The JI PBL Intereron Source
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Becker, C.
Right arrow Articles by Neurath, M. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Becker, C.
Right arrow Articles by Neurath, M. F.
Right arrowPubmed/NCBI databases
*Compound via MeSH
*Substance via MeSH
The Journal of Immunology, 2001, 167: 2608-2618.
Copyright © 2001 by The American Association of Immunologists

Regulation of IL-12 p40 Promoter Activity in Primary Human Monocytes: Roles of NF-{kappa}B, CCAAT/Enhancer-Binding Protein {beta}, and PU.1 and Identification of a Novel Repressor Element (GA-12) That Responds to IL-4 and Prostaglandin E21

Christoph Becker*, Stefan Wirtz*, Xiaojing Ma{dagger}, Manfred Blessing*, Peter R. Galle* and Markus F. Neurath2,*

* Laboratory of Immunology, First Medical Clinic, University of Mainz, Germany; and {dagger} Cornell University, New York, NY 14853


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Appropriate regulation of IL-12 expression is critical for cell-mediated immune responses. In the present study, we have analyzed the regulation of IL-12 p40 promoter activity in primary human monocytes in vivo. Accordingly, we analyzed the p40 promoter by in vivo footprinting in resting and activated primary human blood CD14+ monocytes. Interestingly, footprints at binding sites for trans-activating proteins such as C/EBP, NF-{kappa}B, and ETS were only found upon stimulation with LPS and IFN-{gamma}. In contrast, a footprint over a purine-rich sequence at -155, termed GA-12 (GATA sequence in the IL-12 promoter), was observed in resting, but not activated, cells. Further characterization of this site revealed specific complex formation at a protected GATA core motif in unstimulated primary monocytes and RAW264.7 macrophages. Mutagenesis within the GA-12 sequence caused a significant up-regulation of inducible IL-12 p40 promoter activity in both transient and stable transfection systems, suggesting a repressor function of this site. Furthermore, binding activity of the GA-12 binding protein GAP-12 was increased by treatment with two potent inhibitors of IL-12 expression, IL-4 and PGE2. Finally, we observed that IL-4-mediated repression of IL-12 p40 promoter activity is critically dependent on an intact GA-12 sequence. In summary, our data underline the complex regulation of the human IL-12 p40 promoter and identify GA-12 as a potent, novel repressor element that mediates IL-4-dependent suppression of inducible promoter activity in monocytes. Regulation of GAP-12 binding may thus modulate IL-12 p40 gene expression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Interleukin-12 is a structurally unique cytokine with essential functions in cell-mediated immunity, antimicrobial host responses, and Th1 T cell differentiation (1, 2, 3, 4, 5). It consists of two disulfide-linked subunits, p40 and p35, that form functionally active p40/p35 heterodimers (6, 7, 8). IL-12 is produced mainly by monocytes, macrophages, and dendritic cells. It controls Th1 cell differentiation by binding to a specific receptor comprised of {beta}1 and {beta}2 chains, which are differentially expressed in Th1 and Th2 T cells (9, 10, 11); whereas the {beta}1 chain is expressed in both cell types, the {beta}2 chain is expressed only in Th1 cells. Thus, it is the expression of the {beta}2 chain that accounts for the responsiveness of Th1 cells and the nonresponsiveness of Th2 cells to IL-12. After IL-12 binds to its receptor, it induces activation of specific members of the STAT family of transcription factors (STAT-3 and STAT-4), which then translocate to the nucleus and bind to genomic promoter regions, including that governing IFN-{gamma} (12, 13).

Dysregulated IL-12 gene expression and consecutive Th1 T cell differentiation may contribute to the initiation and perpetuation of various autoimmune and chronic inflammatory diseases, such as rheumatoid arthritis and Crohn’s disease (14, 15, 16). Thus, it is likely that IL-12 gene expression is tightly controlled at the transcriptional or posttranscriptional level. Indeed, recent studies have identified several control elements in the IL-12 p40 promoter that are necessary for inducible p40 gene expression in myeloid cell lines. A NF-{kappa}B element was found to be functionally important for promoter activation in response to LPS and IFN-{gamma} (17). Furthermore, a downstream C/EBP binding site and an ETS-2 element seem essential for high p40 promoter activity, as mutations of these sites caused a strong reduction of inducible IL-12 p40 promoter activity in the RAW264.7 cell line (18, 19, 20). Taken together, these data suggested that the regulated binding of various trans-acting factors mediates p40 promoter activation in monocytic cell lines. However, no repressor elements of the p40 promoter have been identified to date that may be involved in cell type- or activation-specific suppression of IL-12 p40 promoter activity.

In the present study, we have analyzed the human IL-12 p40 promoter in primary human monocytes. An 800-bp fragment of the promoter was found to drive high inducible and cell type-specific expression of a linked reporter gene in transient transfections as well as in transgenic mice. In vivo footprinting and bandshift experiments revealed the binding of NF-{kappa}B p50/p65, C/EBP{beta}, and PU.1 to the promoter in stimulated human monocytes. A footprint over a purine-rich sequence at -155 observed only in resting cells was caused by a specific protein complex, denoted GATA sequence in the IL-12 promoter (GA-12)3 binding protein (GAP-12), thereby repressing inducible IL-12 p40 gene transcription in monocytes. Interestingly, GAP-12 binding to this site was enhanced by IL-4 and PGE2, which have been previously described as potent inhibitors of IL-12 expression (21, 22, 23, 24, 25). Our data suggest a model in which the stimulation of monocytes and macrophages with bacterial cell wall components such as LPS leads to the displacement of the GAP-12 repressor and subsequently to the binding of activating transcription factors such as p50/p65, C/EBP{beta}, and PU.1.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and culture conditions

The RAW264.7 cell line was a gift from Dr. X. Ma (Cornell University, New York, NY). The J774, P338, U937, Daudi, J558, K562, THP-1, COS-7, Raji, BW5147, and Jurkat cell lines were obtained from American Type Culture Collection (Manassas, VA). Cells were cultured in RPMI 1640 supplemented with 10% FCS (PAA, Linz, Austria), 20 mM HEPES buffer (Life Technologies), 2 mM L-glutamine (Life Technologies, Paisley, Scotland), and 1000 U/ml penicillin/streptomycin (Biochrom, Berlin, Germany).

Plasmids

For generation of the human IL-12 p40 promoter in the pCRII vector (p40/pCRII), the promoter was amplified by PCR from Jurkat genomic DNA using two gene-specific primer sequences (upstream primer, 5'-CTGTATGCCTCCCTGAGGG-3'; downstream primer, 5'-AGTGCTTACCTTGCTCTGGG-3') derived from previously published sequence data (20). The resulting 813-bp promoter fragment ranging from -747 to +66 relative to the transcriptional start site was cloned into the pCRII vector (Invitrogen, Leek, The Netherlands) by TA cloning according to the manufacturer’s recommendations.

For the luciferase reporter gene vector p40/pXP1, the p40 promoter was amplified by PCR as described above, treated with mung bean nuclease (Amersham, Arlington Heights, IL) to create blunt ends, and cloned into the SmaI site of the promoterless pXP1 luciferase reporter gene vector (26).

The expression vectors for C/EBP{beta}, C/EBP{alpha}, and C/EBP{delta} were gifts from Dr. C. Trautwein (27) and Dr. R. Schwartz (28), respectively.

Isolation and culture of primary human CD14+ blood monocytes

Human PBMC were isolated from healthy volunteers using Ficoll-Hypaque gradients. PBMC were then further purified using the MACS system (Miltenyi Biotec, Bergisch-Gladbach, Germany) with immunomagnetic beads specific for CD14 (Miltenyi Biotec). Freshly isolated cells were counted and subjected to FACS analysis using an FITC-labeled CD14 Ab. Only cell populations with a purity >95% were used in the experiments described below. The cells were cultured in RPMI 1640 medium (Biochrom) supplemented as described above in humidified atmosphere with 5% CO2 at 37°C.

Cells were treated with the following reagents as specified in Results: 100 U/ml human IFN-{gamma} (Roche, Mannheim, Germany), 100 U/ml mouse IFN-{gamma} (Genzyme, Cambridge, MA), 1 µg/ml bacterial LPS (Sigma, St. Louis, MO), 5 ng/ml human IL-4 (BD PharMingen, San Diego, CA), and 5 ng/ml mouse IL-4 (BD PharMingen).

