IL-18 is expressed from a variety of cell types. Two promoters located upstream of exon 1 (5′-flanking region) and upstream of exon 2 (intron 1) regulate its expression. Both promoter regions were cloned into pCAT-Basic plasmid to yield p1-2686 for the 5′-flanking promoter and p2-2.3 for the intron 1 promoter. Both promoters showed basal constitutive activity and LPS inducibility when transfected into RAW 264.7 macrophages. To learn the regulatory elements of both promoters, 5′-serial deletion and site-directed mutants were prepared. For the activity of the p1-2686 promoter, the IFN consensus sequence binding protein (ICSBP) binding site between −39 and −22 was critical. EMSA using an oligonucleotide probe encompassing the ICSBP binding site showed that LPS treatment increased the formation of DNA binding complex. In addition, when supershift assays were performed, retardation of the protein-DNA complex was seen after the addition of anti-ICSBP Ab. For the activity of the p2-2.3 promoter, the PU.1 binding site between −31 and −13 was important. EMSA using a PU.1-specific oligonucleotide demonstrated that LPS treatment increased PU.1 binding activity. The addition of PU.1-specific Ab to LPS-treated nuclear extracts resulted in the formation of a supershifted complex. Furthermore, cotransfection of ICSBP or PU.1 expression vector increased p1 promoter activity or IL-18 expression, respectively. Taken together, these results indicate that ICSBP and PU.1 are critical elements for IL-18 gene expression.

Interleukin-18 was first purified from the liver of mice infected by Propionibacteriumacnes and subsequently challenged with LPS (1, 2). Murine cDNA was cloned from murine liver mRNA (3), and subsequently, human cDNA was cloned from normal human liver mRNA (4). Murine IL-18 cDNA encodes a precursor protein of 192 aa that is processed into mature protein of 157 aa by the IL-1β-converting enzyme (ICE)3 (5).

IL-18 increases IFN-γ production from Th1 cells, induces NK cell cytotoxicity, and activates Th1 cell proliferation. IL-18 shares some of its biological activities with IL-12. IL-18 induces the fungicidal activity of murine peritoneal exudate cells against Cryptococcus neoformans through IFN-γ production in the presence of IL-12 (6), and it also inhibits IgE production by induction of IFN-γ production from the activated B cells in the presence of IL-12 (7). Recently, the IL-18R was purified from the Hodgkin’s disease cell line, L428, and identified as IL-1Rrp (8). IL-18 activates NF-κB, p56lck, and mitogen-activated protein kinase in Th1 cells (9, 10). In addition, it shows a variety of in vitro biological activities, such as potentiating T cell development in the presence of IL-12 (11) and initiating production of proinflammatory cytokines (12). In vivo studies also demonstrated that it has diverse effects on liver inflammation and injury (13), cytotoxicity (14, 15), and tumor regression (16).

Like other cytokines, IL-18 is expressed from many different types of cells and tissues, including Kupffer cells, P. acnes-induced peritoneal cells (1), epidermal keratinocytes (17), osteoblastic cells (18), brain (19), and adrenal cortex (20). However, little is known about the physiological stimuli that induce IL-18 gene expression. Recently, Tone et al. (21) reported that IL-18 gene expression is controlled by the activities of two promoters, which have no TATA and G+C-rich region. It was also demonstrated that an upstream promoter is inducible by LPS or PMA plus ionomycin, and a downstream promoter has constitutive activity.

To understand the regulatory elements in both promoters, we assayed the promoter activities of deleted and site-directed mutated promoter constructs. It was found that PU.1 is a critical site for maximal activity of the downstream promoter, and ICSBP is critical for the activity of the upstream promoter. Possible interactions between the two promoters are also discussed.

Bacterial LPS (from Escherichia coli serotype 0127: B8) and TLC plates (silica gel) were purchased from Sigma (St. Louis, MO). Poly(dI-dC)·poly(dI-dC) and dNTPs were obtained from Pharmacia LKB Biotechnology (Piscataway, NJ). 1-Deoxydichloroacetyl-1-[14C]chloramphenicol and [α-32P]dCTP were purchased from Amersham (Aylesbury, U.K.). Restriction enzymes, Klenow fragment of DNA pol. I, BSA, and acetyl-CoA were purchased from Boehringer Mannheim (Mannheim, Germany). The ICSBP cDNA construct was provided by Dr. Ben-Zion Levi (Technion-Israel Institute of Technology, Haifa, Israel). The polyclonal Abs against IRF-1, PU.1, or ICSBP were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

