overnight incubation with a 1:1000-diluted antibody Tris-buffered saline as described above. As secondary antibody, we incubated the blot with 1:5000 peroxidase-conjugated goat anti-rabbit IgG (Upstate Biotechnology) for 1.5 h, which was visualized by an Amersham HyperfilmTMMP (Amersham Life Science).

Determination of intracellular iron concentrations
Intracellular iron concentrations were determined spectrophotometrically using a ferrozine-based assay as described [5 ].

Statistical analysis
Statistical calculations were performed with Systat 7.0. We compared the respective experimental groups with Student’s t-test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Iron perturbations modulate the expression of ICAM-1 and HLA-DR in human PBMC
To investigate a possible effect of iron perturbation on PBMC, we first determined ICAM-1 and HLA-DR surface expression in response to IFN-{gamma} and/or iron treatment. The addition of 50 µM iron ferric chloride resulted in a significant increase of intracellular iron concentrations (19.75±1.558 µM/106 cells) as compared with control cells (16.75±0.845 µM/106 cells, P<0.001) and was also significantly enhanced when comparing cells treated with 250 U/ml IFN-{gamma} (16.406±1.157 µM/106 cells) with IFN-{gamma}-treated cells preincubated with iron (20.656±1.552 µM/106 cells, P<0.004).

Untreated monocytes showed a low constitutive expression of ICAM-1 and HLA-DR. Treatment of cells with iron or the iron chelator DFO did not result in a significantly changed expression of ICAM-1 and HLA-DR, as compared with control monocytes (Table 1 ). Nevertheless, stimulation of cells with IFN-{gamma} strongly enhanced ICAM-1 expression by 2.43-fold, and HLA-DR surface expression was increased by 5.56-fold as compared with controls. Iron perturbation significantly reduced the surface expression of both proteins in IFN-{gamma}-stimulated monocytes (Table 1) . Conversely, combined treatment of cells with IFN-{gamma} and DFO increased FACS staining for ICAM-1 by 125.4% as compared with IFN-{gamma}-treated cells and by 198.4% as compared with cells treated with IFN-{gamma} and iron. It is interesting that HLA-DR expression was not significantly different between cells treated with IFN-{gamma} or IFN-{gamma}/DFO. The impressive regulatory effect of iron but the rather small effect of DFO as compared with cells treated with IFN-{gamma} alone may be referred to as the iron content of media (FCS) [23 ].


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  		Table 1. Effects of Iron Perturbation on IFN-{gamma}-Inducible ICAM-1 and HLA-DR Expression on Human Monocytes

 
GTP-CH activity in IFN-{gamma}-activated THP-1 cells is altered by changes in iron supply
To see whether the observed iron-induced changes in ICAM-1 and HLA-DR expression may also hold true for another IFN-{gamma}-inducible pathway, we investigated GTP-CH activity in the human monocytic cell line THP-1. As is evident from Figure 1A , stimulation of THP-1 cells with 100 or 500 U/ml IFN-{gamma} resulted in a drastic increase of GTP-CH activity. The addition of increasing concentrations of iron chloride significantly reduced GTP-CH activity in a dose-dependent manner [5 ]. It is interesting that a significant reduction of GTP-CH activity was observed when very low amounts of iron (2 µM) were added.



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  		Figure 1. (A) Dose-dependent reduction of the IFN-{gamma} effect on GTP-CH in THP-1 by iron supplementation. GTP-CH activity was determined as described in Materials and Methods in THP-1 cells treated with 100 or 500 U/ml IFN-{gamma} and 2–50 µM iron for 24 h. Means ± SD of three independent experiments performed in triplicates are shown. (B) Dose-dependent reduction of GTP-CH mRNA in IFN-{gamma}-stimulated THP-1 cells. THP-1 were treated with 100 U/ml IFN-{gamma} and increasing concentrations of ferric iron chloride (I; 2–50 µM) or were left untreated (C). The results of one of three Northern blots are shown.