Isolation of mRNA and RT-PCR

Total RNA was isolated with the High Pure RNA isolation kit (Roche) according to the manufacturer’s recommendations. Reverse transcription into cDNA was performed using the Moloney murine leukemia virus reverse transcriptase (Life Technologies) according to the manufacturer’s recommendations. PCR was performed using the following primers derived from previously published sequence data: human IL-12 p40, 5'-TTTTCTGGCATCTCCCCTCGTG-3' and 5'-TGGGTGGGTCAGGTTTGATGATG-3'; and {beta}-actin, 5'-TGACGGGGTCACCCACACTGTGCCCATCTA-3' and 5'-CTAGAAGCATTTGCGGTGGACGATGGAGGG-3'. PCR products were analyzed on 1% agarose gels.

Dimethyl sulfate (DMS)-piperidine treatment of primary human monocytes for in vivo footprinting

DMS (0.1%; Sigma) was added directly to the culture medium and incubated for 2 min. Cells were extensively washed, and DNA extraction was performed by an overnight incubation in cell lysis buffer (1 mM Tris-HCl (pH 7.5), 400 mM NaCl, 2 mM EDTA, 0.2% SDS, and 0.2 mg/ml proteinase K) at 37°C. The strand scission reaction was performed by resuspending the DNA in 1 M piperidine (Sigma) with subsequent incubation for 30 min at 90°C. The DNA was finally resuspended in water and diluted at a concentration of 1 µg/µl. For control reactions, naked genomic DNA was treated with DMS in vitro. Therefore, the DNA was incubated for 30 s with 0.1% DMS at room temperature. In vitro methylated control DNA was subsequently treated with piperidine as described above.

Ligation-mediated PCR (LM-PCR)

In vivo footprinting by LM-PCR was conducted essentially as previously described (29, 30, 31). In brief, primer annealing was performed with 0.5 pmol primer 1 for 1 µg genomic DMS-treated and piperidine-cleaved DNA. For primer extension Sequenase 1.0 (U.S. Biochemical, Cleveland, OH) was used. Linker ligation was performed overnight at 15°C. Exponential PCR amplification was performed with primer 2 and the linker primer for 15–22 cycles (94°C for 1 min, 1°C for 2 min, and 76°C for 3 min). Finally, the 32P-labeled third primer (106 cpm) was added together with 2 U Taq DNA polymerase and 2 µl dNTPs (5 mM each), and a final PCR cycle was performed, followed by phenol/chloroform extraction of the samples, ethanol precipitation, and analysis on a 6% denaturing urea/polyacrylamide gel.

Primer sequences specific for LM-PCR of the human IL12 p40 promoter were as follows: primer 1, 5'-GGCTTGGGAAGTGCTTACC-3'; primer 2, 5'-CTCTGGGCAGGACGGAGAGTCC-3'; primer 3, 5'-GGGCAGGACGGAGAGTCCAATGGC-3'; linker (top strand), 5'-GCGGTGACCCGGGAGATCTGAATTC-3'; and linker (bottom strand), 5'-GAATTCAGATC-3'.

The in vivo footprinting ladders were verified by comparison to sequencing ladders of cloned IL-12 p40 promoter DNA (p40/pCRII). Therefore, the p40 promoter was sequenced with the Sequenase sequencing kit (U.S. Biochemical) using LM-PCR primer 3 according to the manufacturer’s instructions.

Isolation of nuclear proteins and in vitro translation

Extraction of nuclear proteins was conducted by the method of Schreiber et al. (32). Protein concentrations were measured with a protein assay kit (Bio-Rad, Munich, Germany). For in vitro translation of C/EBP transcription factors, wheat germ extract, T7 polymerase, and expression vectors as DNA templates were used according to the manufacturer’s recommendations (Promega, Madison, WI).

The EMSA

Oligonucleotides for EMSA were synthesized, annealed, gel-purified, and end-labeled with [{gamma}-32P]ATP (5000 Ci/mmol; Amersham) using T4 polynucleotide kinase (New England Biolabs, Beverly, MA). Radiolabeled DNA probe (25,000 cpm) were added to the binding reaction that also contained 1 µg synthetic DNA duplex of poly(dI-dC) (Pharmacia, Peapack, NJ), 3 µg nuclear proteins, and binding buffer (25 mM HEPES (pH 7.5), 150 mM KCl, 5 mM DTT, and 10% glycerol). For supershift assays, 2 µg specific Abs (Santa Cruz Biotechnology, Santa Cruz, CA) were used. For competition analysis, an excess of unlabeled oligonucleotides containing consensus binding sites for transcription factors was added to the binding reaction. Complex formation was allowed to proceed for 30 min at room temperature. Finally, the complexes were separated from unbound DNA by native PAGE on 5% gels. The gels were exposed to Kodak MS films (Eastman Kodak, Rochester, NY) on intensifying screens at -80°C.

The sequences of oligonucleotides (top strands) for EMSA were as follows: GA-12, 5'-CCTCGTTATTGATACACACACAGAGA-3'; GA-12 mutant (m)1, 5'-CCTCGTTATTTCTACACACACAGAGA-3'; GA-12 m2, 5'-CCTCGTTATTTCTACACACACAGAGA-3'; GA-12 m3, 5'-CCTCGTTATTTCTACACACACAGAGA-3'; GA-12 m4, 5'-CCTCGTTATTTCTACACACACAGAGA-3'; GATA (Santa Cruz Biotechnology), 5'-CACTTGATAACAGAAAGTGATAACTCT-3'; OCT (Stratagene, Heidelberg, Germany), 5'-GATCGAATGCAAATCACTAGCT-3'; p40 NF-{kappa}B, 5'-GAACTTCTTGAAATTCCCCCAGAAGG-3'; p40 NF-{kappa}B m1, 5'-GAACTTCTTGAAATTAGCCCAGAAGG-3'; NF-{kappa}B1 (Stratagene), 5'-GATCGAGGGGACTTTCCCTAGC-3'; NF-{kappa}B2 (Santa Cruz Biotechnology), 5'-AGTTGAGGGGACTTTCCCAGGC-3'; AP-1 (Stratagene), 5'-CTAGTGATGAGTCAGCCGGATC-3'; p40 C/EBP, 5'-TGTTTTCAATGTTGCAACAAGTCAGT-3'; p40 C/EBP m1, 5'-TGTTTTCAATGTTCTAACAAGTCAGT-3'; C/EBP (Santa Cruz Biotechnology), 5'-TGCAGATTGCGCAATCTGCA-3'; p40 ETS-L, 5'-GATGTAAACCCAGAGAAATTAGCATCTCCATCTCCTTCCTTATT-CCCCACCCAAAAGTCATTTCCTCTTAGTTCATTA-3'; p40 ETS-S, 5'-CCCAAAAGTCATTTCCTCTTAGTTC-3'; p40 ETS-S m1, 5'-CCCAAAAGTCATTAACTCTTAGTTC-3'; CREB (Stratagene), 5'-GATTGGCTGACGTCAGAGAGCT-3'; ETS (Santa Cruz Biotechnology), 5'-GGGCTGCTTGAGGAAGTATAAGAAT-3'; IFN regulatory factor-1 (Santa Cruz Biotechnology), 5'-GGAAGCGAAAATGAAATTGACT-3'; STAT-1 (Santa Cruz Biotechnology), 5'-CATGTTATGCATATTCCTGTAAGTG-3'; STAT-3 (Santa Cruz Biotechnology), 5'-GATCCTTCTGGGAATCCTAGATC-3'; STAT5/6 (Santa Cruz Biotechnology) 5'-AGATTTCTAGGAATTCAATCC-3'; and E box (33), 5'-AGCTTGAACCTGCAGCTGCAGGTGGGGGAGTA -3'.

ELISA for IL-12 p40 and p70

To measure IL-12 protein production, 106 primary monocytes/well were seeded out in 1 ml culture medium in triplicate in 48-well tissue culture plates and incubated at 37°C in humidified 5% CO2 atmosphere in the presence or absence of different stimuli as indicated above. After 48 h cell-free culture supernatants were removed and assayed for p70 and p40 concentrations by ELISA (34).