The mouse macrophage-like cell line RAW 264.7 was obtained from American Type Culture Collection (Manassas, VA). Cells were cultured in DMEM supplemented with 2 mM l-glutamine, antibiotics (100 U/ml penicillin G and 100 μg/ml streptomycin), and 10% heat-inactivated FBS (Life Technologies/BRL, Gaithersburg, MD; complete medium) and were maintained at 37°C in a humidified incubator containing 5% CO2. Isolation of starting populations of hemopoietic cells from normal or PU.1 null liver were obtained from 1-day-old neonates. The generation of myeloid cells and neutrophils from normal and PU.1 null myeloid cells has been described (22, 23). Macrophages were obtained from the identical source and were separated by virtue of adherence and subsequent growth in 5000 U/ml of recombinant mouse M-CSF. The generation and characterization of the PU.1-deficient myeloid cell line, 503, as well as the generation of sublines of the 503 line restored with PU.1 or M-CSF receptor have been described previously (23). Primary PU.1 null myeloid cells were restored with PU.1 using retroviral transduction. Briefly, the wells of a 24-well tissue culture plate (Costar, Cambridge, MA) were incubated with a 10 μg/ml solution of fibronectin (Sigma) at 37oC for 2 h. Fibronectin was aspirated, wells were washed, and 0.5 × 106 CFU/ml of the amphotropic retroviral vector containing the complete coding region of PU.1 was added. The plate was further incubated for 1 h at 37oC to allow adherence of viral particles to the fibronectin-coated surface. Hemopoietic cells (0.5 × 106) were then added in a minimal volume of medium plus Polybrene (final concentration, 6–8 μg/ml), and the plate was centrifuged at 1000 × g for 3 h at room temperature. Afterward, cells were resuspended and allowed to recover for 24 h in growth factor-containing medium. The transduction protocol was repeated a total of three times, and cells were assessed 1–2 wk later for IL-18 message.

Total cellular RNA was isolated from RAW 264.7 cells using the RNAzol B reagent (Tel-Test, Friendswood, TX) according to the manufacturer’s instructions. RNA samples (20 μg) were size fractionated on 1.2% agarose/formaldehyde gels and transferred to nylon membranes. The filter was hybridized with radiolabeled IL-18 cDNA probe, washed, and autoradiographed at −70°C.

A mouse genomic DNA library in EMBL3 SP6/T7 vector (Clontech, Palo Alto, CA) was screened for cloning of the two promoter regions of IL-18 gene by using the 5′ fragment of IL-18 cDNA (nt 1–524). Ten positive clones were selected, and their genomic DNAs were digested with XhoI. The products were subjected to Southern blot with another DNA probe (nt 1–194). By comparing with the restriction map and partial DNA sequencing, a clone of these products was identified as containing about 20 kb encompassing from the 5′-flanking region to exon 2 of IL-18 genome. To make reporter constructs, pCAT-basic plasmid (Promega, Madison, WI) was used to form p1-2686 for the 5′-flanking promoter or to form p2-2.3 for the intron 1 promoter. Deletion constructs of two promoter regions were obtained by digesting either p1-2686 or p2-2.3 plasmid using restriction enzymes or the PCR method. Site-directed mutants of ICSBP binding site or PU.1 binding site were constructed by PCR mutagenesis. Then, all constructs were confirmed by DNA sequencing.

RAW 264.7 cells (1 × 107) were transfected by electroporation with 20 μg of CAT constructs in 250 μl of complete medium using Electro Cell Manipulator (BTX, San Diego, CA) at 230 V and 975 μF capacitance. Twenty-four hours later, transfected cells were further treated with LPS for 24 h, and then harvested. The cells were washed with ice-cold PBS, resuspended in 0.25 M Tris (pH 7.8), and subjected to three cycles of freezing and thawing. Cell lysates were heated for 10 min to inactivate CAT inhibitors and centrifuged. Then, the supernatant of the cell lysates was assayed for CAT enzyme activity by the TLC method (24). As an internal control for transfection efficiency, all cells were cotransfected with 5 μg of pCH110 plasmid (Pharmacia, Piscataway, NJ) for β-galactosidase assay.

Single-stranded oligonucleotides were annealed to form the oligomers shown below. To prepare a probe for EMSA, each oligomer was filled with [α-32P]dCTP and three other nonradiolabeled dNTPs by the Klenow fragment. The ICSBP and PU.1 binding sites are underlined; mutations are italicized: ICSBP, 5′-GGGAAGCTTGCTTTCACTTCTCCC-3′ and 3′-TTCGAACGAAAGTGAAGAGGGGACAGG-5′; mICSBP, 5′-GGGAAGCTTGCTCCCACTTCTCCC-3′ and 3′-TTCGAACGAGGGTGAAGAGGGGACAGG-3′; PU.1, 5′-GGGTTCTTCCTCATTCTT-3′ and 3′-AAGAAGGAGTAAGAAGGG-3′; and mPU.1, 5′-GGGTTCTCTCTCATTCTT-3′ and 3′-AAGAGAGAGTAAGAAGGG-3′.

