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* Department of Microbiology and Immunology and
Digestive Disease Research Institute, School of Medicine, and
Department of Pathology, School of Dentistry, Wonkwang University, Iksan, Chonbuk, Korea;
Central Genome Research Institute, Korea National Institute of Health, Seoul, Korea; and
¶ Department of Biology, Chonbuk National University, Jeonju, Chonbuk, Korea
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B activation by itself nor affected IL-1
- or TNF-
-mediated NF-B activation, suggesting a NF-B-independent mechanism in DFO-induced IL-8 production. Global gene expression profiling revealed that DFO significantly up-regulates inflammation-related genes including proinflammatory genes, and that many of those genes are down-modulated by the selective mitogen-activated protein kinase inhibitors. Collectively, these results demonstrate that, in addition to bacterial products or cell wall components, direct chelation of host Fe by infected bacteria may also contribute to the evocation of host inflammatory responses. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Intestinal epithelial cells (IECs)4 that line mucosal surfaces of the intestinal tracts are the first to face the challenge of either normal flora or exogenous putative pathogens. Such IECs play a pivotal role in host defense by sensing very early after microbial infection, resulting production of a variety of proinflammatory cytokines affecting leukocyte activity. Chemokines produced by epithelial cells in response to microbial infection may provide signals essential for the initiation and maintenance of the mucosal inflammatory response. The CXC chemokine IL-8 is expressed in IECs in response to proinflammatory cytokines (12) and cellular stress (13, 14, 15), and is recognized as a major activator of acute inflammation (16).
After infection, microbial pathogens must acquire host iron to survive and replicate on the surface of the mucosal layer. Although bacterial products or cell wall components are well-known inducers of host immune responses, chelation of iron in local environment may also contribute to the initiation of host immune mechanisms. In fact, highly virulent strains possess exceptionally powerful mechanisms for obtaining host iron from healthy hosts (17). An excellent example is bacterial siderophores. These small molecules can withdraw iron from transferrins synthesized by a variety of host species. Therefore, in this study, we investigated whether deferoxamine (DFO), which is an actual component of bacteria to chelate iron for bacterial growth, can trigger inflammatory responses, including IL-8 production, in human IECs as a single stimulus.
In this study, we report that DFO, an iron chelator, induces a strong inflammatory response in IECs as indicated by the production of chemokine IL-8. We provide evidences that p38 and extracellular signal-regulated kinase (ERK)1/2, as well as the membrane protein Na+/H+ exchangers (NHEs), play important roles in mediating the proinflammatory effects of iron chelator. Further, we found that both p38 and ERK1/2 activation by iron chelator contribute to the stabilization of IL-8 mRNA transcripts so as to augment IL-8 protein secretion. By investigating the global gene expression patterns after iron chelator administration, we could find a number of proinflammatory or inflammation-related genes that responded to cellular iron availability in IECs. We also identified a few hundred genes whose expression is down-modulated in the presence of p38 and/or ERK1/2 pathway inhibitors.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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DFO, mimosine (MIM), ferric citrate (FC), amiloride (N-amidino-3,5-diamino-6-chloropyrazine carboxamide), cimetidine, clonidine (2-[2,6-dichloroaniline]-2-imidazoline), alkaline-phosphatase-conjugated monoclonal mouse anti-rabbit IgG, and p-nitrophenyl phosphate tablets were purchased from Sigma-Aldrich (St. Louis, MO). All reagents and media for tissue culture experiments were tested for their LPS contents with a colorimetric Limulus amebocyte lysate assay (detection limit 10 pg/ml; Sigma-Aldrich). Human IL-1 was obtained from Invitrogen (Carlsbad, CA). TNF- and polyclonal goat anti-human IL-8 were obtained from R&D Systems (Minneapolis, MN). SB203580 and PD098059 were purchased from Calbiochem (La Jolla, CA). Polyclonal rabbit anti-human IL-8 was from Endogen (Woburn, MA). Abs against p38 kinase, ERK1/2, and the Abs specific to the phosphorylated forms of these proteins were purchased from Cell Signaling Technology (Beverly, MA). HRP-conjugated anti-rabbit IgG was from Amersham Biosciences (Little Chalfont, U.K.). Anti-human I-B was from Santa Cruz Biotechnology (Santa Cruz, CA).