Site-directed mutagenesis

Site-directed mutagenesis was performed with the QuikChange Site-Directed Mutagenesis Kit (catalog no. 200518, Stratagene) according to the manufacturer’s instructions. Mutant primer sequences (top strands) were as described above.

Transfections and reporter gene analysis

Thee p40/pXP1 reporter gene vector (10 µg) along with 2 µg of a {beta}-galactosidase expression vector were transfected into 107 RAW264.7 cells using the DEAE transfection method. For cotransfection studies, 0.5–4 µg expression vector was used. After 18 h, the cells were stimulated as described above. The stimulation was allowed to proceed for 8 h before the cells were harvested, washed in PBS, and lysed in cell lysis buffer (Promega). Luciferase activity was measured as light emission over a period of 10 s after addition of luciferase assay buffer (Promega) with a standard luminometer (Sirius, Berthold Detection Systems, Pforzheim, Germany). Luciferase activity was normalized to the {beta}-galactosidase expression level of the lysate or where applicable to the protein content of the solution.

To generate cell lines stably transfected with p40/pXP1 constructs, a cotransfection strategy with the pEGFP-C1 vector (CLONTECH Laboratories, Heidelberg, Germany) followed by geneticin-based selection of clones was used. In an initial series of experiments we performed dose-response studies with geneticin (G418, obtained from Roche) in RAW264.7 cells and observed that the lethal concentration of geneticin for these cells was 500 µg/ml. Stable transfectants were obtained by electroporating (Bio-Rad apparatus; 500 V, 2.5 ms) RAW264.7 cells with 20 µg linearized p40/pXP1 reporter gene vectors along with 2 µg of the linearized pEGFP-C1 vector carrying the expression cassettes for neomycin resistance and enhanced green fluorescent protein. Individual clones were identified by fluorescence microscopy (IX 70, Olympus, New Hyde Park, NY), transferred to new petri dishes, and subcultivated. Finally, cells of each clone were lysed and analyzed for luciferase expression.

Statistical analysis

Data from transfection experiments were analyzed by Student’s t test using the program Excel.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
In vivo genomic footprinting demonstrates occupancy of previously described and unrecognized IL-12 p40 promoter elements in primary human CD14+ monocytes

To analyze functional aspects of the IL-12 p40 promoter, we cloned an 800-bp fragment of the human IL-12 p40 promoter upstream of the luciferase gene of the promoterless pXP1 vector yielding the p40/pXP1 construct. Consistent with previous observations in tumor cell lines (17, 18, 19, 20), transient transfection assays showed that this fragment of the promoter is synergistically induced in RAW264.7 cells upon stimulation with LPS plus IFN-{gamma} (9.5 ± 1.5-fold compared with unstimulated cells). These studies suggested that the proximal promoter directs temporal and spatial expression of IL-12 p40. However, as the reporter gene vector remains episomal, transient transfections do not necessarily reflect the complex biochemical processes involved in endogenous IL-12 p40 gene regulation in the nucleus (35). Furthermore, recent studies indicate that promoter regulation in primary cells and tissue culture cell lines may differ significantly (13, 31). To explore whether potential binding sites for regulatory nuclear proteins in the 800-bp p40 promoter fragment were occupied in intact primary human CD14+ monocytes in vivo, we next performed in vivo genomic footprinting studies via LM-PCR. In these studies CD14+ monocytes were highly purified from peripheral blood by immunomagnetic beads. These primary monocytes were analyzed by ELISA and RT-PCR analysis and displayed a highly inducible and transient expression of IL-12 p40 protein and mRNA in response to LPS plus IFN-{gamma} (data not shown). In vivo methylated DNA from unstimulated and LPS- plus IFN-{gamma}-stimulated CD14+ monocytes was then isolated and subjected to the LM-PCR procedure (see Materials and Methods). An altered DMS reactivity in stimulated monocytes, characterized by hyperreactive A and G and protected G nucleotides (as shown by densitometric analysis), was found at the previously described NF-{kappa}B, C/EBP, and ETS sites, suggesting that these sites contribute to promoter regulation in primary monocytes in vivo (Fig. 1Go). In contrast, we observed protected G residues at a potentially novel DNA binding site at -155 bp relative to the transcriptional start site in unstimulated, but not in stimulated, CD14+ monocytes (Fig. 1GoA), as discussed below. No altered DMS reactivities were detected between -260 and -555 bp upstream of the transcriptional start site (Fig. 1GoB) in primary CD14+ monocytes, indicating that the proximal (up to -260 bp) rather than the distal part of the IL-12 p40 promoter is the target for multiple protein/DNA interactions in vivo.



View larger version (58K):
[in this window]
[in a new window]
  		FIGURE 1. Genomic footprinting of the human IL-12 p40 promoter in freshly isolated monocytes. Freshly isolated CD14+ human monocytes were cultured with or without LPS and IFN-{gamma} for 4 h. In vivo footprinting was performed as described in Materials and Methods. Footprinting ladders between -65 and -262 bp (A) and between -250 and -555 bp (B) were aligned to sequencing ladders of cloned IL-12 p40 promoter DNA (p40/pCRII) to identify protected or hyperreactive nucleotides. Protected nucleotides were detected in unstimulated ({circ}) and stimulated (•) primary human monocytes. Hyperreactivities are marked with an arrowhead. One representative experiment of three using the same LM-PCR amplification products in gels A and B is shown. C, Densitometric analysis of protected G residues around the GA-12 core motif (left panel) and the NF-{kappa}B and C/EBP sites (right panel) in primary human monocytes. Data are shown as the percentage of inhibition of band intensity (bands 1–11; see Fig. 2GoA for sequence comparison) compared with naked DNA.

 
Taken together, the in vivo footprinting data suggested that the induction of IL-12 p40 expression by LPS/IFN-{gamma} in primary human monocytes coincides with inducible protein/DNA interactions at the proximal p40 promoter (Fig. 2GoA).



View larger version (39K):
[in this window]
[in a new window]
  		FIGURE 2. Summary of protein/DNA interactions and reporter gene analysis of mutations at in vivo-protected binding sites. A, Summary of protein/DNA interactions in the 5'-flanking promoter region of the IL-12 p40 gene in monocytes (the positions of the bands 1–11 of Fig. 1GoA are indicated). B, RAW264.7 macrophages were transfected (DEAE) with the wild-type p40/pXP1 reporter gene construct and mutant constructs carrying 2-bp exchanges at the indicated promoter sites. Cells were left untreated or were stimulated for 8 h iwth LPS/IFN-{gamma} prior to luciferase measurement. Data represent mean values ± SD of five independent experiments after normalization for transfection efficiency. Luciferase activity is reported as fold induction over unstimulated wild-type p40/pXP1.

 
In human monocytes, p50/p65 and C/EBP{beta} are key activators of IL-12 p40 gene expression

To assess the potential functional relevance of the in vivo-protected DNA sequences, reporter gene experiments were performed. Therefore, we generated various constructs of the IL-12 p40 promoter carrying 2-bp mutations at in vivo-protected sites. These mutant promoter constructs were then transfected into RAW264.7 macrophages, and the resulting luciferase activity was compared with that of the p40 wild-type construct (Fig. 2GoB). Site-directed mutagenesis of the C/EBP, NF-{kappa}B, and ETS sites of the human IL-12 p40 promoter caused a significant reduction of inducible promoter activity in RAW264.7 cells. Furthermore, double mutations of the C/EBP and NF-{kappa}B sites or the C/EBP and ETS sites (but not the NF-{kappa}B plus ETS sites) resulted in lower reporter gene activity compared with the single mutants, suggesting synergistic effects of these sites on p40 promoter activity. Finally, we observed that in a construct carrying mutations at all three sites, LPS- plus IFN-{gamma}-dependent p40 promoter activity was almost completely abrogated, suggesting that proteins binding to the C/EBP, ETS, and NF-{kappa}B sites are synergistic key regulators of IL-12 p40 promoter activity.

