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The Journal of Immunology, 2000, 165: 271-279.
Copyright © 2000 by The American Association of Immunologists

An IFN-{gamma}-Inducible Transcription Factor, IFN Consensus Sequence Binding Protein (ICSBP), Stimulates IL-12 p40 Expression in Macrophages

I-Ming Wang1,*, Cristina Contursi*, Atsuko Masumi2,*, Xiaojing Ma{dagger}, Giorgio Trinchieri{dagger} and Keiko Ozato3,*

* Laboratory of Molecular Growth Regulation, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892; and {dagger} Wistar Institute of Anatomy and Biology, Philadelphia, PA 19104


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
IL-12 is a cytokine that links innate and adaptive immunity. Its subunit p40 is induced in macrophages following IFN-{gamma}/LPS stimulation. Here we studied the role for IFN consensus sequence binding protein (ICSBP), an IFN-{gamma}/LPS-inducible transcription factor of the IFN regulatory factor (IRF) family in IL-12 p40 transcription. Macrophage-like cells established from ICSBP-/- mice did not induce IL-12 p40 transcripts, nor stimulated IL-12 p40 promoter activity after IFN-{gamma}/LPS stimulation, although induction of other inducible genes was normal in these cells. Transfection of ICSBP led to a marked induction of both human and mouse IL-12 p40 promoter activities in ICSBP+/+ and ICSBP-/- cells, even in the absence of IFN-{gamma}/LPS stimulation. Whereas IRF-1 alone was without effect, synergistic enhancement of promoter activity was observed following cotransfection of ICSBP and IRF-1. Deletion analysis of the human promoter indicated that the Ets site, known to be important for activation by IFN-{gamma}/LPS, also plays a role in the ICSBP activation of IL-12 p40. A DNA affinity binding assay revealed that endogenous ICSBP is recruited to the Ets site through protein-protein interaction. Last, transfection of ISCBP alone led to induction of the endogenous IL-12 p40 mRNA in the absence of IFN-{gamma} and LPS. Taken together, our results show that ICSBP induced by IFN-{gamma}/LPS, acts as a principal activator of IL-12p40 transcription in macrophages.


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Interleukin-12 is a cytokine that governs production of IFN-{gamma} in NK cells and CD4+ T cells. It is required for resistance against various infectious agents including viruses, bacteria, and parasites. The cytokine also promotes differentiation of the Th1 type of Th cells, thereby coupling innate and adaptive immunity (1, 2, 3, 4). Many additional studies showed that IL-12 stimulates T cell-dependent and -independent macrophage activation, affects autoimmune processes, and exerts anti-tumor activity (5, 6).

IL-12 is composed of a heterodimer of the p35 and p40 subunits. Whereas IL-12 p35 expression is constitutive and ubiquitous, IL-12 p40 is expressed specifically in macrophages/dendritic cells as well as B cells and neutrophiles (1). Further, IL-12 p40 is strongly induced by pathogens and their components, such as bacterial LPS (7, 8, 9). Although it does not induce IL-12 by itself, IFN-{gamma} is a powerful potentiator of IL-12 induction and is thought to provide a basis for a positive feedback regulation of the cytokine (10, 11). Consistent with this, IFN-{gamma} exerts a potent priming effect on IL-12 p40 induction in cultured macrophages stimulated with LPS. IL-12 p40 is also induced by a T cell dependent mechanism, through the CD40 ligand-CD40 interaction (12, 13).

For its preeminent role in host defense and implications for clinical use, regulation of IL-12 expression has been extensively investigated in the past, many of which focusing on the inducible IL-12 p40 gene. It has been shown that IL-12 p40 induction is regulated at the level of transcription, which requires new protein synthesis (10, 14).