Cell culture
HT-29 human colon epithelial cells (ATCC HTB 38; American Type Culture Collection (ATCC), Manassas, VA) and THP-1 human acute monocytic leukemia cells (ATCC TIB-202) were grown in RPMI 1640 medium supplemented with 10% heat-inactivated FBS, penicillin G (100 IU/ml), streptomycin (100 µg/ml), and L-glutamine (2 mM). T84 human colon epithelial cells (ATCC CCL-284) and Caco-2 human ileocecal epithelial cells (ATCC HTB 37) were grown in DMEM containing the supplements as mentioned above.
Human PBMC and polymorphonucleocytes (PMNs) were isolated from normal donors by dextran sedimentation followed by centrifugation through a discontinuous Ficoll gradient (Amersham Biosciences). All the cell lines or primary cell fractions mentioned above were incubated at 37°C in a humidified atmosphere containing 5% CO2 and 95% air. Log-phase cells or primary cells were seeded at 0.2–5 x 106 per 12-well plate, 24-well plate, or 60-mm culture dish, and used for various experimental purposes.
IL-8 measurement
Adherent cells (HT-29, Caco-2, and T84) were seeded at 2 x 105 into 24-well plates (Nalge Nunc International, Rochester, NY) and grown until formation of confluent monolayers, reaching a final density of 
5 x 105 cells/well. Monolayers were then incubated for 4–24 h in a fresh medium containing stimuli as indicated. Nonadherent cells (peripheral blood cells) were seeded at 5 x 105 into 24-well plates, and incubated for 24 h in a fresh medium containing stimuli as indicated. The supernatants were collected, cleared by centrifugation, and kept at –20°C until evaluation by ELISA. For measurement of IL-8 concentrations in cell culture supernatants, 96-well microtiter plates (MaxiSorp; Nunc) were coated with 0.2 µg/well goat anti-human IL-8 Abs (R&D Systems) in 50 µl of PBS at 4°C overnight. All further steps were conducted at room temperature. After washing three times with PBS, nonspecific binding sites were blocked by incubation with 150 µl PBS + 1% BSA/0.05% Tween 20/well for 2 h. After three washes with PBS, 50 µl of samples or IL-8 standards were added and incubated for 2 h. As a second Ab, 0.05 µg/well polyclonal rabbit anti-human IL-8 (Endogen) was added and incubated for 2 h. As a third Ab, alkaline phosphatase-labeled monoclonal mouse anti-rabbit IgG (Sigma-Aldrich) was diluted in 50 µl of PBS + 0.1% BSA/0.05% Tween 20 to 1:50,000 and incubated for 2 h. Finally, alkaline phosphatase substrate p-nitrophenyl phosphate (Sigma-Aldrich) was added at a concentration of 1 mg/ml in 0.1 M glycine buffer (pH 10.4) containing 1 mM MgCl2 and 1 mM ZnCl2. After overnight incubation, plates were read at 405 nm on a microplate reader (Molecular Devices, Sunnyvale, CA). The detection limit of the ELISA was 30 pg/ml.