Mutation of the C/EBP site led to a strongly impaired promoter activation in response to LPS and IFN-{gamma}, suggesting a crucial role for C/EBP in IL-12 p40 expression. Cotransfection studies were performed to verify whether ectopic overexpression of C/EBP proteins could trans-activate the human IL-12 p40 promoter (Fig. 3Go). Strikingly, overexpression of C/EBP{beta}, but not C/EBP{delta}, trans-activated the p40 promoter 25-fold in unstimulated and 6-fold in LPS- plus IFN-{gamma}-stimulated cells (Fig. 3GoA). In contrast, overexpression of C/EBP{alpha} had adverse effects. The trans-activation of unstimulated macrophages by C/EBP{beta} was strongly dose dependent (Fig. 3GoB), indicating that C/EBP{beta} alone may efficiently drive the human IL-12 p40 promoter without the need of further stimulation. To rule out the possibility that C/EBP binding sites other than the site at -80 bp may contribute to the trans-activation potential of C/EBP{beta}, C/EBP{beta} was cotransfected together with the wild-type and mutant promoter constructs. Strikingly, the promoter carrying a mutation in the -80 C/EBP site was not trans-activated by overexpressing C/EBP{beta}, thus providing evidence for a single functional C/EBP{beta} binding site in the human IL-12 p40 promoter (Fig. 3GoC).



View larger version (49K):
[in this window]
[in a new window]
  		FIGURE 3. Ectopic overexpression of C/EBP{beta} is sufficient to drive the human IL-12 p40 promoter. A, RAW264.7 macrophages were transfected with 2.5 µg C/EBP expression vectors (or pcDNA1vector as control) along with 10 µg of the p40/pXP1 reporter gene vector using the DEAE method. Cells were left untreated or were stimulated for 8 h with LPS and IFN-{gamma}. B, Increasing amounts of a C/EBP{beta} expression vector or a control plasmid (pcDNA1) were transfected along with 10 µg of the wild-type and mutant p40/pXP1 reporter gene vector. C, A total of 2.5 µg C/EBP{beta} expression vector or a control plasmid (pcDNA1) was transfected along with 10 µg of the wild-type and mutant p40/pXP1 reporter gene vectors. Cells were left untreated or were stimulated for 8 h with LPS and IFN-{gamma}. Data represent mean values ± SD of three experiments after normalization for transfection efficiency. Luciferase activity is reported as fold induction over unstimulated control-transfected wild-type p40/pXP1.

 
In vivo footprinting demonstrates protection of a novel sequence element in resting, but not activated, primary human CD14+ monocytes

In vivo footprinting at the IL-12 p40 promoter indicated the simultaneous occupancy of the NF-{kappa}B, C/EBP, and ETS sites in stimulated human monocytes (Fig. 1GoA). In contrast, we observed protected G residues around -155 bp relative to the transcriptional start site in unstimulated CD14+ monocytes (Fig. 1GoA). However, this protection was not observed after stimulation of monocytes with LPS plus IFN-{gamma} in several independent experiments, suggesting the potential binding of a regulatory protein to this site in resting, but not activated, human monocytes. Interestingly, sequence alignment revealed that the observed footprint corresponds to a consensus motif that is characteristic for binding of proteins of the GATA family of transcription factors. This novel promoter element was denoted GA-12.

Mutagenesis of the -155 GA-12 motif results in increased IL-12 p40 promoter activity in both transient and stable transfection systems

Our in vivo footprinting results implicated the possible binding of a regulatory protein to the GATA core of the GA-12 motif at -155 of the IL-12 p40 promoter in unstimulated cells, but not in cells stimulated with LPS/IFN-{gamma}. To explore whether the protected promoter sequence could be functionally important for inducible promoter activity, reporter gene experiments were performed with the wild-type p40/pXP1 reporter gene vector and the p40/pXP1 vector carrying mutations within the GA-12 motif. Interestingly, in transient transfection assays mutations at the GATA core sequence of the GA-12 motif (GA-12 m1, GA12 m2) resulted in 2-fold higher inducible p40 promoter activity than that of the wild-type construct or mutant constructs with base pair exchanges elsewhere in the p40 promoter (Fig. 4Go, mut 5–7). Interestingly, base pair exchanges directly up- and downstream of the core GATA sequence (GA-12 m3, GA-12 m4) also resulted in an increased promoter activity, suggesting that the complex binding at this site may need some spacing around the core motif. Taken together, these data suggest the potential binding of a repressor protein to the GA-12 site in the IL-12 p40 promoter.



View larger version (49K):
[in this window]
[in a new window]
  		FIGURE 4. The GA-12 site is a potent repressor element. A, Reporter gene analysis of the p40/pXP1 luciferase construct carrying mutations at the in vivo-protected GA-12 site or unrelated sequences elsewhere in the promoter (mut5–7). The wild-type and mutant p40/pXP1 reporter gene constructs were transfected using the DEAE transfection method. Cells were left untreated or were stimulated for 8 h with LPS/IFN-{gamma} before luciferase measurement. Luciferase activity is reported as fold induction over unstimulated wild-type p40/pXP1, and data shown are the mean ± SD of three independent experiments after normalization for transfection efficiency. B, Sequences of wild-type and mutant GA-12 oligonucleotides used for site-directed mutagenesis and bandshift assays. The mutated nucleotides are marked with asterisks. C, Reporter gene analysis of stable transfectants carrying the wild-type or mutant (GA-12 m1) p40/pXP1 luciferase constructs. Stably transfected cell lines were generated as described in Materials and Methods and analyzed for constitutive luciferase expression. Data are the average values from two independent experiments using six independent clones per group; the average value per group is indicated.

 
To verify the functional relevance of the GA-12 site for repression of inducible IL-12 p40 promoter activity, we generated stably transfected cell lines with the wild-type and mutant (GA-12 m1) p40/pXP1 reporter gene constructs. Accordingly, the p40/pXP1 and GA-12 m1-p40/pXP1 vectors were integrated into the genome of RAW264.7 macrophages by electroporation and subsequent selection of stable transfectants with geneticin (see Materials and Methods). Multiple clones were finally analyzed for luciferase expression (Fig. 4GoC). Interestingly, clones carrying the promoter with a mutant GA-12 motif produced, on the average, an almost 100-fold higher luciferase activity compared with wild-type clones. This finding indicates an important function for the GA-12 site for promoter regulation in the nucleosomal context.

A specific nuclear factor, GAP-12, binds to the -155 GATA sequence of the IL-12 p40 promoter

Subsequently, EMSA experiments were performed to determine whether the footprint observed over the GA-12 motif in unstimulated primary monocytes might be caused by binding of a sequence-specific nuclear protein. As shown in Fig. 5GoA, retarded complexes were detected using EMSA. The observed complex was present in nuclear extracts from unstimulated primary monocytes (Fig. 5GoA, lanes 1–3) and RAW264.7 macrophages (Fig. 5GoB), but was only weakly detectable or not detectable in cells stimulated with LPS alone or LPS plus IFN-{gamma}. In contrast, specific complexes at the NF-{kappa}B and C/EBP binding sites (using extracts from primary monocytes or RAW264.7 cells) were unchanged or even increased upon such stimulation (data not shown). The protein complex responsible for the EMSA bands in the unstimulated extracts was termed GAP-12. Because the -155 GA-12 motif contains a consensus binding sequence characteristic for the GATA family of transcription factors we considered the possibility that GAP-12 might be a GATA-like transcription factor. Interestingly, the GAP-12 complex comigrated and was even coregulated with the GATA complex obtained with a GATA reference binding site and extracts of primary human monocytes (Fig. 5GoA). The specificity of the complex was demonstrated by competition studies; GAP-12 binding was specifically competed with unlabeled probe and a reference GATA site, but not with unrelated competitor DNA or a mutated -155 GA-12 probe (Fig. 5GoC). Mutant GA-12 oligonucleotides as probes (for sequences see Fig. 4GoB) resulted in the abrogation of GAP-12 binding (Fig. 5GoD). Therefore, consistent with the results of reporter gene analysis (Fig. 4Go) the binding of GAP-12 to the -155 GATA sequence may need some spacing. Subsequently, complex formation at the IL-12 p40 promoter GA-12 site was analyzed in a variety of cell lines. Binding to the -155 GA-12 site was found in all cell lines analyzed (Fig. 5GoE), implicating a ubiquitous, rather than a tissue-restricted, expression of GAP-12. In an attempt to directly identify the protein binding to this site in primary human monocytes, Abs against GATA-1 to GATA-6 were added to the EMSA reactions. None of them produced a supershift under our experimental conditions (Fig. 5GoF and data not shown). Thus, we were not able to identify previously described GATA transcription factors in the GAP-12 complex.



