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The Journal of Immunology, 2003, 171: 426-431.
Copyright © 2003 by The American Association of Immunologists

Bcl6 Is a Transcriptional Repressor for the IL-18 Gene1

Nobue Takeda*,{dagger}, Masafumi Arima*, Nobuhide Tsuruoka*, Seiji Okada2,*, Masahiko Hatano*, Akemi Sakamoto*, Yoichi Kohno{dagger} and Takeshi Tokuhisa3,*

Departments of * Developmental Genetics (H2) and {dagger} Pediatrics (H4), Graduate School of Medicine, Chiba University, Chiba, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Bcl6 functions as a sequence-specific transcriptional repressor, and Bcl6-deficient (Bcl6-/-) mice have been reported to display Th2-type inflammatory diseases in multiple organs. Since IL-18 is a potent stimulator of Th2 cells, we examined the expression of IL-18 mRNA in bone marrow-derived macrophages from Bcl6-/- mice after LPS stimulation. Here we show that the expression was strikingly up-regulated after stimulation. The expression was also up-regulated in RAW264 cells, a murine macrophage cell line, by transfection with the dominant negative type of Bcl6 gene. We identified a putative Bcl6-binding DNA sequence (IL-18BS) upstream of exon 1 of the murine IL-18 gene and three IL-18BSs in the promoter region of human IL-18 gene. Binding of Bcl6 in nuclear protein from resting RAW264 cells to murine IL-18BS was detected by gel retardation assay and chromatin immunoprecipitation assay. The binding activity was diminished gradually in RAW264 cells after LPS stimulation. However, the amount of Bcl6 protein in these cells was constant over the period examined, suggesting the functional modification of Bcl6 protein after stimulation. Furthermore, murine IL-18BS was required for Bcl6 to repress the expression of the luciferase reporter gene under control of the IL-18 promoter. Taken together, Bcl6 is a key regulator of IL-18 production by macrophages.


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The human proto-oncogene Bcl6 has been identified from chromosomal breakpoints involving 3q27 in diffuse large B cell lymphomas (1, 2, 3). The Bcl6 gene encodes a 92- to 98-kDa nuclear phosphoprotein (4, 5) that contains the BTB/POZ domain in the NH2-terminal region and Krüppel-type zinc finger motifs in the COOH-terminal region (1, 2, 3, 6). Since the NH2-terminal half of Bcl6 can bind to silencing mediator of retinoid and thyroid receptor protein and recruit the silencing mediator of retinoid and thyroid receptor protein/histone deacetylase complex to silencer regions of target genes to repress the expression of these genes (7, 8), Bcl6 can function as a sequence-specific transcriptional repressor (9, 10, 11, 12, 13, 14). To observe the physiological functions of Bcl6, this gene was disrupted in the mouse germline. Bcl6-deficient (Bcl6-/-) mice showed growth retardation (15, 16, 17) and displayed inflammatory responses in multiple organs, especially the heart and lungs, characterized by infiltration of eosinophils at a young adult age (15, 16, 18). IL-5 is an important cytokine involved in controlling the growth, differentiation and activation of eosinophils (19, 20). We have recently reported that the IL-5 gene is one of the molecular targets of Bcl6 (21). Furthermore, the production of Th2 cytokines, including IL-5, by Bcl6-/- T cells was also augmented. Thus, mechanisms of this eosinophilic inflammation could be partly explained by a functional dominance of Th2 cells in Bcl6-/- mice. Since Bcl6-binding DNA sequences resembled the sequence motif bound by the STAT factors, Bcl6 might repress IL-4-induced transcription by competitive binding to DNA sites recognized by the IL-4-responsive STAT factor, STAT6 (15). However, STAT6 and Bcl6 double-deficient mice could display inflammatory responses with infiltration of eosinophils in multiple organs (22), indicating that overproduction of Th2 cytokines by Bcl6-/- T cells cannot be explained by the loss of competitive inhibition of STAT6 activity.