 
Iron does not affect IFN-{gamma}R expression nor IFN-{gamma}R binding
To see whether these regulatory effects of iron toward IFN-{gamma}-inducible molecules could be referred to as an interference of the metal with the interaction of IFN-{gamma} with its receptor, we performed the following experiments. First, we determined IFN-{gamma}R expression in human monocytic cells. Iron/DFO neither changed the surface expression of IFN-{gamma}R as compared with control cells nor altered the IFN-{gamma}R number in IFN-{gamma}-stimulated cells (Fig. 2a and b ). Second, to see whether iron perturbations may affect the binding of IFN-{gamma} to its receptor, we performed a binding study using a biotinylated anti-IFN-{gamma} antibody. As can be seen in Figure 2 c and 2d , iron and DFO did not alter IFN-{gamma} binding in untreated or in IFN-{gamma}-stimulated cells.



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  		Figure 2. IFN-{gamma}R expression and IFN-{gamma}R binding on U937 cells, which were pretreated with ferric iron chloride (red line) or DFO (green line) or were cultured in medium alone (black line). IFN-{gamma} (250 IU/mL) was then added only in b and d for 24 h. (a and b) Surface expression of IFN-{gamma}R was detected by biotinylated anti-IFN-{gamma}R mAb (solid line) or biotinylated, isotype-matched, control mAb (dotted line), followed by streptavidin-PE. (c and d) IFN-{gamma} binding to IFN-{gamma}R was detected by incubation of cells with 1 x 103 IU/mL rhIFN-{gamma} (solid line) or without (dotted line), followed by biotinylated anti-IFN-{gamma} mAb and subsequent addition of streptavidin-PE as detailed in Materials and Methods.

 
Iron perturbations change cellular content of ICAM-1, HLA-DR, and GTP-CH mRNA in IFN-{gamma}-activated PBMC
Based on the observations made so far, we next investigated whether the altered expression of ICAM-1, GTP-CH, and HLA-DR in IFN-{gamma}-stimulated and iron-perturbed monocytes could be referred to as different cytoplasmatic mRNA levels. PBMC and THP-1 cells had very low constitutive amounts of ICAM-1 and HLA-DR mRNAs, which could hardly be detected by Northern blot analysis. Iron supplementation had no obvious effect, whereas DFO caused a slight increase in ICAM-1 mRNA (Fig. 3 ). IFN-{gamma} induced ICAM-1 and HLA-DR mRNA levels in PBMC as compared with untreated controls, and iron supplementation impaired this effect of IFN-{gamma}, thus leading to reduced mRNA expression of ICAM-1 and HLA-DR. Conversely, iron deprivation by DFO increased the expression of ICAM-1 mRNA and HLA-DR mRNA to levels comparable with or higher than cells treated with IFN-{gamma} alone (Fig. 3) . Comparable results with respect to inducibility of these IFN-{gamma}-inducible genes by IFN-{gamma} and its regulation by iron were obtained in THP-1 and human peripheral blood monocytes. Moreover, in THP-1 cells, the progressive decrease in IFN-{gamma}-inducible GTP-CH activity following the addition of increasing dosages of iron to cells was paralleled by a progressive decrease in cytoplasmatic GTP-CH mRNA levels, underscoring the cause–effect relationship of iron for cytokine-inducible regulation of GTP-CH (Fig. 1B) .



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  		Figure 3. Modulation of ICAM-1 and HLA-DR mRNA expression in PBMC by IFN-{gamma} and iron perturbation. The cells were treated with 25 µM ferric iron chloride (I), 50 µM desferrioxamine (D), and/or 250 U/ml IFN-{gamma} for 24 h or were left untreated (C). RNA extraction and Northern blots were performed as described in Materials and Methods. A ratio ICAM-1/GAPDH and HLA-DR/GAPDH mRNA was calculated based on the results of densitometric scanning. Mean percentage in relation to GAPDH mRNA expression (=100%) is shown. A representative blot out of three experiments is shown.