View larger version (53K):
[in this window]
[in a new window]
  		FIGURE 5. Partial characterization of the GA-12 binding complex. A, Bandshift assay showing activation-induced changes at the p40 promoter GA-12 site. Three micrograms of nuclear extract of unstimulated and stimulated (4 h) primary human monocytes (left panel) or RAW264.7 macrophages (B) were incubated with a labeled GA-12 (lanes 1–3) or a consensus GATA oligonucleotide (Santa Cruz Biotechnology; lanes 4–6). A specific complex denoted GAP-12 was observed, which was down-regulated upon stimulation with LPS or LPS/IFN-{gamma}. C, Competition analysis of the p40 promoter GA-12 site. An excess of unlabeled probes for transcription factor binding sites was added to 3 µg nuclear extract of unstimulated human monocytes before addition of labeled GA-12 probe. GAP-12 binding was abrogated by competition with a consensus GATA binding site. D, Binding of GAP-12 to wild-type and mutant GA-12 probes. Labeled probes (25,000 cpm; see Fig. 4GoB for sequences) were incubated together with 3 µg nuclear extract of unstimulated human monocytes. GAP-12 binding was strongly impaired using GA12 m1–3 and moderately impaired using GA-12 m4 as probe. E, Complex formation at the GA-12 site in primary monocytes and cell lines of different origin. Three micrograms of each extract was incubated with the labeled wild-type GA-12 probe. F, Supershift analysis of GAP-12. As indicated, 0.5, 1, or 2 µg of each Ab was incubated with nuclear extract of unstimulated human monocytes before the addition of labeled GA-12 probe.

 
GAP-12 DNA binding activity in primary human monocytes is regulated by IL-4 and PGE2

IL-4 is a potent inhibitor of IL-12 expression (21, 22, 23). To investigate whether IL-4 may act by modulating binding of the GAP-12 repressor to the -155 GATA site, primary blood CD14+ monocytes and RAW264.7 cells were stimulated with LPS plus IFN-{gamma} in the presence or absence of IL-4. IL-4 stimulation led to suppression of inducible IL-12 p40 promoter activity that was accompanied by reduced IL-12 p40 mRNA and protein levels (Fig. 6Go). Moreover, after 1 h of LPS/IFN-{gamma} stimulation no GAP-12 binding was detectable, but after 3 h (and even more pronounced after 5 h), binding of GAP-12 to the -155 GATA site was increased in IL-4-treated cells compared with controls (Fig. 6DGo, left panel). In contrast, binding at the IL-12 p40 promoter C/EBP and NF-{kappa}B sites was unchanged after IL-4 treatment, as assessed by EMSA (data not shown). Furthermore, the addition of PGE2, another inhibitor of IL-12 expression (24, 25) produced in Th2-type immune responses, to the culture medium of human monocytes strongly increased GAP-12 binding to its site (Fig. 6GoD, right panel). Our data thus provide evidence that the inhibitory pathway initiated by IL-4 and PGE2 may involve the modulation of GAP-12 binding to the -155 GA-12 site.



View larger version (47K):
[in this window]
[in a new window]
  		FIGURE 6. The GA-12 site is a target of IL-4-mediated repression of the IL-12 p40 promoter. A, ELISA analysis of IL-4-mediated repression of IL-12 p40. One million blood CD14+ cells per well were plated out and stimulated as indicated. After 48 h, supernatants were analyzed for IL-12 p40. For RT-PCR (B), 10 million blood CD14+ cells were stimulated as indicated. Subsequently, RNA was extracted and subjected to RT-PCR using a set of primers specific for human IL-12 p40 and {beta}-actin cDNA. C, IL-4 induced repression of IL-12 p40 promoter activity in RAW264.7 macrophages. RAW264.7 macrophages were transfected (DEAE) with the wild-type p40/pXP1 reporter gene construct. Cells were left untreated or were stimulated for 8 h as indicated. Data are the mean ± SD of three independent experiments after normalization for transfection efficiency. D, GAP-12 binding is induced by IL-4 and PGE2. Left panel, LPS/IFN-{gamma}-stimulated primary human monocytes were cultured with or without IL-4 (5 ng/ml). Right panel, Monocytes were left untreated or were incubated with PGE2 (1 µg/ml) for 4 h. The position of the GAP-12 complex is indicated by an arrow. E, Reporter gene analysis of stable transfectants carrying the wild-type or mutant (GA-12 m1) p40/pXP1 luciferase construct. Cells were stimulated for 30 h with LPS/IFN-{gamma} in the presence or absence of 20 ng/ml IL-4. Data represent the average IL-4-mediated repression (percentage) ± SD of luciferase activity compared to cells cultured with LPS plus IFN-{gamma} in the absence of IL-4 in three to six independent experiments using three independent clones per construct.

 
IL-4-mediated suppression of IL-12 p40 promoter activity is critically dependent on an intact GA-12 site

To directly test the above hypothesis that IL-4 mediates suppression of IL-12 p40 promoter activity by modulating GAP-12 binding to the GA-12 sequence element, we performed additional experiments using stably transfected RAW264.7 cell lines. RAW264.7 macrophages that were transfected with the wild-type or GA-12 m1 mutant IL-12 p40 reporter gene constructs were stimulated with LPS plus IFN-{gamma} in the presence or absence of IL-4, and luciferase activity was analyzed in the cell lysates (Fig. 6GoE). It was found that IL-4 inhibited luciferase expression from the wild-type p40 promoter construct by ~60% (Fig. 6GoE). In contrast, luciferase expression driven by the p40 promoter carrying a mutation in the GA-12 motif was repressed by only 5–20%, suggesting an important function for the GA-12 site in IL-4-mediated IL-12 p40 promoter repression.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The ability to produce IL-12 is a fundamental property of monocytes and dendritic cells in various infectious and autoimmune diseases (2, 35, 36, 37, 38). In the present study, we provide evidence that the IL-12 p40 promoter in primary CD14+ blood monocytes is tightly regulated in vivo by the coordinated binding of various trans-acting proteins such as C/EBP{beta}, NF-{kappa}B p50/p65, and PU.1. Furthermore, we have identified a novel repressor element in the IL-12 p40 promoter, bound by a specific nuclear complex termed GAP-12 in monocytes and macrophages, that is critical for IL-4-mediated suppression of inducible IL-12 p40 promoter activity. Suppression of monocytic GAP-12 binding may thus be relevant for activating IL-12 p40 cytokine gene transcription in response to bacterial Ags and autoimmune diseases.