Several laboratories have studied transcriptional regulation of IL-12 p40. Studying the mouse promoter, Murphy et al. reported that the Rel/NF-{kappa}B site is important for promoter activity stimulated by IFN-{gamma} and LPS (15). A later study by Plevy et al. indicated the role for the C/EBP site, located downstream from the Rel/NF-{kappa}B site, which is shown to cooperate with the Rel/NF-{kappa}B site (16). In contrast, Ma et al., studying the human promoter, showed that the Ets site residing upstream from the Rel/NF-{kappa}B site is critical for promoter stimulation by IFN-{gamma} and LPS (10). These authors showed that a sequence containing the Ets site binds to an inducible protein complex containing multiple factors (17).

A separate series of investigations using knockout mice indicates that proteins belonging to the IFN regulatory factor (IRF)4 family play a significant role in regulating IL-12 expression. IFN consensus sequence binding protein (ICSBP), an immune cell-specific member (18, 19, 20), has attracted special interests in this regard. Like IL-12 p40, ICSBP is induced in macrophages by IFN-{gamma} and LPS in a synergistic manner (this study and Ref. 21). This induction is most likely a consequence of STAT1 activation, which leads to stimulation of ICSBP promoter activity through the IFN-{gamma} activation site (GAS) element (21). ICSBP in turn potentiates STAT1-dependent activation of IFN {gamma}-responsive promoters through the GAS element. Furthermore, ICSBP negatively regulates transcription of IFN-responsive genes through the IFN-stimulated response element (ISRE) sequence (22). Further, ICSBP-/- mice express a very low level of constitutive IL-12 p40 transcripts and are unable to induce the transcripts in response to infections (23, 24). Accordingly, ICSBP-/- mice are highly susceptible to infection by Listeria monocytogenes, Toxoplasma gondii, Leishmania major, and lymphocytic choriomeningitis virus and vaccinia viruses (23, 24, 25, 26). The study performed with the Toxoplasma model attributed this susceptibility to a specific defect in macrophages to induce IL-12 p40 mRNA (24). Recently, Wu et al. showed that ICSBP-/- APCs are unresponsive to the stimulation by CD40/CD40 ligand interactions, indicating that T cell-dependent induction of IL-12 p40 also requires ICSBP (27), consistent with an earlier work (26). Supporting a role for the IRF family, mice lacking IRF-1 are also deficient in IL-12 production and do not efficiently develop Th1 immune responses (28, 29).

The present work began with the observation that ICSBP-/- macrophage-like cells established from the knockout mice display a selective defect in IL-12 p40 mRNA induction. Supporting the view that ICSBP is an activator of IL-12 p40 transcription, we show that transfection of ICSBP markedly enhances IL-12 p40 promoter activity both in ICSBP-/- and ICSBP+/+ macrophages, for which neither IFN-{gamma} nor LPS was required. In addition, although IRF-1 alone was unable to stimulate promoter activity, cotransfection of IRF-1 and ICSBP led to a greater promoter stimulation, indicating that ICSBP and IRF-1 cooperate to induce IL-12 p40 transcription. Further, ICSBP activation of human IL-12 p40 promoter activity was, at least partly, mediated by the Ets site, to which multiple factors including ICSBP and IRF-1 are recruited. We suggest that ICSBP generates an IFN-{gamma}/LPS-inducible transcription pathway that selectively activates the IL-12 p40 gene in macrophages.


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

The murine macrophage-like RAW 264.7 cells (RAW cells hereafter) (10) were maintained in RPMI 1640 supplemented with glutamine, antibiotics, and 10% FBS (Atlanta Biologicals, Norcross, GA; endotoxin <1 ng/ml). CL-2 cells were established from bone marrow cells obtained from ICSBP-/- mice following coincubation with the murine J2 retrovirus, harboring v-raf and v-myc genes (30) (kindly provided by G. Gusella, National Cancer Institute-Frederick Cancer Research Facility, Frederick, MD). Cells were cloned by limiting dilution and maintained in the same media as above, supplemented with recombinant mouse M-CSF (6 ng/ml, R&D Systems, Minneapolis, MN) and recombinant GM-CSF (6 ng/ml, Peprotech, Rocky Hill, NJ).