 
Modulation of mRNA half-life in IFN-{gamma}-inducible genes by iron
As the results obtained so far suggested a transcriptional or post-transcriptional mechanism as being responsible for iron-mediated regulation of IFN-{gamma}-inducible genes, we first investigated mRNA half-lives for these target genes in THP-1 cells. For this aim, THP-1 cells were pretreated with 250 U/ml IFN-{gamma} for 24 h (alone or in combination with 100 µM FeCl3 or 200 µM DFO), and then actinomycin D was added for 1.5, 3, 4.5, and 6 h. As can be seen in Figure 4A , combined treatment of THP-1 cells with IFN-{gamma} plus ferric iron chloride reduced half-lives of ICAM-1, HLA-DR, and GTP-CH mRNA to means between 23.4 and 65.7% as compared with cells treated with IFN-{gamma} alone, and the most prominent reduction was observed for an ICAM-1 mRNA half-life. Conversely, DFO increased the half-life of the target mRNAs significantly as compared with cells treated with IFN-{gamma} alone (=100%; ICAM-1 mRNA half-life was increased to 155.3±16%, P=0.044, n=3; GTP-CH to 97.4±10.5%, P=0.87, n=3; and HLA-DR to 148.6±18.9%, P=0.047, n=3; significance determined by Student’s t-test). However, there was also a slight increase in GAPDH mRNA half-life when cells were treated with a combination of IFN-{gamma} and DFO, which is in accordance with previous observations indicating dependence of GAPDH expression on cellular iron availability [24 ].



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  		Figure 4. (A) Half-life of ICAM-1, HLA-DR, and GTP-CH mRNA in IFN-{gamma}-stimulated and iron-perturbed THP-1 cells. The half-life of mRNA was determined by addition of 5 µg/ml actinomycin D for 0, 1.5, 3, 4.5, and 6 h to cells stimulated with IFN-{gamma} for 24 h in the presence/absence of iron (I) or desferrioxamine (D). Northern blots were performed as described. One out of four representative experiments is shown. Mean half-life ± SD is plotted. (B) Effect of timing of iron treatment on ICAM-1 mRNA half-life. THP-1 cells were stimulated with IFN-{gamma} (250 U/ml), IFN-{gamma} (250 U/ml) and 50 µM iron for 24 h (pre), or IFN-{gamma} (250 U/ml) for 24 h and subsequent addition of iron (50 µM) 1 h after cytokine stimulation (post), respectively. Actinomycin D was added for up to 6 h, and mRNA analysis was performed by Northern blotting. One out of three representative experiments is shown. Mean mRNA half-life ± SD is plotted.

 
To investigate the possibility that iron per se affects the mRNA half-life rather than interfering with the IFN-{gamma} signal-transduction cascade, we studied ICAM-1 mRNA stability in THP-1 cells treated with IFN-{gamma} in the absence or presence of iron for 24 h and in cells treated with IFN-{gamma} for 24 h where iron was added 1 h after cytokine stimulation (Fig. 4B) . In accordance with the previous experiments, addition of iron before IFN-{gamma} stimulation of THP-1 cells decreased mRNA half-life of ICAM-1 from 4.5 ± 1.1 h (IFN-{gamma}) to 1.5 ± 0.4 h (IFN-{gamma} and iron pretreatment), whereas the addition of iron after IFN-{gamma} stimulation did not significantly change the mRNA half-life (4.7±1.2 h) as compared with cells treated with IFN-{gamma} alone. Neither treatment affected the mRNA stability of the housekeeping gene ß-actin.