Recent reports indicated that a functional dominance of Th2 cells in Bcl6-/- mice is due to nonlymphoid cells, including macrophages from Bcl6-/- mice (23). Furthermore, we have recently shown that augmentation of Th1-type responses, including IFN-{gamma} production, was observed in Bcl6-/- mice (21). Thus, Bcl6 may regulate the functions of both Th1 and Th2 cells. IL-18, originally called IFN-{gamma}-inducing factor (24), is a proinflammatory cytokine produced by a wide range of cells, including macrophages (25, 26, 27, 28), that augments the cytotoxic activity of NK (29) and is a potent stimulator of both Th1 (30) and Th2 (31) responses. The expression of the IL-18 gene is controlled by two distinct TATA-less promoters, the LPS-regulated promoter located upstream of exon 1 (P1 promoter) and the constitutive promoter in intron 1 (P2 promoter) (32). It was shown that IFN consensus sequence binding protein and AP-1 (33, 34) as well as PU.1 (33) are critical transcription factors for activation of the P1 promoter and the P2 promoter, respectively. However, a negative regulator of IL-18 expression has never been reported. In this study we found that the expression of IL-18 was augmented in macrophages derived from Bcl6-/- bone marrow cells. We identified the Bcl6-binding sequence in the P1 promoter region. We discuss a physiological role of Bcl6 as a putative silencer molecule of the IL-18 gene.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

C57BL/6 mice were purchased from Japan SLC (Hamamatsu, Japan). Bcl6-deficient (Bcl6-/-) mice were described previously (17). These mice were maintained under specific pathogen-free conditions in the animal center of Graduate School of Medicine, Chiba University (Chiba, Japan).

Macrophage cultures and LPS stimulations

Bone marrow was flushed from the femurs of mice using needles and syringes filled with PBS buffer. Bone marrow-derived macrophages were generated by culturing nonadherent bone marrow cells with IL-3 (200 U/ml) and 10% L cell-conditioned medium (as the source of M-CSF) for 10 days. IL-3 was prepared from culture supernatant of X63Ag8-653 cells transfected with the murine IL-3 gene (35). After the culture, nonadherent cells were removed by extensive washing, and adherent cells were harvested by trypsinization and used as macrophages. Macrophages were cultured in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO) supplemented with streptomycin sulfate (100 µg/ml; Wako Chemical Co., Osaka, Japan), penicillin G potassium (100 U/ml; Banyu Pharmaceutical Co., Tokyo, Japan), and 10% heat-inactivated FCS (Sigma-Aldrich). Macrophages (2 x 105/ml) were plated in a 12-well tissue culture plate. The cells were cultured overnight and then stimulated with LPS (1 µg/ml; Escherichia coli, serotype 111: B4; Sigma-Aldrich) the next day. RAW264, a murine macrophage cell line obtained from RIKEN Cell Bank (Tsukuba, Japan), and Ramos (36) cells were cultured in RPMI 1640 medium supplemented with streptomycin sulfate (100 µg/ml), penicillin G potassium (100 U/ml), and 10% heat-inactivated FCS.

RT-PCR analysis

RNA was prepared from macrophages using TRIzol reagent (Life Technologies, Gaithersburg, MD). RT-PCR was performed using Superscript (Life Technologies) and oligo(dT) (Amersham Pharmacia Biotech, Piscataway, NJ) as previously described (37). The primers used were as follows: {beta}-actin, 5'-GTTTGAGACCTTCAACACC-3' and 5'-GTGGCCATCTCCTGCTCGAAGTC-3'; monocyte chemoattractant protein 1 (MCP-1),4 5'-TCCAGAGATAGCAGCTTAGCGG-3' and 5'-CATTGGGATCATCTTGCTGGTG-3'; murine IL-18, 5'-CCTCCAGCATCAGGACAAAGAAAG-3' and 5'-GCATCATCTTCCTTTTGGCAAGC-3'; IL-12, 5'-CCTGCTGAAGACCACAGATGACATG-3' and 5'-TGCTTCTCCCACAGGAGGTTTCTG-3'; IFN-{gamma}, 5'-AGCGGCTGACTGAACTCAGATTGTAG-3' and 5'-GTCACAGTTTTCAGCTGTATAGGG-3'; and caspase-1, 5'-ATACTCTAATGAAGTTTCAGA-3' and 5'-AGAGGTAGAAACGTTTTGTCA-3'. The PCR products were separated on a 1.5% agarose gel and stained with ethidium bromide.