Flow cytometry analysis

Flow cytometry analysis was performed with untreated CL-2 cells stained with PE-labeled Ly6G (GR-1) and FITC-labeled CD11b (Mac-1) in the presence of a blocking Ab for CD16/CD32 (Fc{gamma}R) (all obtained from PharMingen, San Diego, CA). For induction of MHC class I and class II Ags expression, CL-2 cells were treated with IFN-{gamma} (200 U/ml) for 24 h and cells were stained with FITC-labeled anti-H-2Kb or PE-labeled anti-IAb Ab (PharMingen).

RNase protection assay

RAW and CL-2 cells were first incubate with 1.2% DMSO for 18 h and then pretreated with IFN-{gamma} for 8 h followed by treatment with LPS plus IFN-{gamma} for an additional 8 h (10). Five micrograms of total RNA was subjected to multiprobe RNase protection kit using the cytokine/chemokine template set (mck-2) (PharMingen) as a template. Riboprobes were synthesized with the T7 RNA polymerase and annealed with RNA overnight at 55°C in 0.3 M NaCl. The reactions were digested with 40 µg/ml of RNase A and 2 µg/ml of RNase T1 in buffer containing 10 mM Tris-HCl, pH 7.5, 5 mM EDTA, 0.3 M NaCl for 1 h at 30°C. Samples were purified by phenol-chloroform and resolved in 6% PAGE. Details of RNase protection assays were described (24).

RT-PCR

Quantitative PCR was performed as previously described (22). cDNAs were prepared from 1 µg of total RNA using Superscript (Life Technologies, Gaithersburg, MD). Serially diluted cDNA was subjected to PCR by using appropriate primers: mIL-12 p40, 5'-ATGGCCATGTGGGAGCTGGAG-3' and 5'-TTTGGTGCTTCACACTTCAGG-3'; mHPRT, 5'-GTTGGATACAGGCCAGACTTTGTTG-3' and 5'-GAGGGTAGGCTGGCCTATGGCT-3'. PCRs (35 cycles) were performed and the products were fractionated on a 1.5% agarose gel.

Plasmids

Human IL-12 p40 promoter fragments cloned in pXP2 were described (10, 17) A 350-bp and a 40-bp PCR fragment generated from the promoter region of murine IL-12 gene (15, 16, 31) was cloned into the pGL2 luciferase vector (Promega, Madison, WI). Deletion constructs were generated by PCR and sequenced by the dideoxy method. Expression vectors for ICSBP (pU5), IRF-1 (pACT-1), and IRF-2 (pACT-2) under the control of the ß-actin promoter were described (18, 32). All plasmids used for transfection assays were prepared by CsCl double banding to minimize endotoxin contamination.

Transfection assay

RAW or CL-2 cells (107 cells) were transfected with 10 µg of luciferase reporter or indicated amounts of expression vectors (up to 20 µg DNA) by electroporation in Cell Porter (Life Technologies, Grand Island, NY) at the setting of 300 V, 800 µF (for RAW cells) or 250 V, 1180 µF (CL-2 cells), each for 2 s. The amount of transfected DNA was adjusted with LK440. Cells were incubated with the complete medium for 6 h, then stimulated with murine recombinant IFN-{gamma} (100–1000 U/ml, a gift from Dr. G. Adolf, Boehringer Ingelheim, Bender, Austria) for 10 h followed by further incubation with IFN-{gamma} and LPS (from Escherichia coli, 1 µg/ml, Sigma, St. Louis, MO) for an additional 8 h (10). For the expression of endogenous IL-12 p40, transfected cells were incubated in the medium containing 1.2% DMSO for 6 h before treatment with IFN-{gamma}/LPS. Luciferase activities were normalized by protein concentrations. Unless otherwise indicated, reporter activity is shown as the average of three determinations ± SD.