RNA isolation and RT-PCR
Human epithelial cells (5 x 106) or THP-1 cells (5 x 106) were grown in 60-mm culture dishes and incubated for 4–24 h in a fresh medium containing stimuli as indicated. After discarding growth medium, total RNA was isolated from cells using easy-Blue (iNtRON Biotechnology, Daejon, Korea), following the manufacturer’s instructions. Reverse transcription of the RNA was performed using AccuPower RT PreMix (Bioneer, Daejon, Korea). One microgram of RNA and 20 pmol primers were preincubated at 70°C for 5 min and transferred to a mixture tube. The reaction volume was 20 µl. cDNA synthesis was performed at 42°C for 60 min, followed by RT inactivation at 94°C for 5 min. Thereafter, the RT-generated DNA (2–5 µl) was amplified using AccuPower PCR PreMix (Bioneer). The primers used for cDNA amplification and PCR conditions were as follows: IL-8 (18), 5'-ATGACTTCCAAGCTGGCCGTGGCT-3' (sense) and 5'-TCTCAGCCCTCTTCAAAAACTTCTC-3' (antisense); GAPDH, 5'-CGGAGTCAACGGATTTGGTCGTAT-3' (sense) and 5'-AGCCTTCTCCATGGTGGTGAAGAC-3' (antisense); an initial denaturation at 94°C for 5 min; 20 cycles being conducted for the time course measurements of IL-8 and GAPDH and 30 cycles for the detection of mRNA decay, each cycle with 30 s of denaturation at 94°C, 30 s of annealing at 62°C and 30 s of extension at 72°C; and a final dwell at 72°C for 7 min. The expected PCR products were 289 bp (for IL-8) and 306 bp (for GAPDH). PCR products were resolved on a 1.5% agarose gel and stained with ethidium bromide.
Real-time quantitative RT-PCR
For some experiments, the expression levels of IL-8 mRNA were evaluated by real-time RT-PCR. Total RNA was isolated and cDNA was synthesized as described above. PCR amplification was performed in DNA Engine Opticon for continuous fluorescence detection system (MJ Research, Waltham, MA) in a total volume of 20 µl containing 1 µl of cDNA/control and gene specific primers using DyNAmo SYBR Green qPCR kit (MJ Research). Each PCR was performed in triplicate using the following conditions: 94°C for 30 s, 62°C for 30 s, 72°C for 30 s, plate read (detection of fluorescent product) for 40 cycles, followed by 7 min extension at 72°C. Melting curve analysis was done to characterize the dsDNA product by slowly raising the temperature (0.2°C/s) from 65°C to 95°C with fluorescence data collected at 0.2°C intervals. The levels of IL-8 mRNA normalized for GAPDH were expressed as fold changes relative to untreated controls. The fold change in gene expression was calculated using the following equation: fold change = 2–
CT, where CT = (CT,Target – CT,GAPDH)time x – (CT,Target – CT,GAPDH)time 0, where time x is any time point and time 0 represents the 1x expression of the target gene of untreated cells, which was normalized to GAPDH (19).
Cell viability assay
Cell viability was examined by MTT assay as described elsewhere (20). A stock solution of MTT was prepared in PBS, diluted in RPMI 1640 medium, and added to cell-containing wells at a concentration of 0.5 mg/ml after removing culture medium. The plates were then incubated for 4 h at 37°C in 5% CO2. At the end of incubation, the medium was aspirated, and the formazan product was solubilized with DMSO. Absorbency was measured on a multiscan reader with a 570-nm wavelength filter. All experiments were performed at least three times. In some experiments, monolayers were fixed in 4% neutral buffered paraformaldehyde, and permeabilized with PBS/0.5% Triton X-100, and their nuclei were stained for 5 min with 4',6'-diamidino-2-phenylindole (DAPI) dye. The stained cells were then washed, and viewed with an inverted fluorescence microscope.
Cell extract preparation and Western blot analysis
For the analysis of phosphorylated or total protein levels of mitogen-activated protein kinases (MAPKs) and the I-B degradation, stimulated cells were rinsed twice with ice-cold PBS and then lysed in ice-cold lysis buffer (50 mM Tris-HCl (pH 7.4), containing 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, 0.1% deoxycholate, 5 mM sodium fluoride, 1 mM sodium orthovanadate, 1 mM 4-nitrophenyl phosphate, 10 µg/ml leupeptin, 10 µg/ml pepstatin A, and 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride). Cell lysates were centrifuged at 15,000 rpm for 20 min at 4°C, and the supernatant was mixed with a one-fourth volume of 4x SDS sample buffer, boiled for 5 min, and then separated through a 12% SDS-PAGE gel. After electrophoresis, proteins were transferred to a nylon membrane by means of Trans-Blot SD semidry transfer cell (Bio-Rad, Hercules, CA). The membrane was blocked in 5% skim milk (1 h), rinsed, and incubated with primary Ab (for phosphorylated MAPKs or I-B) in TBST and 3% skim milk overnight at 4°C. Excess primary Ab was then removed by washing the membrane four times in TBST, and the membrane was incubated with 0.1 µg/ml peroxidase-labeled secondary Ab (against rabbit) for 1 h. Following three washes in TBST, bands were visualized by ECL Western blotting detection reagents and exposed to x-ray film.