Northern blot analysis

The expression of genes related to macrophage functions was detected by Northern blot as previously described (38). Briefly, total RNA (10–20 µg) was loaded on 1.0% agarose and transferred to a nylon membrane (Roche, Mannheim, Germany). The filters were hybridized with digoxigenin (DIG)-labeled probes overnight at 50°C. The probe on the filter was detected with sheep anti-DIG Ab conjugated with alkaline phosphatase (Roche). Murine IL-18 and MCP-1 cDNAs were obtained from mRNAs of bone marrow-derived macrophages by RT-PCR. These cDNAs were subcloned into a pGEM-T Easy vector and labeled by DIG, using PCR with T7 and SP6 primers, then used as a probe. PCR primers for the cDNA amplification were described above.

Transfection of the dominant negative Bcl6 gene

The dominant negative Bcl6 (DNBcl6) cDNA (39) was subcloned into pcDNA3 to generate pcDNA3-DNBcl6. The pcDNA3-DNBcl6 (10 µg) was transfected into RAW264 cells. Transfection was performed by electroporation with a Gene Pulser (Bio-Rad, Hercules, CA) at 370 mV. The stable transfectants were selected by culturing with geneticin G418 (0.5 mg/ml; Invitrogen Life Technologies, Carlsbad, CA).

Isolation of nuclear proteins and Western blot analysis

Nuclear proteins were isolated from RAW264 and Ramos cells according to the method described previously (40), with slight modification. Briefly, cells (1 x 107) were resuspended in 400 µl of cold hypotonic buffer (10 mM HEPES (pH 7.9), 10 mM KCl, 1.5 mM MgCl2, 0.1 mM EDTA, 0.1% Triton X-100, 1 mM DTT, 100 mM PMSF, and 5 µg/ml aprotinin). Nuclei were collected by centrifugation and disrupted by sonication in 100 µl of immunoprecipitation buffer (50 mM HEPES (pH 7.5), 150 mM NaCl, 2.5 mM EGTA, 1 mM EDTA, 0.1% Tween 20, 10% glycerol, 1 mM DTT, 100 mM PMSF, and 5 µg/ml aprotinin) at 4°C. Nuclear extracts were immediately stored at -80°C. The amount of protein was determined using the Bio-Rad protein assay (Bio-Rad). Bcl6 in nuclear proteins from RAW264 cells was detected by Western blot as previously described (41). Briefly, 30 µg of nuclear proteins were resolved by SDS-PAGE and transferred to a polyvinyldifluoride membrane (Immobilon-P; Millipore, Bedford, MA). Blots were incubated with rabbit anti-Bcl6 Abs, followed by HRP-conjugated donkey anti-rabbit Ig Abs (Amersham Pharmacia Biotech, Arlington Heights, IL) for 1 h at each step and developed with enhanced chemiluminescence reagents (Amersham Pharmacia Biotech).