DNA affinity binding assay

Biotinylated DNA fragments encompassing the IL-12 p40 Ets site (-292 to -196) (17) were synthesized from the 3.3-kb wild-type human IL-12 p40 reporter or the Ets mutant (3.3 kb {Delta}Ets, see Fig. 4GoA) by PCR using a biotinylated primer as detailed in (33). PCR products were purified by the Qiaquick Kit (Qiagen, Chatsworth, CA). Two micrograms of biotinylated DNA were conjugated to 100 µl of streptavidin-bound magnetic beads (Dynabeads, M280, Dynal, Lake Success, NY) in buffer containing 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.1 M NaCl. Conjugation of the ISRE fragment to magnetic beads was described previously (34). Ten microliters of beads conjugated to 2 µg of DNA were equilibrated with TGEDN buffer (120 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.1 M NaCl, 1 mM DTT, 0.1% Triton X-100, 10% glycerol) and incubated with 500 µg of RAW cell nuclear extracts and 20 µg of Herring sperm DNA (Sigma) at 4°C for 2 h. Beads were washed in TGEDN buffer, and bound materials were eluted in 20 µl of the same buffer supplemented with 0.5% SDS and 1 M NaCl. Eluted materials were separated by 10% SDS-PAGE and detected by immunoblot analysis using rabbit anti-ICSBP or anti-IRF-1 Ab (19) with the enhanced chemiluminescence kit (Amersham). Nuclear extracts were prepared from RAW cells treated with LPS (1 µg/ml) or IFN-{gamma} (500 U/ml) for 8 h or treated first with IFN-{gamma} for 8 h followed by incubation with LPS plus IFN-{gamma} for an additional 8 h as described (34). Recombinant ICSBP, IRF-1, or IRF-2 produced from baculovirus vectors were described (34, 35).



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  		FIGURE 4. Comparison of IRF members for the ability to stimulate IL-12 p40 promoter activity. RAW cells (A and B) or CL-2 cells (C and D) were transfected with 10 µg of 0.243-kb human IL-12 p40 reporter or GBP-ISRE reporter, along with 20 µg of IRF-1, IRF-2, ICSBP, or empty vector and treated with IFN-{gamma}, LPS, or both as in Fig. 2Go. E, Synergistic stimulation of IL-12 p40 promoter activity by IRF-1 and ICSBP. RAW cells were transfected with 10 µg of 0.243-kb human IL-12 p40 reporter along with10 µg of IRF-1, IRF-2, or 5 µg of ICSBP (lanes 1–4) alone or a combination of 5 µg of ICSBP plus 5 or 10 µg of IRF-1 (lanes 5 and 6) or 5 µg of ICSBP plus 5 or 10 µg of IRF-2 (lanes 7 and 8) as in Fig. 2Go.

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
ICSBP-/- macrophage-like cells fail to induce IL-12 p40 transcripts: the selectivity of the failure

Several cell lines were established from ICSBP-/- bone marrow cells following transformation with the J2 retrovirus (30). Among them, a clone designated CL-2 expressed Mac-1 (CD11b), but not GR-1 (Ly-6G), a feature shared with macrophages in vivo (Fig. 1GoA). The ability of CL-2 cells to induce IL-12 p40 mRNA by IFN-{gamma} and LPS stimulation was tested, along with a ICSBP+/+ macrophage cell line RAW264.7 (RAW), used as a control. Cells were pretreated with IFN-{gamma} (which provides a proper priming effect), followed by the subsequent treatment with IFN-{gamma} plus LPS (10, 11). Results of RNase protection assays are shown in Fig. 1GoC. IL-12 p40 transcripts were induced in RAW cells when treated with both IFN-{gamma} and LPS, although not with either stimulus alone. In contrast, no IL-12 p40 mRNA induction was seen in CL-2 cells. Contrary to the lack of IL-12p40 induction, CL-2 cells induced IL-1{alpha}, IL- 1ß, as well as IL-1 receptor antagonist mRNAs (36, 37) after IFN-{gamma} and LPS stimulation, often more robustly than RAW cells. While IL-1 receptor antagonist was induced by IFN alone, IL-1{alpha} and IL-1ß transcripts were induced by LPS and IFN-{gamma}/LPS. These results are similar to those observed with fresh peritoneal macrophages of ICSBP-/- mice (24) and indicate that early signaling pathways activated by IFN-{gamma} and LPS are largely intact, but the capacity to induce IL-12 p40 mRNA is selectively lost in ICSBP-/- cells.