The IL-12 p40 promoter was previously characterized by transfection studies in various cell lines (17, 18, 19, 20, 39, 40, 41). Data derived from these studies suggested that several important regulatory proteins, such as C/EBP, NF-{kappa}B, and ETS-2/GLp109, have functionally active binding sites in the promoter. However, insights into the contribution of these sites to promoter regulation in primary monocytes and macrophages in vivo have been limited. Interestingly, recent studies have shown that promoter regulation in primary cells in vivo may differ significantly compared with data obtained from studies in tissue culture cell lines in vitro (13, 31). Moreover, results often differ depending on the cell line used for these promoter studies. In an approach toward the goal to understand p40 promoter regulation in vivo, we have used in vivo genomic footprinting in primary CD14+ blood monocytes to investigate occupancy of cis-activating elements of the IL-12 p40 promoter in living cells. We observed in vivo-protected and hyperreactive residues in LPS- plus IFN-{gamma} activated primary monocytes at an NF-{kappa}B half site (-122 to -132) and a downstream C/EBP site, suggesting that these sites may contribute to promoter regulation in vivo in monocytes. Depending of the cell line used, recent investigation of these sites have demonstrated the binding of p50/c-Rel, p50/p65, and C/EBP{alpha} and -{beta}, respectively (17, 18, 42). Because in vivo footprinting suggested protein/DNA interactions at the C/EBP site after cell stimulation only, we assume that in vivo, upon stimulation, C/EBP{beta} has to be modified post-translationally to bind to the IL-12 p40 promoter site at -80. Recent studies have indeed revealed that C/EBP{beta} is targeted by several signaling pathways, including LPS-mediated signaling and that phosphorylation of serine and threonine residues is essential for its DNA binding and trans-activation (27, 28, 43, 44, 45, 46). The functional relevance of the NF-{kappa}B and C/EBP sites in monocytes was further underlined by the striking reduction of inducible p40 promoter activity in RAW264.7 macrophages upon specific site-directed mutation of these sites described by different groups (17, 18) and in the present manuscript. Furthermore, we observed that simultaneous mutations of these sites resulted in lower inducible promoter activity compared with the single mutant constructs, suggesting synergistic effects of both sites on LPS- plus IFN-{gamma}-dependent p40 promoter activity. Such functional synergy of NF-{kappa}B and C/EBP has recently been described for several promoters, including that of the IL-6 and IL-8 genes (47, 48). In our experiments overexpression of C/EBP{beta} strongly trans-activated the human IL-12 p40 promoter even without the need of further stimulation. The protein appears to act via the previously described C/EBP site on IL-12 p40 promoter activity, because mutation of this motif completely abolished the trans-activation potential of overexpressed C/EBP{beta}. In contrast, promoter activity was suppressed upon overexpression of C/EBP{alpha}, another member of the C/EBP family of transcription factors. Because C/EBP{alpha} was recently shown to be highly expressed in proliferating myelomonocytic cells and down-regulated during cell maturation (49, 50), one may speculate that C/EBP{alpha} might serve to repress the IL-12 p40 promoter in undifferentiated cells, whereas upon maturation, C/EBP{alpha} is replaced by C/EBP{beta}. Further support for an important function of C/EBP{beta} for IL-12 p40 promoter regulation comes from studies in C/EBP{beta}-knockout mice. In these mice, defective activation of macrophages and diminished IL-12 production was observed (51).

Dysregulated IL-12 levels have a strong impact on cell-mediated immune responses, suggesting the potential existence of negative regulatory elements that control IL-12 gene expression. In the present study, using in vivo footprinting in live monocytes, we have identified a novel purine-rich sequence element denoted GA-12 in the human IL-12 p40 promoter. GA-12 was protected from in vivo methylation in unstimulated, but not activated primary human monocytes suggesting protein binding to this site in the former, but not the latter cells. Sequence alignments showed that the protected G residues correspond to a GATA consensus sequence at -155 bp that is well preserved between the human and murine p40 promoter. Gel retardation assays demonstrated sequence specific binding of a complex, termed GAP-12, to this site in monocytes and macrophages. Interestingly, activation of both RAW264.7 cells and primary human CD14+ monocytes resulted in a strong reduction of nuclear GAP-12 binding activity associated with an abrogation of protein/DNA interactions at the -155 bp GA-12 site in vivo. The GAP-12 complex in human monocytes was competed by a reference GATA site. Furthermore, using a reference GATA site as probe in bandshift experiments with extracts of unstimulated human monocytes resulted in the appearance of a complex that comigrated and even was coregulated with the GAP-12 complex. Because GAP-12 can bind to a reference GATA site, one may speculate that GAP-12 might be a GATA-related protein or at least a protein that can bind to a GATA site. Unfortunately, only little is known about GATA proteins in monocytes and macrophages, and down-regulation of GATA-1 and GATA-2 seems to be critical for myeloid development (52, 53, 54, 55). Interestingly, several myeloid promoters appear to have GATA sites that bind GATA proteins (56). The PU.1 promoter also contains a site that can bind GATA proteins, and cotransfection studies overexpressing GATA-1 and GATA-2 have shown to repress the PU.1 promoter 2-fold (57). In primary CD14+ human monocytes neither GATA-1 nor GATA-2 mRNA was detectable by RT-PCR (our unpublished observations). However, using RT-PCR with degenerated primers specific for the highly conserved GATA zinc finger region we are currently investigating potentially novel GATA proteins expressed in monocytes.

The repressor function of GAP-12 on p40 promoter activity in monocytes was shown by several findings. In initial experiments we observed that site-directed mutagenesis within the GA-12 motif resulted in a considerable up-regulation of inducible promoter activity compared with the wild-type construct in both transient transfection assays and stable transfection experiments. In particular, promoter activity was induced nearly 100-fold in stably transfected cell lines carrying mutations at the GA-12 site, strongly suggesting that GA-12 plays an important role in controlling inducible IL-12 p40 promoter activity in the nucleosomal context. Mutations a few base pairs up- and downstream of the GATA core sequence were also effective, suggesting that GAP-12 binding to the GATA sequence may need some spacing around the core motif. This hypothesis was supported by bandshift experiments using mutated GA-12 oligonucleotides as probes. Furthermore, we found that binding of the GAP-12 complex was strongly increased in extracts of monocytes stimulated with IL-4 or PGE2, two mediators of Th2-like immune responses that have been shown to suppress IL-12 production (21, 22, 23, 24, 25). Our data demonstrated that IL-4-mediated repression of promoter activity was strongly impaired in stable transfectants carrying a mutant GA-12 site, implicating an important function of this site in the regulation of IL-12 gene expression. The correlation between GAP-12 binding observed in the bandshift experiments and repressor function as determined in reporter gene assays underlines the role of GAP-12 for the inhibition of IL-12 p40 promoter activity.

Taken together, our data support a model in which in unstimulated human monocytes GAP-12 binds to the IL-12 p40 promoter, thereby suppressing gene expression. Upon stimulation, GAP-12 is displaced from the promoter, and NF-{kappa}B p50/p65, C/EBP{beta}, and PU.1 promote the induction of IL-12 p40 gene expression. Because LPS stimulation led to suppression of GAP-12 binding activity in RAW264.7 without inducing p40 promoter activity, one can assume that other elements (outside the proximal promoter) must exist that function to prevent LPS alone from driving p40 gene transcription. However, in any case, the GA-12 element is the first regulatory site identified in the IL-12 p40 promoter that functions as a repressor element. Furthermore, this site is critical for IL-4-mediated suppression of promoter activity and thus appears to play an important role in regulating IL-12 p40 gene transcription.


   Acknowledgments
 
We thank E. Schmitt (Institute of Immunology, University of Mainz, Mainz, Germany) for critical reading of the manuscript.


   Footnotes
 
1 This work was supported by grants from the Deutsche Forschungsgemeinschaft (Ne 490/2-1) and the Gerhard-Hess program (Ne 490/3-1) of the Deutsche Forschungsgemeinschaft (to M.F.N.). M.F.N. is the recipient of a Fullbright scholarship for advanced scientists, and C.B. is the recipient of a scholarship from the Graduiertenkolleg of the Deutsche Forschungsgemeinschaft. Back

2 Address correspondence and reprint requests to Dr. Markus F. Neurath, Laboratory of Immunology, First Medical Clinic, University of Mainz, Langenbeckstrasse 1, 55101 Mainz, Germany. E-mail address: neurath{at}1-med.klinik.uni-mainz.de Back

3 Abbreviations used in this paper: GA-12, GATA sequence in the IL-12 promoter; GAP-12, GA-12 binding protein; LM-PCR, ligation-mediated PCR; DMS, dimethyl sulfate; m, mutant. Back