EMSA

Double-stranded oligonucleotides corresponding to a putative Bcl6-binding sequence in the murine IL-18 gene (5'-TATTTTAGTTTTTCAAGGAAGAGCTAGACA-3'; mIL-18BS) were synthesized. The binding activity of Bcl6 to mIL-18BS was determined by EMSA as described previously (42). Briefly, mIL-18BS was labeled with DIG using DIG Oligonucleotide 3'-End-Labeling Kits (Roche, Indianapolis, IN). Binding reactions were performed in the mixture containing purified GST-Bcl6 zinc finger protein (50 ng) or nuclear proteins (1 µg), poly(dI-dC) (0.5 µg; Amersham Pharmacia Biotech, Piscataway, NJ), and the DIG-labeled probe (15 fM) in 10 µl of reaction buffer (10 mM HEPES (pH 7.8), 50 mM KCl, 1 mM DTT, and 50 µg/ml BSA). This mixture was separated by electrophoresis on a 6% nondenaturing polyacrylamide gel and transferred to a nylon membrane (Roche) using an electroblot (Bio-Rad). The DIG-labeled probe was detected with sheep anti-DIG Abs conjugated with alkaline phosphatase. The Ab detection reaction was performed using an ECL detection system (Roche). Competitive EMSA was performed by adding a 10- to 50-fold molar excess of unlabeled, double-stranded oligonucleotide to the mixture. Sequences of the mutant oligonucleotide (one-base mismatch; underlined) were as follows: Mut1, 5'-TATTTTAGTTTTTAAAGGAAGAGCTAGACA; and Mut2, 5'-TATTTTAGTTTTTCACGGAAGAGCTAGACA. To detect Bcl6 in the mixture, Bcl6-specific rabbit polyclonal Abs (rabbit IgG, Santa Cruz Biotechnology, Santa Cruz, CA) was preincubated with nuclear proteins for 30 min at 4°C, followed by incubation with the DIG-labeled mIL-18BS.

Luciferase assay

The luciferase reporter plasmid (P1D1 and P1D2) (32) was a gift from Dr. Tone (Oxford University, Oxford, U.K.). The P1D1was constructed using pGL3-Basic vector (Promega, Madison, WI) and 5'-flanking fragments (-2686 bp) of exon 1 of the murine IL-18 gene, which contains the mIL-18BS (-2574 to -2565 bp). P1D2 was a truncated reporter plasmid (-2504 bp) in which mIL-18BS is deleted (32). The luciferase reporter assay was described previously (21). Briefly, RAW264 cells (5 x 105) were transfected with 10 µg of the luciferase reporter plasmid and 3 µg of Bcl6 expression vector (pcDNA3-Bcl6) or control vector (pcDNA3). For all transfections, 1.5 µg of pRL-thymidine kinase vector were cotransfected as an internal control for transfection efficiency. Electroporation was conducted using a Gene Pulser (Bio-Rad) at 0.30 V and 960 µF. To stimulate cells, 20 µg/ml of LPS was added to the culture in the presence or the absence of trichostatin A (TSA; 150 nM; Biomol, Plymouth Meeting, PA) 15 h after transfection. These cells were harvested 24 h after stimulation. Luciferase activity in cell extracts was determined using a luciferase assay kit (Promega) and was standardized using luciferase activity by pRL-thymidine kinase vector.

Chromatin immunoprecipitation (ChIP) assay

Chromatin immunoprecipitation was performed using the ChIP assay kit (Upstate Biotechnology, Lake Placid, NY) and was then conducted according to the manufacturer’s recommendations. Briefly, formaldehyde solution (37%; Fisher Scientific, Pittsburgh, PA) at a final concentration of 1% was added directly to RAW264 cells before or 6 h after stimulation with LPS (20 µg/ml). Cross-linking of proteins on chromatin was allowed to occur at 37°C for 10 min, and the cells were lysed by SDS lysis buffer with protease inhibitors. Chromatin in the lysate was sonicated to an average length of 200–500 bp as determined by agarose gel electrophoresis. The suspension was precleared with salmon sperm DNA/protein A/agarose (50% slurry) for 30 min at 4°C and incubated with 2 µg of rabbit polyclonal anti-Bcl6 Abs (Santa Cruz Biotechnology), 5 µg of rabbit polyclonal anti-acetylated histone H4 Abs (Santa Cruz Biotechnology), or no Ab overnight at 4°C with mild shaking. The immune complexes were incubated with salmon sperm DNA/protein A/agarose (50% slurry) with mild shaking for 1 h at 4°C, washed, and eluted. After precipitation, the supernatant from the no Ab sample was processed to the cross-link reversal step and analyzed as an unfractionated input chromatin. Cross-links were reversed by 0.5 M NaCl. After proteinase K digestion, DNA in samples was phenol-extracted, ethanol-precipitated, and resuspended in 50 µl of TE buffer. Two microliters of DNA solution was used for 28 cycles of PCR amplification. PCR products were analyzed by electrophoresis on a 2% agarose gel and visualized by ethidium bromide staining. The following primers were used in the ChIP assay: murine IL-18BS, 5'-GCTTCAGAACACAATACATAAGCC-3' and 5'-TGTGCCAGAGAGTAATGGATTG-3' (271 bp).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of IL-18 mRNA is augmented in macrophages from Bcl6-/- mice