Transient transfection and luciferase activity assay
For transient transfections, HT-29 cells were seeded at 5 x 105 in a 12-well plate 1 day before transient transfection (90–95% cell confluency). Cells were transfected with serum- and antibiotics-free RPMI 1640 medium containing 4 µl/ml Lipofectamine 2000 reagent (Invitrogen) and 1.6 µg/ml NF-B luciferase reporter construct, which was kindly provided by Prof. J.-S. Chun (Kwangju Institute of Science and Technology, Gwangju, Korea). After 5 h of incubation, medium was replaced with RPMI 1640 medium containing 10% FBS and antibiotics. Cells were allowed to recover at 37°C for 20 h and subsequently were stimulated as indicated. Cell lysates were prepared and assayed for luciferase activity using Luciferase Assay System (Promega, Madison, WI), according to the manufacturer’s instructions.
NF-B DNA-binding activity
Human intestinal epithelial HT-29 cells were grown to 80% confluency in 100-mm culture dishes and incubated for various times with DFO, IL-1, or combination thereof. Nuclear extracts were prepared according to the protocol provided by BD Mercury TransFactor Kits User Manual (BD Biosciences, Palo Alto, CA). Briefly, cells were rinsed in cold PBS, scraped, and centrifuged. The cells were lysed by incubation on ice for 15 min in 1x lysis buffer (10 mM HEPES, 1.5 mM MgCl2, 10 mM KCl, 1 mM DTT, and protease inhibitor mixture) and centrifuged. The cell pellets were resuspended in 1x lysis buffer again. Further cell disruption was achieved by repetitive filling and flushing of cell suspensions with a syringe having a narrow-gauge needle. The resulting suspensions were centrifuged. The crude nuclear pellets were then resuspended in extraction buffer (20 mM HEPES, 1.5 mM MgCl2, 0.42 M NaCl, 0.2 mM EDTA, 1 mM DTT, and protease inhibitor mixture) and the nuclei were disrupted using a fresh syringe as above. The nuclear suspensions were incubated on ice for 30 min with occasional tapping, and centrifuged. The nuclear extracts were added to assay wells (BD Mercury TransFactor NF-B p65 Kit, BD Biosciences), which were blocked with 1x TransFactor/blocking buffer, and incubated for 60 min at room temperature. After washing three times with 1x TransFactor/blocking buffer, primary Ab (1:500 diluted) was added to the wells and incubated for 60 min at room temperature. After three washes, second Ab (1:1000 diluted) was added and incubated for 30 min. Then, 1x TransFactor buffer was applied to wash the wells. Finally, tetramethylbenzidine substrate was added and incubated for 10 min at room temperature. The absorbance was read at 650 nm on a microplate reader (Molecular Devices).
cDNA microarray analysis
Human intestinal epithelial HT-29 cells were grown in 100-mm culture dishes and incubated for 16 h with iron chelator DFO alone or DFO in combination with MAPK inhibitors. Total RNA was isolated as described above. The approximate amounts of RNA required for a given experiment were 15–20 µg. Total RNA was subjected to reverse transcription while being labeled with either Cy3 or Cy5 by means of 3DNA indirect labeling kit, according to the protocol provided by the manufacturer (Genisphere, Hatfield, PA). For hybridization, Cy3- and Cy5-labeled cDNAs were applied to a human cDNA chip containing 7500 cDNA clones with 90% known sequences and 10% expressed sequence tags (HSVC V1.0; GenomicTree, Daejon, Korea). Hybridized slide was scanned with ScanArray 3000 (Packard Bioscience, Meriden, CT). Signal quantification and data processing were performed using Imagene 5.1 and GeneSight 3.5 (BioDiscovery, El Segundo, CA). Each experiment was repeated three times to reduce a high risk of false-positive or false-negative results. The results from three independent identical experiments were merged, and the merged data were used for subsequent comparisons. The average intensity values of Cy3 and Cy5 for each gene were calculated. Significant differences in gene expression between untreated and treated cells were determined using the Student’s t test with a significance threshold of p < 0.05. To estimate fold induction, the ratios of Cy5 (treated) to Cy3 (untreated) signal intensities were calculated. The log2 of each ratio was determined to equalize the magnitude of deflection of up-regulated and down-regulated genes, and gene expression differences were ranked based on absolute values.