Accepted for publication July 9, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Hsieh, C. S., S. E. Macatonia, C. S. Tripp, S. F. Wolf, A. O’Garra, K. M. Murphy. 1993. Development of TH1 CD4+ T cells through IL-12 produced by Listeria-induced macrophages. Science 260:547.[Abstract/Free Full Text]
  2. Trinchieri, G.. 1993. Interleukin-12 and its role in the generation of TH1 cells. Immunol. Today 14:335.[Medline]
  3. Manetti, R., P. Parronchi, M. G. Giudizi, M. P. Piccinni, E. Maggi, G. Trinchieri, S. Romagnani. 1993. Natural killer cell stimulatory factor (interleukin 12 [IL-12]) induces T helper type 1 (Th1)-specific immune responses and inhibits the development of IL-4-producing Th cells. J. Exp. Med. 177:1199.[Abstract/Free Full Text]
  4. Magram, J., S. E. Connaughton, R. R. Warrier, D. M. Carvajal, C. Y. Wu, J. Ferrante, C. Stewart, U. Sarmiento, D. A. Faherty, M. K. Gately. 1996. IL-12-deficient mice are defective in IFN-{gamma} production and type 1 cytokine responses. Immunity 4:471.[Medline]
  5. Mattner, F., J. Magram, J. Ferrante, P. Launois, P. K. Di, R. Behin, M. K. Gately, J. A. Louis, G. Alber. 1996. Genetically resistant mice lacking interleukin-12 are susceptible to infection with Leishmania major and mount a polarized Th2 cell response. Eur. J. Immunol. 26:1553.[Medline]
  6. Gubler, U., A. O. Chua, D. S. Schoenhaut, C. M. Dwyer, W. McComas, R. Motyka, N. Nabavi, A. G. Wolitzky, P. M. Quinn, P. C. Familletti, et al 1991. Coexpression of two distinct genes is required to generate secreted bioactive cytotoxic lymphocyte maturation factor. Proc. Natl. Acad. Sci. USA 88:4143.[Abstract/Free Full Text]
  7. Wolf, S. F., P. A. Temple, M. Kobayashi, D. Young, M. Dicig, L. Lowe, R. Dzialo, L. Fitz, C. Ferenz, R. M. Hewick, et al 1991. Cloning of cDNA for natural killer cell stimulatory factor, a heterodimeric cytokine with multiple biologic effects on T and natural killer cells. J. Immunol. 146:3074.[Abstract]
  8. Schoenhaut, D. S., A. O. Chua, A. G. Wolitzky, P. M. Quinn, C. M. Dwyer, W. McComas, P. C. Familletti, M. K. Gately, U. Gubler. 1992. Cloning and expression of murine IL-12. J. Immunol. 148:3433.[Abstract]
  9. Gillespie, K. M., C. C. Szeto, V. M. Betin, P. W. Mathieson. 2000. Role of {beta}1 and {beta}2 subunits of the interleukin-12 receptor in determining T helper 1/T helper 2 responses in vivo in the rat. Immunology 99:109.[Medline]
  10. Rogge, L., A. Papi, D. H. Presky, M. Biffi, L. J. Minetti, D. Miotto, C. Agostini, G. Semenzato, L. M. Fabbri, F. Sinigaglia. 1999. Antibodies to the IL-12 receptor {beta}2 chain mark human Th1 but not Th2 cells in vitro and in vivo. J. Immunol. 162:3926.[Abstract/Free Full Text]
  11. Rogge, L., M. L. Barberis, M. Biffi, N. Passini, D. H. Presky, U. Gubler, F. Sinigaglia. 1997. Selective expression of an interleukin-12 receptor component by human T helper 1 cells. J. Exp. Med. 185:825.[Abstract/Free Full Text]
  12. Jacobson, N. G., S. J. Szabo, N. R. Weber, Z. Zhong, R. D. Schreiber, Jr J. E. Darnell, K. M. Murphy. 1995. Interleukin 12 signaling in T helper type 1 (Th1) cells involves tyrosine phosphorylation of signal transducer and activator of transcription (Stat)3 and Stat4. J. Exp. Med. 181:1755.[Abstract/Free Full Text]
  13. Barbulescu, K., C. Becker, J. F. Schlaak, E. Schmitt, K. H. Meyer zum Buschenfelde, M. F. Neurath. 1998. IL-12 and IL-18 differentially regulate the transcriptional activity of the human IFN-{gamma} promoter in primary CD4+ T lymphocytes. J. Immunol. 160:3642.[Abstract/Free Full Text]
  14. Monteleone, G., L. Biancone, R. Marasco, G. Morrone, O. Marasco, F. Luzza, F. Pallone. 1997. Interleukin 12 is expressed and actively released by Crohn’s disease intestinal lamina propria mononuclear cells. Gastroenterology 112:1169.[Medline]
  15. McIntyre, K. W., D. J. Shuster, K. M. Gillooly, R. R. Warrier, S. E. Connaughton, L. B. Hall, L. H. Arp, M. K. Gately, J. Magram. 1996. Reduced incidence and severity of collagen-induced arthritis in interleukin-12-deficient mice. Eur. J. Immunol. 26:2933.[Medline]
  16. Neurath, M. F., I. Fuss, B. L. Kelsall, E. Stuber, W. Strober. 1995. Antibodies to interleukin 12 abrogate established experimental colitis in mice. J. Exp. Med. 182:1281.[Abstract/Free Full Text]
  17. Murphy, T. L., M. G. Cleveland, P. Kulesza, J. Magram, K. M. Murphy. 1995. Regulation of interleukin 12 p40 expression through an NF-{kappa}B half-site. Mol. Cell. Biol. 15:5258.[Abstract]
  18. Plevy, S. E., J. H. Gemberling, S. Hsu, A. J. Dorner, S. T. Smale. 1997. Multiple control elements mediate activation of the murine and human interleukin 12 p40 promoters: evidence of functional synergy between C/EBP and Rel proteins. Mol. Cell. Biol. 17:4572.[Abstract]
  19. Ma, X., G. Gri, G. Trinchieri. 1996. A novel ets-2-related nuclear factor is involved in transcriptional activation of the human interleukin-12 p40 gene promoter in response to interferon-{gamma} and LPS stimulation of monocytic cells. Ann. NY Acad. Sci. 795:357.[Medline]
  20. Ma, X., J. M. Chow, G. Gri, G. Carra, F. Gerosa, S. F. Wolf, R. Dzialo, G. Trinchieri. 1996. The interleukin 12 p40 gene promoter is primed by interferon-{gamma} in monocytic cells. J. Exp. Med. 183:147.[Abstract/Free Full Text]
  21. D’Andrea, A., X. Ma, A. M. Aste, C. Paganin, G. Trinchieri. 1995. Stimulatory and inhibitory effects of interleukin (IL)-4 and IL-13 on the production of cytokines by human peripheral blood mononuclear cells: priming for IL-12 and tumor necrosis factor {alpha} production. J. Exp. Med. 181:537.[Abstract/Free Full Text]
  22. Koch, F., U. Stanzl, P. Jennewein, K. Janke, C. Heufler, E. Kampgen, N. Romani, G. Schuler. 1996. High level IL-12 production by murine dendritic cells: upregulation via MHC class II and CD40 molecules and downregulation by IL-4 and IL-10. [Published erratum appears in 1996 J. Exp. Med. 184:1590.]. J. Exp. Med. 184:741.[Abstract/Free Full Text]
  23. Levings, M. K., J. W. Schrader. 1999. IL-4 inhibits the production of TNF-{alpha} and IL-12 by STAT6-dependent and -independent mechanisms. J. Immunol. 162:5224.[Abstract/Free Full Text]
  24. Monteleone, G., T. Parrello, I. Monteleone, S. Tammaro, F. Luzza, F. Pallone. 1999. Interferon-{gamma} (IFN-{gamma}) and prostaglandin E2 (PGE2) regulate differently IL-12 production in human intestinal lamina propria mononuclear cells (LPMC). Clin. Exp. Immunol. 117:469.[Medline]
  25. van-der-Pouw, K. T., L. C. Boeije, R. J. Smeenk, J. Wijdenes, L. A. Aarden. 1995. Prostaglandin-E2 is a potent inhibitor of human interleukin 12 production. J. Exp. Med. 181:775.[Abstract/Free Full Text]
  26. Nordeen, S. K.. 1988. Luciferase reporter gene vectors for analysis of promoters and enhancers. BioTechniques 6:454.[Medline]
  27. Trautwein, C., C. Caelles, G. P. van-der, T. Hunter, M. Karin, M. Chojkier. 1993. Transactivation by NF-IL6/LAP is enhanced by phosphorylation of its activation domain. Nature 364:544.[Medline]
  28. Hu, H. M., M. Baer, S. C. Williams, P. F. Johnson, R. C. Schwartz. 1998. Redundancy of C/EBP{alpha}, -{beta}, and -{delta} in supporting the lipopolysaccharide-induced transcription of IL-6 and monocyte chemoattractant protein-1. J. Immunol. 160:2334.[Abstract/Free Full Text]