Bone marrow cells from Bcl6-/- mice were cultured with IL-3 and L cell-conditioned medium for 10 days. Macrophages developed in the culture were stimulated with LPS for 24 h. The expression of IL-18, MCP-1, IL-12, IFN-{gamma}, and caspase-1 in these macrophages was analyzed by RT-PCR (Fig. 1A). The expression of IL-18 and MCP-1 in Bcl6-/- macrophages was augmented compared with that in control macrophages. The expression of IL-12, IFN-{gamma}, and caspase-1 was also induced in Bcl6-/- macrophages, but the amount of the messages was similar to that in control macrophages. Similar results were obtained by Northern blot analysis (data not shown).



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  		FIGURE 1. IL-18 expression is strongly augmented in Bcl6-/- macrophages. A, Bone marrow-derived macrophages from Bcl6-/- and control mice were cultured with LPS (1 µg/ml) for 24 h. The expression of several genes related to macrophages was analyzed by RT-PCR. B, RAW264 cells were transfected with the dominant negative Bcl6 (pcDNA3-DNBcl6), and stable transfectants were cultured with LPS (2 µg/ml) for 24 h. The amounts of IL-18 and MCP-1 mRNAs were measured by Northern blot.

 
To confirm the regulation of IL-18 expression by Bcl6, we used the dominant negative form of the Bcl6 gene (pcDNA3-DNBcl6). We transfected pcDNA3-DNBcl6 into RAW264 cells, and these cells were stimulated with LPS for 24 h. The expression of IL-18 mRNA in these cells was analyzed by Northern blot (Fig. 1B). The amount of IL-18 mRNA in RAW264 cells transfected with pcDNA3-DNBcl6 was 3- to 4-fold higher than that in RAW264 cells transfected with pcDNA3. MCP-1, a molecular target of Bcl6 (23), was also augmented in RAW264 cells transfected with pcDNA3-DNBcl6. These results suggest that IL-18 is a molecular target of Bcl6.

Bcl6 binds to a DNA sequence in the promoter region of the IL-18 gene

We tried to identify a DNA sequence similar to Bcl6 binding sequences in the IL-18 gene by computer analysis. The Bcl6 binding sequence (IL-18BS) was found ~2.6 kb upstream of exon 1, which is in the P1 promoter, of the murine IL-18 gene (Fig. 2A). IL-18BS was also identified in the human IL-18 gene (hIL-18BS1, hIL-18BS2, hIL-18BS3). These three hIL-18BSs distribute within the promoter region (data not shown). These IL-18BS sequences were compared with the Bcl6-binding sequences of the known Bcl6 target genes, CD23, MCP-1, and MIP-1{alpha}.