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  		FIGURE 1. A and B, Flow cytometry analysis of CL-2 cells. A, Expression of Mac-1/GR-1. CL-2 cells were stained with control, anti-Mac-1, or anti-GR-1 Ab. B, Induction of MHC class I and class II expression by IFN-{gamma}. CL-2 cells were treated with IFN-{gamma} (200 U/ml) for 24 h and stained with FITC labeled anti-Kb or PE-labeled anti-IAb Ab. C, RNase protection assay for IL-12 p40 mRNA expression. RAW and CL-2 Cells were first treated with IFN-{gamma} for 8 h and then with IFN-{gamma} plus LPS (IFN-{gamma}/LPS) for an additional 8 h. Multiprobe RNase protection analysis was performed with 5 µg of total RNA using the mouse cytokine/chemokine template set. IL-1Ra, IL-1 receptor antagonist.

 
To further investigate the ability of CL-2 cells to respond to IFN-{gamma}, cell-surface expression of MHC class I and class II Ags was examined. Flow cytometry results in Fig. 1GoB show that both H-2Kb and I-Ab Ags are induced in CL-2 cells upon IFN-{gamma} treatment, confirming that IFN-{gamma} signaling is functional in CL-2 cells.

Transfection of ICSBP leads to activation of IL-12 p40 promoter activity

Analysis of the human promoter. To assess the role for ICSBP in IL-12 p40 transcription, transient tranfection analysis was performed using a luciferase reporter connected to 243 bp of the human p40 promoter (10) (see Fig. 6GoA for diagram). RAW and CL-2 cells transfected with the reporter were then stimulated with IFN-{gamma}, LPS alone, or IFN-{gamma}/LPS. As seen in Fig. 2GoA, lanes 1–4, treatment of RAW cells with IFN-{gamma}/LPS enhanced IL-12 p40 promoter activity by ~6-fold. However, treatment with IFN-{gamma} or LPS alone did not significantly enhance promoter activity, in line with the requirement of IFN-{gamma}/LPS stimulation for IL-12 p40 induction in vivo (Fig. 1GoC). In contrast, promoter activity remained at a background level in CL-2 cells after stimulation by the two agents. The absence of IL-12 p40 promoter stimulation in CL-2 cells was not due to the general absence of IFN-{gamma} responsiveness, because activity of IFN-{gamma} binding protein (GBP)-ISRE reporter, known to be stimulated by IFN-{gamma} (35), was enhanced in CL-2 as well as RAW cells by IFN-{gamma} (lanes 9 and 10). Although some luciferase promoters are reported to be nonspecifically stimulated in some RAW cells (16), we did not observe significant stimulation of PGL2, PGL3, or a basal IL-12 p40 promoter by IFN-{gamma}/LPS treatment under these conditions (Fig. 3Go and our unpublished observations, see also Fig. 6Go).