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Results |
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We first examined whether chelation of iron from human IECs gives a signal to produce IL-8 as a single stimulus. For this reason, we primarily chose undifferentiated HT-29 human colon epithelial cells (ATCC HTB 38), and cultured under the standard conditions as described in Materials and Methods. Treatment of HT-29 cells with DFO, an iron chelator, markedly induced IL-8 secretion (Fig. 1A). The effect of DFO was concentration-dependent in the range of 0–1 mM. However, higher concentrations of DFO failed to further increase the production of IL-8. Maximal increase of IL-8 production was achieved at the concentration of 0.1 mM. MIM, an iron chelator structurally distinct from DFO, also induced IL-8 secretion in HT-29 cells. Conversely, the addition of FC (Fe3+, 0.5 mM) significantly prevented the production of IL-8 induced by DFO (Fig. 1B), indicating that the target of DFO is specific for the intracellular iron in HT-29 cells. The IL-8 concentrations induced by DFO were comparable with those elicited by IL-1 (1 ng/ml) or TNF- (1 ng/ml) and the combinations of DFO and either IL-1 or TNF- showed additive effects on IL-8 secretion (Fig. 1C).
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Time-dependent experiment revealed that DFO slowly induces IL-8 secretion and maximal induction is seen after 8–16 h of incubation (Fig. 2A). This is different from that observed in IL-1-elicited IL-8 secretion, where a plateau of IL-8 concentration reached within 4 h after IL-1 treatment. We also found that the increase of IL-8 protein secretion appears to correspond to that of IL-8 mRNA accumulation levels (Fig. 2B). To further quantify the level of expression, IL-8 mRNA were assessed by real-time PCR using a SYBR green dye (Fig. 2, C and D). As shown in Fig. 2D, relative value of IL-8 mRNA as normalized to internal control GAPDH were well matched with the results of RT-PCR.
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Iron chelator induces IL-8 secretion in human IECs via NF-B-independent mechanism
Because NF-B is the central regulator of IL-8 gene expression in IECs (26), we examined the effect of DFO on NF-B activation by measuring I-B degradation, NF-B DNA binding, and NF-B-dependent transcriptional activity. As Fig. 4A shows, there was no evidence for I-B degradation after DFO (0.2 mM) treatment, while treatment with IL-1 significantly induced I-B degradation. Thus, we next measured the NF-B-binding activity by ELISA-based method. In contrast to IL-1, surprisingly, treatment with DFO failed to induce NF-B binding to NF-B consensus oligonucleotides absorbed on ELISA plate (Fig. 4B). No significant NF-B-binding activity was seen up to 9 h (data not shown). In addition, DFO had no effect on IL-1-mediated NF-B-binding activity (Fig. 4B). Finally, we investigated whether DFO increases NF-B-dependent gene transcription in HT-29 cells. To this end, HT-29 cells were transiently transfected with a NF-B-luciferase reporter construct or the empty vector. Exposure of HT-29 cells to DFO for 24 h showed no sign of luciferase activity, while IL-1 or TNF- significantly increased the luciferase activity in the cells transfected with the NF-B-luciferase reporter construct (Fig. 4C). Taken together, we could conclude that iron chelator-mediated IL-8 production is independently regulated from NF-B-dependent transcriptional activity in HT-29 cells.