Nuclear extracts for EMSA were prepared from RAW 264.7 cells as previously described (25) with minor modifications. For binding reactions, 7 μg of nuclear extract was incubated with reaction buffer (10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 1 mM DTT, 1 mM EDTA, 5% glycerol, 2 μg of poly(dI-dC)·poly(dI-dC), and 1 μg of BSA) in the presence or the absence of competitor or Ab for 20 min at room temperature. Then, the radiolabeled probe (≥20,000 cpm) was added to the reaction mixture for an additional 10 min at room temperature. Protein-DNA complexes were separated from the free probe by gel electrophoresis on 6% polyacrylamide gels in 0.5× TBE buffer. The gel was dried and analyzed by autoradiography.

Total RNA was isolated from 0.5–5 × 106 cultured cells using Trizol (Life Technologies) as directed and was subjected to DNase I treatment (10 U for 30 min at 37°C; Boehringer Mannheim). Total RNA (1.0 μg) was reverse transcribed using Superscript II (Life Technologies), and 1/10th of the reaction was subjected to PCR using the following conditions: 94oC for 1 min, 55–65oC for 1 min, 72oC for 1 min for 30 cycles in a Perkin-Elmer thermocycler (GeneAmp 9600, Perkin-Elmer, Norwalk, CT). PCR primers for IL-18 were previously designed (3). Negative control reactions for RT-PCR contained RNA template that had not undergone RT. An aliquot (25 μl) of each 50-μl PCR reaction was run in a 1.5% agarose gel with ethidium bromide and photographed.

When RAW 264.7 macrophages were treated with LPS, IL-18 mRNA expression was up-regulated in a dose-dependent manner (Fig. 1,A). IL-18 expression was clearly detectable at 6 h and was maintained up to 24 h after LPS treatment (Fig. 1,B). Cycloheximide had no effect on IL-18 expression at 9 h, suggesting that its expression was directly induced by LPS rather than by other LPS-induced cytokines. In untreated cells, constitutive expression of IL-18 mRNA was detected, indicating that IL-18 gene expression is regulated by two different mechanisms: constitutively and inducibly. A previous study by Tone et al. (21) showed that murine IL-18 gene expression is regulated by two distinct promoters located upstream of exon 1 (5′-flanking region) and upstream of exon 2 (intron 1), being analyzed as an inducible or a constitutive promoter in terms of responsibilities to LPS. We cloned two promoters: one is positioned from −2686 to +67 of the 5′-flanking region, and the other is positioned from approximately −2300 to +72 of the intron 1 of murine IL-18 gene from a BALB/c mouse-derived leukocyte genomic library. Each promoter region was cloned into the upstream of the CAT gene in pCAT-Basic plasmid to yield p1-2686 for the 5′-flanking promoter and p2-2.3 for the intron 1 promoter. To assay the inducibility by LPS, each p1-2686 and p2-2.3 plasmid was transfected into RAW 264.7 macrophages. Without LPS stimulation, each promoter showed basal constitutive promoter activities (Fig. 1 C). However, in contrast to the previous report, both promoters conferred LPS-inducible promoter activities, although p1-2686 promoter exhibits a little higher LPS inducibility. These results indicate that constitutive or LPS-inducible mouse IL-18 gene expression in RAW 264.7 macrophages is regulated simultaneously, not by each promoter separately, even though the relative contribution of each promoter to inducibility may be different.

FIGURE 1.

Mouse IL-18 mRNA expression and CAT activity of IL-18 two promoters in RAW 264.7 macrophages. A, Northern blot analysis. RAW 264.7 cells were stimulated with the indicated concentrations of LPS for 9 h. Following incubation, cells were harvested, and total RNA (20 μg) was subjected to Northern blot analysis using 32P-labeled probes as described in Materials and Methods. Equal RNA loading was confirmed by ethidium bromide staining of 28S and 18S ribosomal RNA. B, Time-dependent expression of IL-18. RAW 264.7 cells were stimulated with 1 μg/ml for various times, and Northern blot was performed as described above. CHX, 1 μg/ml cycloheximide C, CAT activity was determined in RAW 264.7 cells transfected with a full-length promoter 1-CAT construct (p1-2686) or a promoter 2-CAT construct (p2–2.3) of IL-18. RAW 264.7 cells were transfected with the indicated CAT constructs, treated with or without LPS (1 μg/ml) for 24 h, and assayed for CAT activity. Transfection efficiency was normalized using β-galactosidase as described in Materials and Methods. Data represent the percent acetylation and the mean ± SD of four independent experiments.

FIGURE 1.

Mouse IL-18 mRNA expression and CAT activity of IL-18 two promoters in RAW 264.7 macrophages. A, Northern blot analysis. RAW 264.7 cells were stimulated with the indicated concentrations of LPS for 9 h. Following incubation, cells were harvested, and total RNA (20 μg) was subjected to Northern blot analysis using 32P-labeled probes as described in Materials and Methods. Equal RNA loading was confirmed by ethidium bromide staining of 28S and 18S ribosomal RNA. B, Time-dependent expression of IL-18. RAW 264.7 cells were stimulated with 1 μg/ml for various times, and Northern blot was performed as described above. CHX, 1 μg/ml cycloheximide C, CAT activity was determined in RAW 264.7 cells transfected with a full-length promoter 1-CAT construct (p1-2686) or a promoter 2-CAT construct (p2–2.3) of IL-18. RAW 264.7 cells were transfected with the indicated CAT constructs, treated with or without LPS (1 μg/ml) for 24 h, and assayed for CAT activity. Transfection efficiency was normalized using β-galactosidase as described in Materials and Methods. Data represent the percent acetylation and the mean ± SD of four independent experiments.