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  		FIGURE 2. Bcl6 binds to the Bcl6 binding sequence in the IL-18 gene. A, A genomic map of the 5'-flanking region of the murine IL-18 gene. The putative Bcl6-binding sequence is compared with the consensus Bcl6-binding sequence, the Bcl6-binding sequences of other genes. B, Nuclear protein from Ramos cells was incubated with DIG-labeled mIL-18BS, and the retardation band was detected by EMSA. Mut1 and Mut2 did not inhibit formation of the gel retardation band. The band was destroyed by the addition of anti-Bcl6 Abs in the mixture of the nuclear proteins and mIL-18BS probe.

 
Next, EMSA was performed to examine the binding activity of Bcl6 to the Bcl6-binding sequence in the murine IL-18 gene (mIL-18BS). When nuclear extracts from Ramos cells, which are human B cell lymphoma cells with high expression of Bcl6 protein, were incubated with the DIG-labeled mIL-18BS as a probe, gel retardation bands were observed (Fig. 2B). The major retarded band was obviously removed by a nonlabeled competitor with the same sequence (WT). In contrast, mutated cold probes (one-base mismatch; underlined) substituted from TTTCAA to TTTAAA (Mut1) or to TTTCAC (Mut2) did not inhibit formation of the gel retardation band, indicating sequence-specific binding of Bcl6 to mIL-18BS. Furthermore, the band was also destroyed by the addition of anti-Bcl6 Abs in the mixture of nuclear proteins and mIL-18BS probe.

Bcl6 represses LPS-induced activation of the IL-18 promoter

To examine whether Bcl6 repressed transcription from the P1 promoter of the IL-18 gene, RAW264 cells were transiently transfected with the luciferase reporter plasmid containing the P1 promoter. The promoter activity was very low without LPS stimulation, and became strong after LPS stimulation (Fig. 3). When RAW264 cells were transfected with the reporter plasmid deleted with the mIL-18BS and stimulated with LPS, promoter activity was augmented to 3-fold over that of the P1 promoter. To examine the repressor activity of Bcl6, the reporter gene and pcDNA3-Bcl6 were costransfected into RAW264 cells, and the transfectants were stimulated with LPS. The promoter activity was suppressed to 20% the promoter activity without pcDNA3-Bcl6. In contrast, pcDNA3-Bcl6 did not suppress the activity of the P1 promoter deleted with mIL-18BS. Furthermore, this suppression was abrogated by the addition of TSA, an inhibitor of histone deacetylase, to the culture. However, the addition did not modulate the activity of P1 promoter deleted with mIL-18BS. These results suggest that Bcl6 functions as a potent repressor for the IL-18 gene in macrophages.



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  		FIGURE 3. Bcl6 can repress the expression of the reporter gene controlled by the P1 promoter with mIL-18BS. The reporter plasmid with or without mIL-18BS (P1D1 or P1D2, respectively) was cotransfected with or without pcDNA3-Bcl6 into RAW264 cells. These cells were stimulated with LPS (20 µg/ml) in the presence or the absence of TSA (150 nM) for 24 h after transfection, and the luciferase activity in RAW264 cells was measured. Results represent the mean ± SD of triplicate cultures per group. These results are representative of four independent experiments. *, p < 0.05.

 
Binding activity of Bcl6 in RAW264 cells to the mIL-18BS decreases after LPS stimulation

We have shown that Bcl6 specifically binds to the Bcl6 binding sequence in exon 4 of the IL-5 gene, and that the binding activity of endogenous Bcl6 was transiently diminished in Th2, but not in Th1, clones after anti-CD3 stimulation (21). Simultaneously, we investigated the binding activity of Bcl6 in RAW264 cells to mIL-18BS after LPS stimulation by EMSA. Fig. 4 shows that the binding activity of Bcl6 was detected before stimulation. However, activity decreased as time passed and disappeared 6 h after stimulation. We examined the amount of Bcl6 in nuclear proteins of RAW264 cells after LPS stimulation by Western blot. The amount of Bcl6 in the RAW264 cells did not change after stimulation, suggesting the functional modification of Bcl6 protein in activated RAW264 cells. This binding activity was inversely correlated with the amount of IL-18 mRNA in these cells. The expression of IL-18 mRNA was induced 2–4 h, increased 6 h, and diminished 24 h after LPS stimulation.