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  		FIGURE 6. DNA affinity binding assay. A, Diagram of assay. A total of 200 ng of biotinylated DNA containing the Ets site (-292 to -196) or DNA lacking the core Ets element ({Delta}Ets -207 to -212, Fig. 4GoA) were conjugated to 10 µl of magnetic beads and incubated with 500 µg of nuclear extracts from RAW cells. Beads were washed, and bound materials were eluted and detected by immunoblot assays. B, Wild-type Ets DNA conjugated to the beads were incubated with nuclear extracts from RAW cells treated with IFN-{gamma}, LPS for 16 h, or sequentially with IFN-{gamma} and IFN-{gamma} plus LPS. Unprocessed extracts (input, 10 µg) or bound materials were immunoblotted with Abs for ICSBP, TFIIB, or STAT1. C, Wild-type and mutant Ets DNA conjugated to the beads were incubated with extracts as above, and bound materials were tested with Abs for ICSBP and IRF-1. Control is input nuclear extract from cells stimulated with IFN-{gamma} /LPS. D, A total of 200 ng of biotinylated ISRE or wild-type Ets conjugated to the beads were incubated with 50 or 100 ng of rICSBP with or without 100 ng of recombinant IRF-1 or IRF-2, and bound ICSBP was detected by anti-ICSBP Ab. E, The wild-type Ets conjugated to beads were incubated with 10 ng of rICSBP alone (lane 1) or mixed with 500 µg of nuclear extracts from RAW cells (lanes 2 and 3) or nuclear extracts alone (lanes 4 and 5), and bound materials were detected by Ab for ICSBP. Due to the his-tag, rICSBP migrated above the position of the endogenous ICSBP.

 


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  		FIGURE 2. Stimulation of human IL-12 p40 promoter activity by ICSBP. A, RAW or CL-2 cells (107) were transfected with 10 µg of 0.243-kb human (h) IL-12 p40 luciferase reporter or GBP-ISRE luciferase reporter along with 20 µg of control empty vector (C) or ICSBP expression vector. Six hours after transfection, cells were treated with IFN-{gamma} or LPS alone for 16 h, or first with IFN-{gamma}, then with IFN-{gamma} plus LPS for 8 h each (as described in Materials and Methods). Values represent the average of three assays ± SD. B, ICSBP dose dependence. RAW cells and CL-2 cells were transfected with 0.243-kb hIL-12 p40 reporter, along with 1.5, 2.5, 5,10, or 20 µg of ICSBP plasmid. The amount of transfected DNA was adjusted to 30 µg using control vector. Cells were incubated with medium alone or treated IFN-{gamma} and LPS as in A.

 


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  		FIGURE 3. Stimulation of murine (m) IL-12 p40 promoter activity by ICSBP. RAW and CL-2 cells were transfected with 350-bp mIL-12 p40 luciferase reporter (350) or 40-bp mIL-12 p40 reporter (Basal), along with control vector (C) or ICSBP vector as in Fig. 2Go.

 
IL-12 p40 promoter activity was then tested following cotransfection of ICSBP. ICSBP markedly enhanced IL-12 p40 promoter activity both in RAW and CL-2 cells even in the absence of IFN-{gamma}/LPS stimulation (Fig. 2GoA, lane 5). In RAW cells, promoter activity was further enhanced when cells were treated with IFN-{gamma}/LPS (lanes 6 and 7), with the greatest promoter activity seen when cells were transfected with ICSBP and treated with IFN-{gamma}/LPS (lane 8). In either case, IL-12 p40 promoter activity was not stimulated when cells were cotransfected with the control vector without insert. In contrast, treatment with IFN-{gamma}/LPS did not change the level of promoter activity in CL-2 cells after transfection with ICSBP. These results indicate that ICSBP itself can activate IL-12 p40 promoter activity.

Results in Fig. 2GoB show that IL-12 p40 promoter activation is ICSBP dose dependent in both CL-2 and RAW cells. At each ICSBP dose tested, treatment with IFN-{gamma}/LPS further increased promoter activity in RAW cells, but not in CL-2 cells (see Discussion).