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To identify the regulatory elements of the p1 promoter responsive to LPS, sequential 5′ deletion constructs of plasmid p1-2686 were prepared (Fig. 2,A). These constructs were transfected into RAW 264.7 cells by electroporation, and the cells were treated with or without LPS (1 μg/ml) for 24 h. As shown in Fig. 2 B, deletion of the region from −2686 to −1528 did not affect the promoter activity significantly, but deletion of the region from −1528 to −954 (p1-954) gave rise to a significant decrease in LPS-induced transcriptional activity (70% reduction) and in basal transcriptional activity (40% reduction), indicating the existence of a positive element in this region. Also, further deleting the promoter to −22 bp (p1-22) abolished the response to LPS (70% reduction) as well as basal transcriptional activity (50% reduction) compared with the p1-39 construct, suggesting the presence of a positive regulatory element in the region from −39 to −22. Transfection of the p1-438, p1-590, and p1-954 constructs resulted in lower levels of CAT activity by LPS (each 50%) compared with the p1-139, suggesting the presence of negative cis-acting elements located between −438 and −139 bp. Taken together, these results indicate that the two regions, positions from −39 to −22 bp and from −1528 to −954 bp, contain elements that are responsible for the LPS responsiveness as well as for the basal transcriptional activity of the p1 promoter.

FIGURE 2.

CAT activity of 5′ deletion mutants of the mouse IL-18 promoter 1-CAT constructs in RAW 264.7 cells. A, Schematic representation of the mouse IL-18 promoter 1-CAT deletion constructs. B, The indicated deletion CAT constructs were transfected into RAW 264.7 cells by electroporation. CAT activity was measured in transfected cells stimulated with or without LPS (1 μg/ml) for 24 h. Data represent the percent acetylation and the mean ± SD of three independent experiments.

FIGURE 2.

CAT activity of 5′ deletion mutants of the mouse IL-18 promoter 1-CAT constructs in RAW 264.7 cells. A, Schematic representation of the mouse IL-18 promoter 1-CAT deletion constructs. B, The indicated deletion CAT constructs were transfected into RAW 264.7 cells by electroporation. CAT activity was measured in transfected cells stimulated with or without LPS (1 μg/ml) for 24 h. Data represent the percent acetylation and the mean ± SD of three independent experiments.

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Analysis of the sequence in the region from −39 to −22 revealed the presence of a potential binding site for binding of IFN-activated transcription factor, ICSBP, or IRF-E, which differed by 1 bp from their consensus sequence (26, 27). To determine whether this site was responsible for the LPS inducibility of the proximal region of p1 promoter identified above, site-directed mutagenesis of the ICSBP binding site (TGCTTTCACTTCTC→TGCTCCCACTTCTC) was introduced into each p1-39 and p1-2686 to generate the plasmid p1-m39 (mICSBP) and p1-m2686 (mICSBP), respectively (Fig. 3,A). The constructs were then transiently transfected into RAW 264.7 cells, and their activity was examined following 24-h treatment with LPS (1 μg/ml). As shown in Fig. 3 B, RAW 264.7 cells transfected with the p1-m39 (mICSBP) showed a significant 2- to 3-fold decrease in CAT activity by LPS compared with that of cells transfected with the wild-type construct. The reduction of CAT activity was more prominent when mutation was introduced in the full-length p1 promoter (3- to 4-fold reduction). These results indicate that the ICSBP binding site is essential for p1 promoter activity.

FIGURE 3.

Effects of site-directed mutation in the ICSBP binding site on IL-18 promoter 1 activity. A, CAT constructs (−39 to +67 or −2686 to +67) carrying mutations of the ICSBP binding site. Italicized bold letters indicate the specific mutated sequences. B, After transfection with the indicated CAT constructs, RAW 264.7 cells were treated with or without LPS (1 μg/ml) for 24 h, and CAT activities were measured. The results represent the percent acetylation and the mean ± SD of three independent experiments.

FIGURE 3.

Effects of site-directed mutation in the ICSBP binding site on IL-18 promoter 1 activity. A, CAT constructs (−39 to +67 or −2686 to +67) carrying mutations of the ICSBP binding site. Italicized bold letters indicate the specific mutated sequences. B, After transfection with the indicated CAT constructs, RAW 264.7 cells were treated with or without LPS (1 μg/ml) for 24 h, and CAT activities were measured. The results represent the percent acetylation and the mean ± SD of three independent experiments.