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Previously we documented that iron chelator activates MAPK pathways as well as multiple caspases that are involved in the apoptotic cascade of human myeloid leukemic HL-60 cells (23). We also found that p38 kinase plays a crucial role in iron chelator-induced apoptosis. Because activation of the p38 and ERK1/2 is also known as an important step in the cascade of cellular events leading to IL-8 production in IECs (26, 27, 28), we examined whether DFO enhances IL-8 production by a mechanism involving p38 and ERK1/2. Exposure of HT-29 cells to DFO (0.2 mM) caused strong induction of ERK1/2 phosphorylation as early as 10 min, which remained increased during a 6-h observation period (Fig. 5A). As compared with ERK1/2, the DFO-induced phosphorylation of p38 was relatively delayed, reaching maximal phosphorylation at 3 h after DFO treatment (Fig. 5A).
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Activation of p38 kinase lies upstream of ERK1/2
Because single treatment of either p38 or ERK1/2 inhibitor could completely inhibit DFO-induced IL-8 secretion in HT-29 cells, we next asked whether there is a signaling interaction between p38 and ERK1/2. For this purpose, cells were treated with either SB203580 or PD098059, stimulated with DFO, and analyzed for p38 and ERK1/2 activation by Western blotting. The results are shown in Fig. 6. Fig. 6A shows that treatment with 10-µM SB203580 significantly reduced ERK1/2 phosphorylation induced by DFO. This is consistent with the expected reduction of ERK1/2 phosphorylation by PD098059 (Fig. 6A). In contrast, treatment with 10-µM PD098059 showed little effect on DFO-induced p38 phosphorylation, while it slightly increased p38 kinase phosphorylation in the control HT-29 cells (Fig. 6B). Taken together, although we did not elucidate whether p38 kinase directly influences the phosphorylation of ERK1/2, the current results strongly demonstrate that p38 appears to be upstream of ERK1/2 in the DFO-induced signaling in the process of IL-8 production in HT-29 cells.
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mRNA accumulation is the result of the balance between mRNA synthesis and degradation (29). Because our results demonstrated that both NF-B (Fig. 4) and AP-1 (data not shown) are not directly involved in iron chelator-mediated IL-8 production in HT-29 cells, we next examined whether MAPKs activation by DFO is involved in the stabilization of IL-8 mRNA transcripts so as to augment IL-8 protein secretion. HT-29 cells were treated with DFO (0.2 mM) for 8 h, followed by treatment with actinomycin D (5 µg/ml), an inhibitor of mRNA synthesis, in the presence or absence of MAPK inhibitors. Total RNA was isolated at various time points, and remaining IL-8 mRNA was measured by semiquantitative RT-PCR. As shown in Fig. 7, p38 and ERK inhibition resulted in the rapid disappearance of IL-8 mRNA relative to actinomycin D-treated control cells, suggesting a posttranscriptional regulation of IL-8 gene transcript by iron chelator.
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Recent evidence indicates that NHEs (antiporters) regulate inflammatory processes. NHEs are rapidly activated in response to a variety of inflammatory signals, such as IL-1 (30), TNF- (31), IFN-
(32), LPS (33), and lithium (18). In this regard, inhibition of NHEs suppresses inflammatory responses, including IL-8 production by monocytes and respiratory epithelial cells (34). Therefore, we examined whether DFO induces IL-8 production via an NHE-dependent mechanism. Inhibition of NHE activation with a selective inhibitor, amiloride, or nonselective NHE inhibitors, cimetidine and clonidine, significantly abrogated the stimulatory effect of DFO on IL-8 production in HT-29 cells (Fig. 8). These inhibitors at dosages tested in this study showed no effects on cell viability, as determined by MTT assay (data not shown). These data indicate that NHEs play a central role in the DFO-induced IL-8 response in HT-29 cells.
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To determine whether iron chelator could also modulate the expression of inflammation-related genes, including proinflammatory genes, we used a high throughput cDNA microarray approach and investigated the global gene expression patterns of HT-29 cells after DFO treatment. Because we observed that both p38 and ERK1/2 play pivotal roles in iron chelator-induced IL-8 secretion, we also searched for the genes whose expression is down-modulated in the presence of the SB203580 or PD098059. HT-29 cells were incubated for 16 h with DFO (0.2 mM) or DFO in combination with SB203580 (10 µM) or PD098059 (10 µM), and the total RNA was isolated to perform microarray analysis using a human 8.0K cDNA chip (HSVC V1.0; GenomicTree). Gene expression levels were analyzed using the Imagene 5.1 and GeneSight 3.5 software tool.