Analysis of mouse IL-12 p40 promoter activity. To further substantiate ICSBP stimulation of IL-12 p40 promoter activity, a luciferase reporter containing a 350-bp murine IL-12 p40 promoter was tested in cotransfection assays. As seen in Fig. 3Go (left panel), activity of the murine promoter was strongly enhanced by ICSBP in both cell types, again without requiring IFN-{gamma}/LPS stimulation. Similar to human promoter activity, treatment with IFN-{gamma}/LPS markedly enhanced murine promoter activity in RAW cells but not in CL-2 cells. A control reporter containing a minimum promoter region of the murine IL-12 p40 gene was not stimulated by ICSBP in either cells (Fig. 3Go, right panel).

Specificity of ICSBP activation

It was of importance to determine whether other members of the IRF family are capable of stimulating IL-12 p40 promoter activity. In light of a defect in IL-12 production reported for IRF-1-/- mice (28, 29), we were interested in testing the activity of IRF-1. We also felt it important to test IRF-2, because ICSBP and IRF-2 repress transcription from IFN-{alpha}ß-inducible promoters in a similar manner, suggesting a shared function by the two factors (18, 38). Results are shown in Fig. 4Go. Whereas ICSBP led to IL-12 p40 promoter stimulation both in CL-2 and in RAW cells, neither IRF-1 nor IRF-2 stimulated promoter activity in the presence or absence of IFN-{gamma}, LPS, or IFN-{gamma}/LPS in CL-2 cells (Fig. 4GoC). In RAW cells, IRF-1 also stimulated IL-12 p40 promoter activity, which might be due to a cooperation with ICSBP (see Fig. 4GoE). Instead, cotransfection of IRF-1 or IRF-2 led to a slight decrease in promoter activation in RAW cells after stimulation by IFN-{gamma}/LPS. The basis for this reduction has not been studied (Fig. 4GoA). Although unable to enhance IL-12 p40 promoter activity, IRF-1 did enhance activity of the GBP-ISRE reporter in the presence and absence of IFN-{gamma}, confirming the activator function of this factor (Fig. 4Go, B and D, lanes 3 and 4) (39, 40). As seen in lanes 1 and 2 of Fig. 4Go, B and D, GBP-ISRE promoter activity was stimulated by IFN-{gamma}, but this stimulation was repressed by cotransfection of IRF-2 or ICSBP both in RAW and CL-2 cells (lanes 5–8), as would have been expected of their repressor function (18). These results show that ICSBP, but not IRF-1 or IRF-2, enhances IL-12 p40 promoter activity.

Synergistic activation of IL-12 p40 transcription by ICSBP and IRF-1

To further evaluate the potential role for IRF-1, we examined whether it could cooperate with ICSBP in stimulating IL-12 p40 promoter activity. In experiments shown in Fig. 4GoE, RAW cells were cotransfected with a suboptimal amount of ICSBP in combination with IRF-1, or IRF-2 along with a IL-12 p40 reporter. Whereas ICSBP alone led to only a modest level of stimulation, cotransfection of IRF-1 and ICSBP led to a much greater level of stimulation, reaching almost 10-fold higher promoter activity than that by ICSBP alone (lanes 5 and 6 vs lane 4). In contrast, cotransfection of IRF-2 and ICSBP gave no stimulation, but rather it appeared to slightly reduce promoter activation by ICSBP (lanes 7 and 8). These results indicate that ICSBP and IRF-1 cooperatively enhance IL-12 p40 promoter activity, although IRF-1, by itself, does not function as an activator. Similar cooperative stimulation was seen with the 350-bp murine Il-12 p40 promoter (not shown).