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  		FIGURE 4. Functional modification of Bcl6 in RAW264 cells after LPS stimulation. RAW264 cells were stimulated with LPS (20 µg/ml). Nuclear proteins from these cells were incubated with DIG-labeled mIL-18BS, and the retardation band was detected by EMSA. The amount of Bcl6 in the nuclear proteins was measured by Western blot. The amounts of IL-18 and Bcl6 mRNAs in these cells were measured by Northern blot.

 
We further confirmed the binding of Bcl6 to mIL-18BS in RAW264 cells by the ChIP assay. Bcl6 and chromatin complexes in RAW264 cells were immunoprecipitated with anti-Bcl6 Abs. The PCR products including the mIL-18BS sequence were detected in the complexes from unstimulated RAW264 cells, but not in those from the stimulated cells (Fig. 5). We also examined the acetylation of histone in the P1 promoter region of the IL-18 gene. The PCR products including the Bcl6 binding sequence were observed in the same complexes from activated RAW264 cells, but not in the unstimulated RAW264 cells. The chromatin precipitation from cells with rabbit polyclonal IgG Abs did not show any significant PCR products (data not shown). These results indicate that Bcl6 binds to mIL-18BS in resting macrophages, and histones in these regions are deacetylated, and that the binding of Bcl6 decreases and histones in the P1 promoter encompassing the Bcl6 binding site are acetylated in macrophages after LPS stimulation.



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  		FIGURE 5. Binding of Bcl6 to the mIL-18BS and acetylated histones in the P1 promoter was detected by ChIP assay. RAW264 cells were stimulated with LPS (20 µg/ml) for 6 h. Bcl6 on the chromatin in RAW264 cells after stimulation was immunoprecipitated by polyclonal anti-Bcl6 Abs (left) or polyclonal anti-acetylated histone H4 Abs (right). The IL-18BS in the precipitated chromatin was detected by PCR. These results are representative of three independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The expression of murine IL-18 was known to be controlled by two distinct TATA-less promoters, the LPS-regulated promoter located upstream of exon 1 (P1 promoter) and the constitutive promoter within intron 1 (P2 promoter). It was shown that PU.1 is a critical transcription factor for maximal activation of the P2 promoter, and IFN consensus sequence binding protein and AP-1 are a critical transcription factors for the P1 promoter (32, 33, 34). Tone et al. (32) reported that there was greater inducibility of the P1 promoter upon stimulation with LPS and PMA/ionomycin than the P2 promoter. Kalina et al. (43) have reported specific binding of activated STAT5 to three consensus sequences upstream in the promoter region of the human IL-18 gene, and cotransfection of STAT5 resulted in increased induction of the human IL-18 promoter. In this study we demonstrate that Bcl6 is a negative regulator of IL-18 expression in macrophages. We found mIL-18BS in the P1 promoter, but could not find the Bcl6 binding sequences in the P2 promoter. This mIL-18BS is required for Bcl6-mediated repression of the exogenous reporter gene. Therefore, mIL-18BS is a novel silencer element in the IL-18 gene. Furthermore, we have found three highly homologous sequences to the Bcl6 binding sequence in the promoter region of the human IL-18 gene, one of which is in one of the STAT5-binding sites. Thus, Bcl6 may also competitively inhibit STAT5-induced activation of the human IL-18 gene.