Alterations in iron availability modify IFN-{gamma}-induced mRNA transcription of ICAM-1 and HLA-DR genes
To examine whether altered transcriptional regulation of IFN-{gamma}-inducible target genes by iron may also be involved, we performed nuclear runoff transcription assays (Fig. 5 ). IFN-{gamma} enhanced the transcription of the investigated genes, and the addition of iron strongly decreased ICAM-1 and HLA-DR transcription in IFN-{gamma}-activated myelomonocytic cells. Opposite, the application of the iron chelator DFO did not change very much ICAM-1 but further increased HLA-DR transcription as compared with cells treated with IFN-{gamma} alone. All changes were specific, as no effect of either treatment on the transcription of ß-actin could be observed. We also examined the impact of iron perturbations on GTP-CH transcription; however, GTP-CH transcription was very weak so that we were not able to investigate possible changes.



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  		Figure 5. Effects of iron perturbation on ICAM-1 and HLA-DR transcription. Nuclear runoff assays were performed with THP-1 cells treated with 250 U/ml IFN-{gamma} and/or iron (I; 100 µM) and DFO (D; 200 µM) for 4 h, as described in Materials and Methods, and were hybridized with ICAM-1, HLA-DR, and ß-actin containing plasmids. One out of four representative experiments is shown. Transcriptional induction was calculated after densitometric scanning of the films with the unstimulated control set as 1.

 
Iron perturbations do not modulate IFN{gamma}-activated STAT1{alpha} phosphorylation
We performed Western blot analysis to examine the effect of iron perturbations on phosphorylation of STAT1{alpha} after stimulation with IFN-{gamma}. It was possible to show an impressive increase in phosphorylated STAT1{alpha} after IFN-{gamma} stimulation (250 U/ml) of THP-1 cells, but there were no changes as compared with a combined stimulation with IFN-{gamma}/DFO 200 µM or IFN-{gamma}/iron 100 µM (Fig. 6 ).



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  		Figure 6. Iron perturbations do not change phosphorylation of STAT1{alpha}. Cells were left untreated (control, C) or incubated with ferric iron chloride (I; 100 µM) or desferrioxamine (D; 200 µM) with/without subsequent stimulation with IFN-{gamma} (250 U/ml) for 15 min. STAT1{alpha} phosphorylation was then investigated by means of Western blot techniques as detailed in Materials and Methods. One of three representative experiments is shown above.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Iron perturbation is capable of modulating IFN-{gamma} activity as we demonstrated for three IFN-{gamma}-inducible genes by at least two different pathways. First, modulation of cellular iron homeostasis alters the IFN-{gamma}-induced transcription of the three cytokine-inducible genes, ICAM-1, HLA-DR, and presumably of GTP-CH. In addition, treatment with DFO also resulted in post-transcriptional stabilization of these mRNAs in IFN-{gamma}-treated monocytic cells. However, this latter effect was most pronounced for ICAM-1 and to a lesser extent for GTP-CH and HLA-DR mRNA. It is well established that IFN-{gamma} induces ICAM-1 expression by a transcriptional mechanism involving activation and binding of transcription factors to consensus motifs within the ICAM-1 promoter such as IFN-{gamma}-responsive elements, NF-{kappa}B consensus sequences, and activated protein-1 (AP1)/12-O-tetradecanoylphorbol 13-acetate-responsive elements or AP2- and AP3-like sites [10 ,25 26 27 ]. Iron perturbations decrease ICAM-1 mRNA transcription, which should be a result of alterations in the binding activities of these transcription factors. This could be referred to as direct effects of the metal toward the binding activity of the transcription factors, as shown for NF-IL-6 or hypoxia-inducible factor with the iNOS promoter [6 ,21 ,28 ], or a result of catalyzation of hydroxyl radical formation by iron with subsequent changes in transcription factor binding [29 ]. More likely, as iron also affected ICAM-1 and GTP-CH transcription, iron may rather modulate IFN-{gamma} activity by generally affecting the signal-transduction process of this cytokine. This hypothesis is also supported by our experiments showing that addition of iron after IFN-{gamma} treatment did not significantly alter the mRNA half-life as compared with cells treated with IFN-{gamma} alone, whereas the mRNA half-life was remarkably reduced by a combined stimulation with iron and IFN-{gamma} for 24 h. Moreover, iron neither affected binding of IFN-{gamma} to its receptor nor modulated IFN-{gamma}R expression on resting and cytokine-stimulated human monocytes.