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To determine whether LPS induces DNA binding activity to a putative ICSBP binding site of the p1 promoter, we performed EMSA using an oligonucleotide probe encompassing the ICSBP binding site. Control nuclear extract of RAW 264.7 cells showed minimal binding activity. Treatment with LPS, however, markedly increased the formation of a DNA binding complex (Fig. 4,A). When a 100-fold excess of a wild-type probe was added, it efficiently competed with the protein-DNA complex, whereas the probe containing a mutated ICSBP binding sequence did not compete. LPS-induced ICSBP binding was time dependent, with maximal binding at 3–6 h after treatment (Fig. 4,B). The ICSBP binding complex was also detected at 0 h in longer exposure (data not shown). In addition, when supershift assays were performed using Abs against ICSBP or IRF-1, retardation of the protein-DNA complex was seen only by the addition of ICSBP, but not IRF-1 (Fig. 4 C).

FIGURE 4.

LPS induces the DNA binding activity of ICSBP to promoter 1. A, Binding specificity of ICSBP. Nuclear extracts from RAW 264.7 cells treated with or without LPS (1 μg/ml) for 6 h were incubated with 32P-labeled oligomers containing the ICSBP binding site in the absence or the presence of a 100-fold molar excess of unlabeled oligomers (ICSBP) or oligomers containing the mutated ICSBP binding site (mICSBP) and were analyzed by EMSA as described in Materials and Methods. B, Time-dependent activation of ICSBP binding. RAW 264.7 cells were treated with LPS (1 μg/ml) at various times. EMSA was performed as described above. C, Identification of ICSBP. Supershift assays were performed by incubating the LPS-treated nuclear extracts with 2 μg of antiserum specific for ICSBP or IRF-1 before the addition of 32P-labeled oligomers. The supershifted (arrowhead) and specific binding (arrow) complexes are indicated. F, free probe.

FIGURE 4.

LPS induces the DNA binding activity of ICSBP to promoter 1. A, Binding specificity of ICSBP. Nuclear extracts from RAW 264.7 cells treated with or without LPS (1 μg/ml) for 6 h were incubated with 32P-labeled oligomers containing the ICSBP binding site in the absence or the presence of a 100-fold molar excess of unlabeled oligomers (ICSBP) or oligomers containing the mutated ICSBP binding site (mICSBP) and were analyzed by EMSA as described in Materials and Methods. B, Time-dependent activation of ICSBP binding. RAW 264.7 cells were treated with LPS (1 μg/ml) at various times. EMSA was performed as described above. C, Identification of ICSBP. Supershift assays were performed by incubating the LPS-treated nuclear extracts with 2 μg of antiserum specific for ICSBP or IRF-1 before the addition of 32P-labeled oligomers. The supershifted (arrowhead) and specific binding (arrow) complexes are indicated. F, free probe.

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Next, p1 promoter was cotransfected with ICSBP expression vector (28) to determine the direct activation of p1 promoter by ICSBP. As shown in Fig. 5, cotransfection with ICSBP expression vector increased p1 promoter activity (∼2-fold) compared with cotransfection with control vector. Taken together, these results show that ICSBP binding activity is critical for inducing p1 promoter activity.

FIGURE 5.

Effect of cotransfection with ICSBP expression vector on IL-18 promoter 1 activity. HeLa cells were cotransfected with 5 μg of p1-39 CAT construct, 5 μg of human ICSBP expression vector (pT-ICSBP) or control vector (pT), and pCMV-gal plasmid as an internal control. After 36 h, cells were harvested and assayed for CAT activity. Transfection efficiency was normalized using β-galactosidase activity. The results represent the percent acetylation and the mean ± SD of three independent experiments.

FIGURE 5.

Effect of cotransfection with ICSBP expression vector on IL-18 promoter 1 activity. HeLa cells were cotransfected with 5 μg of p1-39 CAT construct, 5 μg of human ICSBP expression vector (pT-ICSBP) or control vector (pT), and pCMV-gal plasmid as an internal control. After 36 h, cells were harvested and assayed for CAT activity. Transfection efficiency was normalized using β-galactosidase activity. The results represent the percent acetylation and the mean ± SD of three independent experiments.

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To identify the important regulatory elements responsible for the constitutive or LPS activation of p2 promoter described above, a series of deletion mutants linked to CAT reporter gene were constructed from the full-length p2-2.3 plasmid (Fig. 6,A). These constructs were transiently transfected into RAW 264.7 cells, and the expression of CAT was measured in the presence or the absence of LPS. As shown in Fig. 6 B, RAW 264.7 cells transfected with the p1-31 construct were almost sufficient to confer basal level activity, but they showed the reduced relative LPS inducibility (<2 fold). Transfection of the pC-13 construct resulted in a significant decrease in CAT activity by medium (70% reduction) or LPS (70% reduction) compared with the pC-31 construct, suggesting that the region from −31 to −13 contains the essential transcription factor binding sites for p2 promoter activity. As shown in the case of p1 promoter, potential negative regulatory sites are located in the region from −72 to −461 and the region from −571 to −1600.

FIGURE 6.