Vasanwala et al. (44) have recently demonstrated that Bcl6 regulates the Blimp-1 promoter through a novel mechanism involving AP-1 elements. They found that Bcl6 is a potent repressor of AP-1-mediated transcriptional activity by the interaction of the zinc finger region of Bcl6 with c-Jun, JunB, and JunD proteins. Since AP-1 is a critical transcription factor for the activity of the P1 promoter, Bcl6 might be a potent repressor of the IL-18 gene transcriptional activity mediated by AP-1 factors as a alternative mechanism. However, we observed that the P1 promoter activity in RAW264 cells cotransfected with the Bcl6 expression vector was up-regulated by treatment of the cells with TSA, a histone deacetylase inhibitor. These results were supported by a previous study on the promoter of the human IL-18 gene by Koyama et al. (45). Furthermore, histones in these promoter regions are deacetylated in macrophages at rest. Thus, Bcl6 that binds to IL-18BS may deacetylate histones of the promoter region of the IL-18 gene to close the chromatin structure. This chromatin remodeling of the P1 promoter may inhibit binding of other important transcriptional activators to the promoter region.

Binding of Bcl6 protein to the mIL-18BS in nuclear extracts from resting RAW264 cells was detected by EMSA. The binding activity of Bcl6 was diminished gradually from 6 h after LPS stimulation. However, the amounts of Bcl6 protein in these cells were not changed over the time examined. These results suggest that Bcl6 may be post-transcriptionally modified to loose its binding activity to the mIL-18BS in RAW264 cells after stimulation. The recent report demonstrated that acetylation of lysine residues in the middle part of Bcl6 disrupts the ability of Bcl6 to recruit histone deacetylases, thereby loosing its capacity to repress transcription (46). Transcriptional activity of several factors is also regulated by post-transcriptional modifications (47, 48, 49, 50, 51). A zinc finger-type transcription factor, GATA-1, increases its binding activity to the target DNA sequence by acetylation of lysine residues in the zinc finger domain (49). Thus, the DNA binding domain of Bcl6 may be deacetylated to lose its binding activity to mIL-18BS in RAW264 cells after stimulation with LPS. Additional work is required to elucidate mechanisms of post-transcriptional modifications of Bcl6 to loose its binding activity to the mIL-18BS in activated macrophages.

This study has given us more insight into how Bcl6 functions to regulate Th2-type inflammation. Although Bcl6-/- mice display severe Th2-type inflammation, mechanisms of the inflammation caused the by loss of Bcl6 were unclear (1, 3, 21). Toney et al. (23) reported that the nonlymphoid cell population in Bcl6-/- mice causes Th2-type inflammation. Here we suggest that deregulated IL-18 expression by nonlymphoid cells, such as macrophages, may be one of the causes of the Th2 inflammation in Bcl6-/- mice. This is supported by the finding that aberrant expression of IL-18 results in the increased production of both Th1 and Th2 cytokines in IL-18 transgenic mice (52). Indeed, the high levels of Th2 cytokines and IFN-{gamma} were produced by CD4+ T cells from Bcl6-/- mice (21). We found that the IL-18 gene is a novel target gene for Bcl6.


   Acknowledgments
 
We are grateful to Dr. M. Tone for the P1D1 and P1D2 plasmid. We also thank H. Satake for skillful technical assistance, and N. Kakinuma for secretarial services.


   Footnotes
 
1 This work was supported in part by Health Sciences Research Grants (Research on Eye and Ear Sciences, Immunology, Allergy, and Organ Transplantation) from the Ministry of Health and Welfare of Japan; Grants-in-Aid from the Ministry of Education, Science, Technology, Sports, and Culture of Japan; and the Uehara Memorial Foundation. Back

2 Current address: Division of Hematopoiesis, Center for AIDS Research, Kumamoto University, Kumamoto 860-0811, Japan Back

3 Address correspondence and reprint requests to Dr. Takeshi Tokuhisa, Department of Developmental Genetics (H2), Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan. E-mail address: tokuhisa{at}med.m.chiba-u.ac.jp Back

4 Abbreviations used in this paper: MCP-1, monocyte chemoattractant protein 1; ChIP, chromatin immunoprecipitation; DIG, digoxigenin; DNBcl6, dominant negative Bcl6; IL-18BS, Bcl6-binding sequence in the IL-18 gene; TSA, trichostatin A. Back

Received for publication December 2, 2002. Accepted for publication April 28, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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