Our data also provide evidence for post-transcriptional regulation of IFN-{gamma}-inducible genes, which was most pronounced for ICAM-1. Half-life of ICAM-1 mRNA is regulated by IFN-{gamma}-responsive AUUUA regions in the 3'-UTR by not-yet identified proteins [30 ]. Iron decreases this mRNA stability, and it is enhanced by the addition of DFO. The background for this observation could be a result of weakening/strengthening of the stabilizing signal toward ICAM-1 mRNA induced by IFN-{gamma} in a complementary manner to what is known about post-transcriptional stabilization of transferrin receptor mRNA by iron-regulatory proteins [31 ,32 ].

Corresponding to the observations made with ICAM-1, iron also inhibited HLA-DR and presumably GTP-CH transcription in IFN-{gamma}-stimulated monocytic cells. However, although HLA-DR transcription has been well-studied [3 ,14 ], significant information on the nature of the GTP-CH promoter and its regulatory transcription factors is still rare [33 ]. Some authors suggest that NF-{kappa}B and AP1 may play a role in GTP-CH transcription, at least in osteoblastic cells [34 ]. Conversely, two proteins, RFX5 and CIITA (class II trans activator) [3 ,14 ,35 ], mainly exerted transcriptional induction of HLA-DR following IFN-{gamma} treatment. This lack of a common regulatory transcription factor induced by IFN-{gamma} within the promoters of the three IFN-{gamma}-inducible genes ICAM-1, HLA-DR, and GTP-CH provides further evidence for our notion that iron may rather affect the IFN-{gamma} signal transduction than the binding of a single transcription factor to its DNA consensus sequence within a IFN-{gamma}-inducible target gene. This assumption is supported by our finding that HLA-DR and GTP-CH mRNA expression is also regulated post-transcriptionally by iron perturbations although to a much lesser extent than observed for ICAM-1 mRNA. Although evidence for post-transcriptional regulation is completely absent for GTP-CH, regulation of MHC class II expression at the post-transcriptional level by IFN-{gamma} has been described [36 ].

The regulation of IFN-{gamma}-inducible genes by iron is of great immunologic relevance, especially in the case of chronic infections or tumor diseases. Iron is an essential factor for the growth and proliferation of tumor cells and microorganisms [1 ,37 ]. Thus, via the action of pro- and anti-inflammatory cytokines, the body has evoked strategies to withhold iron from the pathogens. This is exerted by IFN-{gamma}, IL-1, TNF-{alpha}, and/or IL-10-induced stimulation of iron uptake and storage in the reticuloendothelial system [38 39 40 41 ]. Part of this diversion of iron traffic is maintained by an autoregulatory feedback loop between NO and iron homeostasis involving activation of iron-regulatory proteins by the radical [2 ]. Nevertheless, this withdrawal of iron from the circulation limits the availability of this metal to the pathogens and thus negatively affects their growth [37 ]. As a consequence of this strategy, patients develop anemia, the so-called anemia of chronic disease [42 ,43 ]. However, iron withholding has a beneficial consequence, namely strengthening of a cell-mediated, immune function. As shown previously and herein, this procedure leads to increased IFN-{gamma} activity toward macrophages by stimulating IFN-{gamma}-induced effector pathways such as cell adhesion (via ICAM-1 expression), antigen presentation (via MHC-II expression), or toxic effector mechanisms via NO [5 ,6 ,21 ,28 ]. The relevance of this interaction was sustained by in vivo results demonstrating that iron-loaded macrophages lose their ability to kill intracellular pathogens (such as L. pneumophila, L. monocytogenes, or E. chaffensis) via IFN-{gamma}-mediated pathways, and their killing effectivity is recovered by addition of an iron chelator [7 8 9 , 44 ]. Thus, the development of new iron chelators, which penetrate cells much better than DFO and may thus modulate the immune function more favorably, will be a promising future approach to treat infections with intracellular microorganisms.