CAT activity of 5′ deletion mutants of the mouse IL-18 promoter 2-CAT constructs in transfected RAW 264.7 cells. A, Schematic representation of the mouse IL-18 promoter 2-CAT deletion constructs. B, The indicated deletion CAT constructs were transfected into RAW 264.7 cells by electroporation. CAT activities were measured in transfected cells stimulated with or without LPS (1 μg/ml) for 24 h. The results represent the percent acetylation and the mean ± SD of three independent experiments.

FIGURE 6.

CAT activity of 5′ deletion mutants of the mouse IL-18 promoter 2-CAT constructs in transfected RAW 264.7 cells. A, Schematic representation of the mouse IL-18 promoter 2-CAT deletion constructs. B, The indicated deletion CAT constructs were transfected into RAW 264.7 cells by electroporation. CAT activities were measured in transfected cells stimulated with or without LPS (1 μg/ml) for 24 h. The results represent the percent acetylation and the mean ± SD of three independent experiments.

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Sequence analysis of the region from −31 to −13 for transcription factor binding revealed that this region contained a potential binding site for PU.1 (TTCCTC). To determine whether the putative PU.1 binding site in this region is functional for the constitutive or LPS-inducible activation of p2 promoter, site-specific mutagenesis of the PU.1 binding site (TTCCTC→TCTCTC) was introduced to generate the plasmid p2-m31 (mPU.1; Fig. 7,A). RAW 264.7 cells transfected with the plasmid containing the mutated PU.1 binding site showed a significant decrease in LPS-induced CAT activity as well as basal activity compared with cells transfected with the wild-type construct (Fig. 7 B). When the same PU.1 mutation was introduced into the full-length promoter construct p2-2.3 (p2-m2.3) or p2-571 (p2-m571), promoter activity was also substantially reduced. Therefore, the PU.1 binding site plays an important role in p2 promoter activation.

FIGURE 7.

Effects of site-directed mutation in the PU.1 binding site on the IL-18 promoter 2 activity. CAT constructs carrying site-directed mutations of the PU.1 binding site in −31 to +72, −571 to +72, or −2300 to +72 regions of IL-18 promoter 2. Italicized bold letters indicate the specific mutated sequences. B, After transfection with the indicated CAT constructs, RAW 264.7 cells were treated with or without LPS (1 μg/ml) for 24 h, and CAT activities were measured. The results represent the percent acetylation and the mean ± SD of three independent experiments.

FIGURE 7.

Effects of site-directed mutation in the PU.1 binding site on the IL-18 promoter 2 activity. CAT constructs carrying site-directed mutations of the PU.1 binding site in −31 to +72, −571 to +72, or −2300 to +72 regions of IL-18 promoter 2. Italicized bold letters indicate the specific mutated sequences. B, After transfection with the indicated CAT constructs, RAW 264.7 cells were treated with or without LPS (1 μg/ml) for 24 h, and CAT activities were measured. The results represent the percent acetylation and the mean ± SD of three independent experiments.

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Next, to learn whether LPS induced DNA binding activity to a putative PU.1 binding site of p2 promoter, we performed EMSA for PU.1 binding with nuclear extracts prepared from RAW 264.7 cells either unstimulated or treated with LPS. Although control nuclear extract showed a constitutive binding activity, treatment with LPS (1 μg/ml) increased PU.1 binding activity (Fig. 8,A). The specificity of increased binding activity by LPS was demonstrated by competition experiments with a 20- or 100-fold molar excess of wild-type probe. An oligonucleotide probe containing the mutated sequences, however, did not compete for binding, suggesting that the integrity of the PU.1 binding site was required for the binding activity. Dose-dependant binding of PU.1 in response to LPS was confirmed as shown in Fig. 8,B, comparing constitutive Oct-1 binding as the internal control. As seen in the case of ICSBP binding, PU.1 binding was maximal at 3–6 h after treatment with LPS (Fig. 8,C). The identity of nuclear protein binding to this site was confirmed by supershift assay with specific Ab against PU.1. The addition of PU.1-specific Abs to LPS-treated nuclear extracts resulted in a formation of supershifted complex (Fig. 8 D).

FIGURE 8.

LPS induces DNA binding of PU.1 to the IL-18 promoter 2. A, Binding specificity of PU.1. Nuclear extracts from RAW 264.7 cells treated with LPS (1 μg/ml) for 6 h were incubated with 32P-labeled PU.1 oligomers in the absence or the presence of a 20- and 100-fold molar excess of unlabeled oligomers (PU.1) or oligomers containing the mutated PU.1 binding site (mPU.1) and analyzed by EMSA. B, Dose-dependent effect of LPS on PU.1 binding activity. EMSAs were performed with oligomers and nuclear extracts from RAW 264.7 cells treated with the indicated concentrations of LPS for 6 h. Equal loading of nuclear extracts was assessed by constitutively expressed Oct-1 binding. C, Time-dependent activation of PU.1 binding. EMSA was performed with 32P-labeled PU.1 oligomers and nuclear extracts from RAW 264.7 cells treated with 1 μg/ml LPS for various times. D, Identification of PU.1. Supershift assays were performed by incubating the LPS-treated nuclear extracts with 2 μg of antiserum specific for PU.1. The supershifted (arrowhead) and specific (arrow) complexes are indicated. F, free probe.