  29. Garrity, P. A., B. J. Wold. 1992. Effects of different DNA polymerases in ligation-mediated PCR: enhanced genomic sequencing and in vivo footprinting. Proc. Natl. Acad. Sci. USA 89:1021.[Abstract/Free Full Text]
  30. Mueller, P. R., B. Wold. 1989. In vivo footprinting of a muscle specific enhancer by ligation mediated PCR. Science 246:780.[Abstract/Free Full Text]
  31. Becker, C., K. Barbulescu, S. Wirtz, K. H. Meyer zum Büschenfelde, S. Pettersson, M. F. Neurath. 1999. Constitutive and inducible in vivo protein-DNA interactions at the tumor necrosis factor-{alpha} promoter in primary human T lymphocytes. Gene Expression 8:115.[Medline]
  32. Schreiber, E., P. Matthias, M. M. Muller, W. Schaffner. 1989. Rapid detection of octamer binding proteins with "mini-extracts," prepared from a small number of cells. Nucleic Acids Res. 17:6419.[Free Full Text]
  33. Meyer, K. B., M. Skogberg, C. Margenfeld, J. Ireland, S. Pettersson. 1995. Repression of the immunoglobulin heavy chain 3' enhancer by helix-loop-helix protein Id3 via a functionally important E47/E12 binding site: implications for developmental control of enhancer function. Eur. J. Immunol. 25:1770.[Medline]
  34. Neurath, M. F., S. Pettersson, K. H. Meyer zum Büschenfelde, W. Strober. 1996. Local administration of antisense phosphorothioate oligonucleotides to the p65 subunit of NF-{kappa}B abrogates established experimental colitis in mice. Nat. Med. 2:998.[Medline]
  35. Weinmann, A. S., S. E. Plevy, S. T. Smale. 1999. Rapid and selective remodeling of a positioned nucleosome during the induction of IL-12 p40 transcription. Immunity 11:665.[Medline]
  36. Kobayashi, M., L. Fitz, M. Ryan, R. M. Hewick, S. C. Clark, S. Chan, R. Loudon, F. Sherman, B. Perussia, G. Trinchieri. 1989. Identification and purification of natural killer cell stimulatory factor (NKSF), a cytokine with multiple biologic effects on human lymphocytes. J. Exp. Med. 170:827.[Abstract/Free Full Text]
  37. Benjamin, D., V. Sharma, M. Kubin, J. L. Klein, A. Sartori, J. Holliday, G. Trinchieri. 1996. IL-12 expression in AIDS-related lymphoma B cell lines. J. Immunol. 156:1626.[Abstract]
  38. Yoshimoto, T., H. Nagase, T. Yoneto, J. Inoue, H. Nariuchi. 1998. Interleukin-12 expression in B cells by transformation with Epstein-Barr virus. Biochem. Biophys. Res. Commun. 252:556.[Medline]
  39. Cowdery, J. S., N. J. Boerth, L. A. Norian, P. S. Myung, G. A. Koretzky. 1999. Differential regulation of the IL-12 p40 promoter and of p40 secretion by CpG DNA and lipopolysaccharide. J. Immunol. 162:6770.[Abstract/Free Full Text]
  40. Gri, G., D. Savio, G. Trinchieri, X. Ma. 1998. Synergistic regulation of the human interleukin-12 p40 promoter by NF{kappa}B and Ets transcription factors in Epstein-Barr virus-transformed B cells and macrophages. J. Biol. Chem. 273:6431.[Abstract/Free Full Text]
  41. Ma, X., M. Neurath, G. Gri, G. Trinchieri. 1997. Identification and characterization of a novel Ets-2-related nuclear complex implicated in the activation of the human interleukin-12 p40 gene promoter. J. Biol. Chem. 272:10389.[Abstract/Free Full Text]
  42. Yoshimoto, T., H. Nagase, T. Ishida, J. Inoue, H. Nariuchi. 1997. Induction of interleukin-12 p40 transcript by CD40 ligation via activation of nuclear factor-kappaB. Eur. J. Immunol. 27:3461.[Medline]
  43. An, M. R., C. C. Hsieh, P. D. Reisner, J. P. Rabek, S. G. Scott, D. T. Kuninger, J. Papaconstantinou. 1996. Evidence for posttranscriptional regulation of C/EBP{alpha} and C/EBP{beta} isoform expression during the lipopolysaccharide-mediated acute-phase response. Mol. Cell. Biol. 16:2295.[Abstract]
  44. Mahoney, C. W., J. Shuman, S. L. McKnight, H. C. Chen, K. P. Huang. 1992. Phosphorylation of CCAAT-enhancer binding protein by protein kinase C attenuates site-selective DNA binding. J. Biol. Chem. 267:19396.[Abstract/Free Full Text]
  45. Nakajima, T., S. Kinoshita, T. Sasagawa, K. Sasaki, M. Naruto, T. Kishimoto, S. Akira. 1993. Phosphorylation at threonine-235 by a ras-dependent mitogen-activated protein kinase cascade is essential for transcription factor NF-IL6. Proc. Natl. Acad. Sci. USA 90:2207.[Abstract/Free Full Text]
  46. Wegner, M., Z. Cao, M. G. Rosenfeld. 1992. Calcium-regulated phosphorylation within the leucine zipper of C/EBP{beta}. Science 256:370.[Abstract/Free Full Text]
  47. Matsusaka, T., K. Fujikawa, Y. Nishio, N. Mukaida, K. Matsushima, T. Kishimoto, S. Akira. 1993. Transcription factors NF-IL6 and NF-{kappa}B synergistically activate transcription of the inflammatory cytokines, interleukin 6 and interleukin 8. Proc. Natl. Acad. Sci. USA 90:10193.[Abstract/Free Full Text]
  48. Stein, B., J. Baldwin-AS. 1993. Distinct mechanisms for regulation of the interleukin-8 gene involve synergism and cooperativity between C/EBP and NF-{kappa}B. Mol. Cell. Biol. 13:7191.[Abstract/Free Full Text]
  49. Natsuka, S., S. Akira, Y. Nishio, S. Hashimoto, T. Sugita, H. Isshiki, T. Kishimoto. 1992. Macrophage differentiation-specific expression of NF-IL6, a transcription factor for interleukin-6. Blood 79:460.[Abstract/Free Full Text]
  50. Scott, L. M., C. I. Civin, P. Rorth, A. D. Friedman. 1992. A novel temporal expression pattern of three C/EBP family members in differentiating myelomonocytic cells. Blood 80:1725.[Abstract/Free Full Text]
  51. Screpanti, I., L. Romani, P. Musiani, A. Modesti, E. Fattori, D. Lazzaro, C. Sellitto, S. Scarpa, D. Bellavia, G. Lattanzio, et al 1995. Lymphoproliferative disorder and imbalanced T-helper response in C/EBP{beta}-deficient mice. EMBO J. 14:1932.[Medline]
  52. Lodie, T. A., R. Savedra, D. T. Golenbock, B. C. Van, R. A. Maki, M. J. Fenton. 1997. Stimulation of macrophages by lipopolysaccharide alters the phosphorylation state, conformation, and function of PU.1 via activation of casein kinase II. J. Immunol. 158:1848.[Abstract]
  53. Pongubala, J. M., B. C. Van, S. Nagulapalli, M. J. Klemsz, S. R. McKercher, R. A. Maki, M. L. Atchison. 1993. Effect of PU.1 phosphorylation on interaction with NF-EM5 and transcriptional activation. Science 259:1622.[Abstract/Free Full Text]
  54. Lee, M. E., D. H. Temizer, J. A. Clifford, T. Quertermous. 1991. Cloning of the GATA-binding protein that regulates endothelin-1 gene expression in endothelial cells. J. Biol. Chem. 266:16188.[Abstract/Free Full Text]
  55. Voso, M. T., T. C. Burn, G. Wulf, B. Lim, G. Leone, D. G. Tenen. 1994. Inhibition of hematopoiesis by competitive binding of transcription factor PU.1. Proc. Natl. Acad. Sci. USA 91:7932.[Abstract/Free Full Text]
  56. Tenen, D. G., R. Hromas, J. D. Licht, D. E. Zhang. 1997. Transcription factors, normal myeloid development, and leukemia. Blood 90:489.[Free Full Text]
  57. Chen, H., G. D. Ray, P. Zhang, C. J. Hetherington, D. A. Gonzalez, D. E. Zhang, G. F. Moreau, D. G. Tenen. 1995. PU.1 (Spi-1) autoregulates its expression in myeloid cells. Oncogene 11:1549.[Medline]



This article has been cited by other articles:


Home page
 J. Immunol.