   ACKNOWLEDGEMENTS
 
This work was supported by the Austrian Research Fund "Fonds zur Förderung der wissenschaftlichen Forschung" Project P 14215 and by a grant from the Austrian National Bank, NB-8467. We thank Sabine Engl and Gertraud Malleier for technical assistance and Dr. Nancy Hogg for providing the ICAM-1 vector.

Received August 28, 2002; revised March 7, 2003; accepted March 13, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Brock, J. H. (1994) Iron in infection, immunity, inflammation and neoplasia Brock, J. H. Halliday, J. W. Pippard, M. Powell, L. W. eds. Iron Metabolism in Health and Disease W. B. Saunders London, UK.
  2. Weiss, G., Wachter, H., Fuchs, D. (1995) Linkage of cell-mediated immunity to iron metabolism Immunol. Today 16,495-500[CrossRef][Medline]
  3. Mach, B., Steimle, V., Martinez-Soria, E., Reith, W. (1996) Regulation of MHC class II genes: lessons from a disease Annu. Rev. Immunol. 14,301-331[CrossRef][Medline]
  4. Boehm, U., Klamp, T., Groot, M., Howard, J. C. (1997) Cellular responses to interferon-{gamma} Annu. Rev. Immunol. 15,749-795[CrossRef][Medline]
  5. Weiss, G., Fuchs, D., Hausen, A., Reibnegger, G., Werner, E. R., Werner-Felmayer, G., Wachter, H. (1992) Iron modulates interferon-{gamma} effects in the human myelomonocytic cell line THP-1 Exp. Hematol. 20,605-610[Medline]
  6. Dlaska, M., Weiss, G. (1999) Central role of transcription factor NF-IL6 for cytokine and iron-mediated regulation of murine inducible nitric oxide synthase expression J. Immunol. 162,6171-6177[Abstract/Free Full Text]
  7. Byrd, T. F., Horwitz, M. A. (1991) Lactoferrin inhibits or promotes Legionella pneumophila intracellular multiplication in nonactivated and interferon-{gamma} activated human monocytes depending upon its degree of iron saturation: iron-lactoferrin and nonphysiologic iron chelates reverse monocyte action against Legionella pneumophila J. Clin. Invest. 88,1103-1112
  8. Barnewell, R. E., Rikihisa, Y. (1994) Abrogation of gamma interferon-induced inhibition of Ehrlichia chaffeensis infection in human monocytes with transferrin iron Infect. Immun. 62,4804-4810[Abstract/Free Full Text]
  9. Alford, C. E., King, T. E., Campell, P. A. (1991) Role of transferrin, transferrin receptors, and iron in listericidal activity J. Exp. Med. 174,459-466[Abstract/Free Full Text]
  10. van de Stolpe, A., van der Saag, P. T. (1996) Intercellular adhesion molecule-1 J. Mol. Med. 74,13-33[Medline]
  11. Dustin, M. L., Rothlein, R., Bhan, A. K., Dinarello, C. A., Springer, T. A. (1986) Induction by IL 1 and interferon-{gamma}: tissue distribution, biochemistry and function of a natural adherence molecule (ICAM-1) J. Immunol. 137,245-254[Abstract]
  12. Möst, J., Schwaeble, W., Drach, J., Sommerauer, A., Dierich, M. P. (1992) Regulation of the expression of ICAM-1 on human monocytes and monocytic tumor cell lines J. Immunol. 148,1635-1642[Abstract]
  13. Schneider, S. C., Sercarz, E. E. (1997) Antigen processing differences among APC Hum. Immunol. 54,148-158[CrossRef][Medline]
  14. van den Elsen, P., Gobin, S. J. P., van Eggermond, M. C., Peijnenburg, A. (1998) Regulation of MHC class I and II gene transcription: differences and similarities Immunogenetics 48,208-221[CrossRef][Medline]