FIGURE 8.

LPS induces DNA binding of PU.1 to the IL-18 promoter 2. A, Binding specificity of PU.1. Nuclear extracts from RAW 264.7 cells treated with LPS (1 μg/ml) for 6 h were incubated with 32P-labeled PU.1 oligomers in the absence or the presence of a 20- and 100-fold molar excess of unlabeled oligomers (PU.1) or oligomers containing the mutated PU.1 binding site (mPU.1) and analyzed by EMSA. B, Dose-dependent effect of LPS on PU.1 binding activity. EMSAs were performed with oligomers and nuclear extracts from RAW 264.7 cells treated with the indicated concentrations of LPS for 6 h. Equal loading of nuclear extracts was assessed by constitutively expressed Oct-1 binding. C, Time-dependent activation of PU.1 binding. EMSA was performed with 32P-labeled PU.1 oligomers and nuclear extracts from RAW 264.7 cells treated with 1 μg/ml LPS for various times. D, Identification of PU.1. Supershift assays were performed by incubating the LPS-treated nuclear extracts with 2 μg of antiserum specific for PU.1. The supershifted (arrowhead) and specific (arrow) complexes are indicated. F, free probe.

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Constitutive IL-18 expression was detected in normal mouse macrophages and neutrophils (Fig. 9), but there was no IL-18 expression in the PU.1 null neutrophil cell line 503, strongly indicating that PU.1 has an essential role in constitutive expression of IL-18. In addition, transfection of PU.1 into 503 cells (503-PU.1) restored constitutive IL-18 expression, whereas transfection of M-CSF receptor (503-MR) did not restore it. Restoration of IL-18 expression was also observed when primary liver cells from PU.1 knockout mice were transfected with PU.1.

FIGURE 9.

Restoration of IL-18 expression in PU.1 null cells by transfecting PU.1. PU.1 null cells, 503 cells, or primary liver cells from PU.1 knockout mice were transfected with PU.1 as described in Materials and Methods. Then, cells were harvested, and IL-18 expression was analyzed by RT-PCR (30 cycles) and gel electrophoresis (1.5% agarose gel). 503, PU.1 null neutrophils; 503-MR, 503 cells transfected with M-CSF receptor; 503-PU.1. 503 cells transfected with PU.1; KO cultured liver cells, short term cultures of liver cells from PU.1 knockout mice; KO cultured liver cells + PU.1, knockout cultured liver cells transfected with PU.1; M, molecular size marker.

FIGURE 9.

Restoration of IL-18 expression in PU.1 null cells by transfecting PU.1. PU.1 null cells, 503 cells, or primary liver cells from PU.1 knockout mice were transfected with PU.1 as described in Materials and Methods. Then, cells were harvested, and IL-18 expression was analyzed by RT-PCR (30 cycles) and gel electrophoresis (1.5% agarose gel). 503, PU.1 null neutrophils; 503-MR, 503 cells transfected with M-CSF receptor; 503-PU.1. 503 cells transfected with PU.1; KO cultured liver cells, short term cultures of liver cells from PU.1 knockout mice; KO cultured liver cells + PU.1, knockout cultured liver cells transfected with PU.1; M, molecular size marker.

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Now it is generally accepted that IL-18 is a multifunctional cytokine that has diverse immune regulatory effects on T cells, B cells, NK cells, and nonimmune cells. Based on its known biological activities, IL-18 seems to share or cooperate in immunological functions with IL-12 in vitro and in vivo. However, the regulatory machinery of IL-12 and IL-18 seems to be quite different. IL-12 is a heterodimeric cytokine of p35 and p40. The inducibility of functional IL-12 gene expression is controlled by p40 gene expression. To date, it is speculated that constitutive IL-18 gene is expressed in a far wider range of cell types than IL-12. For the production of active IL-18 gene product, processing by cysteine proteinase such as ICE is required. In addition, a recent report by Tone et al. (21) indicates the structure and transcriptional regulation are quite different from those of other cytokines. It has two distinct promoters, having different inducibilities by stimuli, constitutive or inducible promoters. IL-18 gene has no TATA motif in either promoter and no typical AU-rich sequence in the 3′-untranslated regions.

LPS inducibility of IL-18 seems to be different depending on cell types. In Kupffer cells and peritoneal cells, IL-18 did not respond to LPS (3), but, IL-18 was induced by LPS in RAW cells as shown in our and Tone’s studies (21) and in mouse splenocytes (29). However, there are some differences between our results and Tone’s findings. First, two promoters showed LPS inducibility in our results (Figs. 1 C and 6B), even though the inducibility of p2 promoter (constitutive promoter in Tone’s study) was slightly less than that of p1 promoter. Second, the relative basal promoter activities of the two promoters are different. In our results, the relative basal activities of the two promoters were relatively equal, but much higher in p2 promoter (constitutive promoter) in the case of Tone’s study. It is not clear yet what caused these differences between the two studies, which used similar promoter constructs and the same cell line, but Northern blots supported these differences: more LPS inducibility in our study and strong basal expression in Tone’s study.