The expression levels of each gene from untreated (labeled with Cy3 before hybridization) vs that of DFO-, DFO + PD098059-, or DFO + SB203580-treated epithelia (Cy5 labeled) are represented as fold increase, as compared with the control. The individual genes (>4-fold up-regulated by DFO) are listed in Table SI.5 As expected, DFO by itself, as a single stimulus, significantly up-regulated 133 genes in HT-29 cells (Table SI). Of these 133 up-regulated genes, 73 genes (55%) exhibited reduced induction (by 25% or more) of transcript abundance in the presence of SB203580 or PD098059 (Table SI). Among those genes represented in Table SI, we summarized the genes (37 genes) that are involved in inflammation and listed in Table I. Many other genes also exhibited decreased expression levels in response to DFO in HT-29 cells (data not shown). To validate the accuracy of microarray expression profiling data, we performed semiquantitative RT-PCR with some inflammation-related genes such as CCL19 and CCL20 and confirmed that the two methods show overall qualitative agreement (data not shown). The membrane expression of ICAM-1, which has been known to augment immune responses and leukocyte accumulation in inflamed tissues, was also estimated by flow cytometer (data not shown). Although conclusions regarding effects on individual specific genes require verification via other means, this microarray technique is effective at showing the overall effect of iron deprivation on human IECs.
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Discussion |
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Iron is also important for host immunosurveillance because of its growth-promoting role for immune cells and its interference with cell-mediated immune effector pathways and cytokine activities (40, 41). It has been demonstrated that iron deficiency as well as iron overload can exert subtle effects on immune status by altering the proliferation and activation of T, B, or NK cells (42, 43). In this study, we demonstrated that chelation of iron by bacterial siderophore DFO induces a full-blown inflammatory response in human IECs. Several investigators have reported that both iron and iron chelators may modulate certain inflammatory mediators and regulate inflammatory processes (44, 45). However, our study is the first one to demonstrate that iron chelator activates an inflammatory response even in the absence of conventional immunostimulatory/inflammatory stimuli. Central to this iron chelator-induced inflammatory response of IECs is the early activation (15 min after DFO treatment) of the p38 and ERK1/2 systems. These early pathways play a pivotal role in mediating later events of the IEC inflammatory response, such as the up-regulation of various inflammation-related genes in addition to IL-8 gene expression.
It was interesting to notice that the iron chelator also induces IL-8 in other cell types such as monocyte and human primary blood leukocytes. These indicate that induction of inflammatory signals by iron chelator may have a broader relevance for the cells that are the first to face the challenge of exogenous putative pathogens (25). However, unlike we found in the above cells, our preliminary results revealed that HUVECs show no response against iron chelator even in high concentrations (1 mM) (data not shown). Because HUVEC are also known to induce IL-8 in response to various stimuli (46), this result implies that the effect of iron chelator is somewhat cell-type specific. Further, the extent of iron chelator-responsiveness may also vary depending on the state of cellular differentiation, as evidenced by Caco-2 cells.
NHEs (antiporters) are a family of ubiquitous plasma membrane transport proteins that catalyze the exchange of extracellular Na+ for intracellular H+ (47). Interestingly, it has been noticed that NHEs are activated in response to a variety of inflammatory signals (18, 30, 31, 32, 33). Moreover, inhibition of NHEs has been reported to inhibit inflammatory responses, including IL-8 production by monocytes and epithelial cells as well as macrophage inflammatory protein-1 and -2, and IL-12 production by macrophages (34, 48). In this study, although we did not directly measure the activity of NHEs in iron chelator-treated HT-29 cells, our observation that NHE inhibition almost completely prevented the proinflammatory effects of DFO demonstrates a key role for these proteins in mediating the IEC inflammatory response to iron chelation.