  15. Werner, E. R., Werner-Felmayer, G., Mayer, B. (1998) Tetrahydrobiopterin, cytokines, and nitric oxide synthesis Proc. Soc. Exp. Biol. Med. 219,171-182[Abstract]
  16. Weiss, G., Fuchs, D., Hausen, A., Reibnegger, G., Werner, E. R., Werner-Felmayer, G., Semenitz, E., Dierich, M. P., Wachter, H. (1993) Neopterin modulates toxicity mediated by reactive oxygen and chloride species FEBS Lett 321,89-92[CrossRef][Medline]
  17. Fuchs, D., Hausen, A., Reibnegger, G., Werner, E. R., Dierich, M. P., Wachter, H. (1988) Neopterin as a marker for cell-mediated immunity: application in HIV infection Immunol. Today 9,150-155[CrossRef][Medline]
  18. Tsuchiya, S., Yamabe, M., Yamaguchi, Y., Kobayashi, Y., Konno, T., Tada, K. (1980) Establishment and characterization of a human acute monocytic leukemia cell line (THP-1) Int. J. Cancer 26,171-176[Medline]
  19. Viveros, O. H., Abou-Donia, M. M., Nixon, J. C., Nichol, C. A. (1981) Biopterin cofactor biosynthesis: independent regulation of GTP-cyclohydrolase in adrenal medulla and cortex Science 213,349-350[Abstract/Free Full Text]
  20. Werner, E. R., Fuchs, D., Hausen, A., Reibnegger, G., Wachter, H. (1987) Simultanous determination of neopterin and creatinine in serum with solid phase extraction and on-line-elution liquid chromatography Clin. Chem. 33,2028-2033[Abstract/Free Full Text]
  21. Weiss, G., Werner-Felmayer, G., Werner, E. R., Grünewald, K., Wachter, H., Hentze, M. W. (1994) Iron regulates nitric oxide synthase activity by controlling nuclear transcription J. Exp. Med. 180,969-976[Abstract/Free Full Text]
  22. Farrell, R. E., Jr (1993) Nuclear runoff assay Farrell, R. E. eds. RNA Methodologies: A Laboratory Guide for Isolation and Characterization San Diego CA.
  23. Weiss, G., Goosen, B., Doppler, W., Fuchs, D., Pantopoulos, K., Werner-Felmayer, G., Wachter, H., Hentze, M. W. (1993) Translational regulation via iron-responsive elements by the nitric oxide/NO-synthase pathway EMBO J 12,3651-3657[Medline]
  24. Quail, E. A., Yeoh, G. C. (1995) The effect of iron status on glyceraldehyde 3-phosphate dehydrogenase expression in rat liver FEBS Lett 359,126-128[CrossRef][Medline]
  25. van de Stolpe, A., Caldenhoven, E., Stade, B. G., Koenderman, L., Raaijmakers, J. A., Johnson, J. P., van der Saag, P. T. (1994) 12-O-Tetradecanoylphorbol-13-acetate- and tumor necrosis factor alpha-mediated induction of intercellular adhesion molecule-1 is inhibited by dexamethasone. Functional analysis of the human intercellular adhesion molecular-1 promoter J. Biol. Chem. 269,6185-6192[Abstract/Free Full Text]
  26. Collins, T., Read, M. A., Neish, A. S., Whitley, M. Z., Thanos, D., Maniatis, T. (1995) Transcriptional regulation of endothelial cell adhesion molecules: NF-kappa B and cytokine-inducible enhancers FASEB J 9,899-909[Abstract]
  27. Ohh, M., Takei, F. (1996) New insights into the regulation of ICAM-1 gene expression Leuk. Lymphoma 20,223-228[Medline]
  28. Mellilo, G., Taylor, L. S., Brooks, A., Musso, T., Cox, G. W., Varesio, L. (1997) Functional requirement of the hypoxia responsive element in the activation of the inducible nitric oxide synthase promoter by the iron chelator desferrioxamine J. Biol. Chem. 272,12236-12243[Abstract/Free Full Text]