PU.1, the ETS transcription factor, is expressed exclusively in myeloid and B cells. PU.1 is involved in the regulation of promoters for the genes encoding receptors for M-CSF and GM-CSF. Studies from PU.1 gene-disrupted mice indicated that it is involved in M-CSF- and GM-CSF-mediated proliferation and development. These results indicate that PU.1 functions in the differentiation of multipotential lymphoid and myeloid progenitors. PU.1 binds to the consensus DNA sequence of TTCCTC and is involved in the expression of numerous genes that regulate B cell maturation and macrophage differentiation. It is essential for transcription of Ig light chains, J chains, and the macrophage scavenger receptors (30). For transcriptional activation of PU.1, phosphorylation of serine 148 in PU.1, such as by casein kinase II (31), is required, implying that a proper phosphorylation state and conformation are important for PU.1 activity. It was also reported that LPS and IFN-γ induce DNA binding of PU.1 through a post-translational mechanism(s) in murine tissue macrophages (32).

Deletion and mutagenesis studies (Figs. 6 and 7) indicate that the PU.1 binding site is critical for p2 promoter activity, especially in constitutive activation of p2 promoter. Relative LPS inducibility of p2-31 construct was reduced (<2-fold) compared with that of p2-2.3, and introducing a PU.1 mutation or deletion mutation of the p2-31 construct appeared to affect constitutive expression to a much greater degree than inducible expression. Furthermore, studies using PU.1 null cells (Fig. 9) demonstrated that PU.1 has an essential role in constitutive expression of IL-18. However, it cannot be ruled out that PU.1 also has a minimal role in LPS inducibility, as shown in the case of minimal inducibility of p2-31 construct and its binding activity induced by LPS treatment (Fig. 8).

The deletion and mutagenesis studies of p1 promoter showed that ICSBP is also involved in both basal and inducible activities of p1 promoter. However, mutation of the ICSBP element seemed to affect inducible expression of p1-39 more than constitutive expression (Figs. 2 and 3), suggesting that ICSBP has an essential role in inducible expression of IL-18 with a minor role in constitutive expression. ICSBP has been known as a transcriptional repressor involved in negative regulation of transcription (28). However, recent reports demonstrated that ICSBP is essential for IL-12 p70 (33) and p40 (34) induction. In this regard, our results of cotransfection experiment (Fig. 5) are consistent with these results (33, 34) describing ICSBP as an enhancer of IL-12 production. ICSBP is expressed exclusively in immune cells, such as monocytes and B cells. It has three functional domains: a DNA binding domain, a transcriptional repressor domain, and a domain that enables the association with other IRFs (35). This association domain is conserved among some IRF members and is located near the carboxyl terminus between residues 200 and 377. Therefore, ICSBP might have different effects in different immune cells depending on the milieu of IRFs that are associated with it (36). Our unpublished results indicated that other stimuli, such as IFN-γ, showed a slightly different regulation of promoter activity employing other transcription factor complexes, including IRF-1 in addition to ICSBP.

In addition, several studies (30, 36, 37) reported that PU.1 also interacts with the IRF family to increase target gene expression. This complex includes IRF-1, IRF-4, ICSBP, PU.1 interaction partner, and PU.1. This interaction is controlled by stimuli-induced phosphorylation of these proteins, such as serine 148 phosphorylation in PU.1. It implies a possible interaction between ICSBP in p1 promoter and PU.1 in p2 promoter, but it seems unlikely because there is no substantial difference between Northern analysis and promoter analysis in terms of LPS inducibilities. However, further studies are required to determine the possible interaction between the two promoters using different stimuli or a construct containing both promoters.

Regulation of IL-18 gene expression demonstrates a unique aspect in cytokine gene expression, structurally and functionally. Also, post-transcriptional and translational events, including processing with ICE, are another limiting point for the production of mature IL-18. Further understanding of the relationship between these cascade events will reveal the controlling machinery for IL-18 expression during inflammation and other immune responses. In conclusion, expression of the IL-18 gene is controlled by two promoters that are inducible by LPS. ICSBP (for p1 promoter) and PU.1 (for p2 promoter) are critical factors for IL-18 promoter activity, performing dominant regulatory roles in inducible and constitutive expression of IL-18, respectively.

1

This work was supported by grants from the HAN Project (HS2240 and KM1241) from the Ministry of Science and Technology, Republic of Korea.

3

Abbreviations used in this paper: ICE, IL-1β-converting enzyme; ICSBP, IFN consensus sequence binding protein; IRF-1, IFN regulatory factor-1; CAT, chloramphenicol acetyltransferase.

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