The primary molecular target for iron chelator to induce inflammatory signals is not currently estimated. However, it is unlikely that iron chelator acts on all of those inflammatory events and enzyme cascades described in this paper independently. A more plausible scenario is that DFO targets an early pathway upstream from MAPKs and NHEs. For example, it is conceivable that DFO stimulates a membrane receptor whose ligation by its ligands normally induces a response similar to the one observed with iron chelator. Excellent candidates are the IL-1 and TNF- receptors as well as Toll receptors, which are the major membrane structures responsible for relaying extracellular inflammatory signals toward intracellular effector sites of inflammation in IECs. However, as described in Results, because DFO alone failed to activate NF-B pathway, it appears improbable that the proinflammatory effects of iron chelator in IECs are mediated by inflammatory membrane receptors. Indeed, the additive effects of iron chelator with IL-1 or TNF- to induce IL-8 secretion (Fig. 1C) also suggest the membrane receptor-independent mechanism in iron chelator-mediated IL-8 secretion.
IL-8 expression is initially regulated at the transcriptional level, although posttranscriptional control has been reported (49, 50). After stimulation with IL-1, IL-8 mRNA is rapidly induced and can be detected within 60 min, suggesting that IL-8 mRNA is an early response gene (49). The rapid induction of IL-8 gene expression is mediated by latent transcription factor that binds to the IL-8 promoter. The only major cis-acting elements demonstrated to play a functional role in the regulation of the IL-8 promoter are located in two different regions: a distal promoter element comprised of an AP-1 binding site and a proximal promoter element containing binding sites for NF-IL-6 and NF-B (49). Thus, most previous reports have demonstrated the major roles of NF-B- or AP-1-dependent mechanism for the induction of IL-8 expression in human IECs. In the present paper, however, we observed that neither NF-B nor AP-1 (data not shown) is involved in iron chelator-mediated IL-8 production in HT-29 cells. Instead, we found that iron chelator could increase the IL-8 mRNA stability, presumably through the activation of p38 and ERK1/2 pathways. Importantly, we observed that inhibition of p38 or ERK with SB203580 or PD098059 significantly reduced the IL-8 mRNA half-lives compared with untreated samples. In good agreement with this, a recent report demonstrated that MAPKs contribute to IL-8 secretion by IECs via a posttranscriptional mechanism (51).
To investigate further the spectrum of proinflammatory and inflammation-related genes dependent on cellular iron status, we have used gene expression profiling with cDNA microarrays. Our results revealed a number of both novel and known genes that responded to cellular iron availability. We identified 37 inflammation-related genes whose expression is highly up-regulated in the state of cellular iron deprivation. Further, we identified 21 genes whose expression is significantly decreased by the treatment with MAPK inhibitors (Table I). Although the mechanisms where iron chelator alters the expression of individual-specific genes in human IECs require further verification via other means, these microarray data will be very valuable to form the starting point for future experimental studies.
In summary, our experiments using IECs demonstrate that direct chelation of intracellular iron induces both p38 and ERK1/2 MAPK activation, and that this activation of MAPK results in the development of an inflammatory response in IECs. Our results further imply, for the first time, that modulation of iron status by infected bacteria may also contribute to the evocation of host inflammatory responses even in the absence of conventional immunostimulatory/inflammatory stimuli.
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Footnotes |
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2 E.-Y.C. and E.-Y.K. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Chang-Duk Jun, Department of Microbiology and Immunology, Digestive Disease Research Institute, School of Medicine, Wonkwang University, Iksan, Chonbuk, Korea 570-749. E-mail address: cdjun{at}wonkwang.ac.kr
4 Abbreviations used in this paper: IEC, intestinal epithelial cell; DAPI, 4',6'-diamidino-2-phenylindole; DFO, deferoxamine; ERK, extracellular signal-regulated kinase; FC, ferric citrate; MAPK, mitogen-activated protein kinase; MIM, mimosine; NHE, Na+/H+ exchangers; PMN, polymorphonucleocyte; CT, threshold cycle.
5 The on-line version of this article contains supplemental material.
Received for publication August 18, 2003. Accepted for publication March 23, 2004.
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