  29. Rosen, G. M., Pou, S., Ramos, C. L., Cohen, M. S., Britigan, B. E. (1995) Free radicals and phagocytic cells FASEB J 9,200-209[Abstract]
  30. Ohh, M., Smith, C. A., Carpenito, C., Takei, F. (1994) Regulation of intercellular adhesion molecule-1 gene expression involves mRNA stabilization mechanisms: effects of interferon-{gamma} and phorbol myristate acetate Blood 84,2632-2639[Abstract/Free Full Text]
  31. Rouault, T. A., Klausner, R. D. (1997) Regulation of iron metabolism in eukaryotes Curr. Top. Cell. Regul. 35,1-19[Medline]
  32. Hentze, M. W., Kühn, L. (1996) Molecular control of vertebrate iron metabolism: mRNA based regulatory circuits operated by iron, nitric oxide, and oxidative stress Proc. Natl. Acad. Sci. USA 93,8175-8182[Abstract/Free Full Text]
  33. Witter, K., Werner, T., Blusch, J. H., Schneider, E-M., Riess, O., Ziegler, I., Rödl, W., Bacher, A., Gütlich, M. (1996) Cloning, sequencing and functional studies of the gene encoding human GTP-cyclohydrolase I Gene 171,285-290[CrossRef][Medline]
  34. Togari, A., Arai, M., Mogi, M., Kondo, A., Nagatsu, T. (1998) Coexpression of GTP-cyclohydrolase I and inducible nitric oxide synthase mRNAs in mouse osteoblastic cells activated by proinflammatory cytokines FEBS Lett 428,212-216[CrossRef][Medline]
  35. Kern, I., Steimle, V., Siegrist, C. A., Mach, B. (1995) The two novel MHC class II transactivators RFX5 and CIITA both control expression of HLA-DM genes Int. Immunol. 7,1295-1299[Abstract/Free Full Text]
  36. Del Pozzo, G., Guardiola, J. (1996) The regulation mechanism of HLA class II gene expression at the level of mRNA stability Immunogenetics 44,453-458[CrossRef][Medline]
  37. Weinberg, E. D., Weinberg, G. A. (1995) The role of iron in infection Curr. Opin. Infect. Dis. 8,164-172[CrossRef]
  38. Alvarez-Hernandez, X., Liceaga, J., McKay, I. C., Brock, J. H. (1989) Induction of hypoferremia and modulation of macrophage iron metabolism by tumor necrosis factor Lab. Invest. 61,319-322[Medline]
  39. Miller, L. L., Miller, S. C., Torti, S. V., Tsuji, Y., Torti, F. M. (1991) Iron-dependent induction of ferritin H chain by tumor necrosis factor Proc. Natl. Acad. Sci. USA 88,4946-4950[Abstract/Free Full Text]
  40. Weiss, G., Bogdan, C., Hentze, M. W. (1997) Pathways for the regulation of macrophage iron metabolism by the antiinflammatory cytokines IL-4 and IL-13 J. Immunol. 158,420-425[Abstract]
  41. Fahmy, M., Young, S. P. (1993) Modulation of iron metabolism in the monocyte cell line U937 by inflammatory cytokines: changes in transferrin uptake, iron handling and ferritin mRNA Biochem. J. 296,175-181
  42. Means, R. T., Krantz, S. B. (1992) Progress in understanding the pathogenesis of the anemia of chronic disease Blood 80,1639-1647[Abstract/Free Full Text]
  43. Weiss, G. (1999) Iron and anemia of chronic disease Kidney Int. Suppl. 69,S12-S17[CrossRef][Medline]
  44. Mencacci, A., Cenci, E., Boelaert, J. R., Bucci, P., Mosci, P., Fé de Ostiani, C., Bistoni, F., Romani, L. (1997) Iron overload alters innate and T-helper cell responses to Candida albicans in mice J. Infect. Dis. 175,1467-1476[Medline]



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