The Ets site contributes to ICSBP stimulation of human IL-12 p40 promoter activity

Various deletion reporters constructed from the human IL-12 p40 promoter were tested to assess cis elements through which ICSBP stimulates transcription (Fig. 5GoA). These included the longest 3.3-kb reporter, the shortest reporter with the minimum promoter region (TATA in Fig. 5Go), and {Delta}Ets from which the 5-bp Ets core was deleted from 3.3-kb promoter. RAW or CL-2 cells were transfected with ICSBP or a control vector and treated with or without IFN-{gamma}, LPS alone, or both (Fig. 5Go, B and C). Reporters containing promoters longer than the 0.222-kb were strongly stimulated by ICSBP both in RAW and CL-2 cells, with the exception of {Delta}Ets. These reporters were also stimulated by treatment with IFN-{gamma}/LPS when tested in RAW cells (but not in CL-2 cells). In contrast, reporters with a fragment shorter than 0.204-kb promoter were not significantly stimulated by ICSBP, nor by IFN-{gamma}/LPS treatment. In agreement with these data, additional reporters containing the 1.2-kb, 0.6-kb, or 0.265-kb fragment, but not the 0.122-kb fragment, were stimulated by ICSBP (not shown). These results indicate that the Ets element, previously shown to be critical for IFN-{gamma}/LPS stimulation of promoter activity (10), plays a significant role in mediating ICSBP activation as well. However, we noted that the 0.204-kb and {Delta}Ets reporters gave slightly higher promoter activities than the 0.122-kb and TATA reporter, when cotransfected with ICSBP, suggesting that an additional site between 0.204 kb and 0.122 kb may also play a small role in mediating ICSBP stimulation of IL-12 p40 promoter activity.



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  		FIGURE 5. Promoter deletion analysis. A, Diagram of the human IL-12 p40 promoter and deletions. The positions of the Ets, REL/NF-{kappa}B, and C/EBP sites are marked. B and C, RAW cells or CL-2 cells were transfected with indicated reporters along with control or ICSBP vectors, and then treated with IFN-{gamma} or LPS or both as in Fig. 2Go. Each value represents the average of three determinations.

 
ICSBP is a part of IFN-{gamma}/LPS-inducible complexes assembled on the Ets site

The initial EMSA performed with probes covering a short Ets site (from -204 to -224), an extended Ets region (-292 to -196) (17), and several additional sites including an ISRE-like sequence near the TATA box (-52 to -34) did not reveal ICSBP binding, as assessed with anti-ICSBP Ab or with recombinant ICSBP. Because the Ets element is shown to bind a multiprotein complex (17), which is difficult to detect by standard EMSA, we employed an alternative, DNA affinity binding assay. Previously, we have successfully used this assay and described recruitment of multiple factors to specific regulatory elements, which was otherwise not detectable by EMSA (33, 34). As schematically presented in Fig. 6GoA, a biotinylated DNA from -296 to -199 containing the Ets site was conjugated to magnetic beads and incubated with RAW cell nuclear extracts, and bound materials were detected by immunoblot analysis. As shown in Fig. 6GoB (top panel, input lane), ICSBP expression was very low in untreated RAW cells, but IFN-{gamma} treatment strongly induced the expression, as expected (19, 20). While LPS alone induced ICSBP only modestly, IFN-{gamma}/LPS treatment led to a dramatically higher level of ICSBP expression. Thus, ICSBP was synergistically induced by IFN-{gamma}/LPS in RAW cells, as in peritoneal macrophages (41). Furthermore, ICSBP was recruited to the complexes bound to the Ets-conjugated beads. The amount of recruited ICSBP depended on the level of ICSBP expressed in RAW cells. Other nuclear proteins, TFIIB (expressed at constant levels) and STAT-1 (increased after IFN-{gamma} treatment), were not recruited to the Ets-conjugated beads. Fig. 6GoC shows that in addition to ICSBP, IRF-1 was recruited to the Ets DNA. However, neither protein was recruited to the Ets mutant (the same as that in {Delta}Ets in Fig. 5Go). To assess whether ICSBP directly contacts the Ets DNA, baculovirus rICSBP was tested for binding. Results in Fig. 6GoD show that rICSBP binding to Ets either alone or in the presence of recombinant IRF-1 or IRF-2 was >10 times lower than the one on the ISRE sequence (18). However, when mixed with extracts from RAW cells, rICSBP was recruited to Ets (Fig. 6GoE), indicating that ICSBP does not directly contact Ets but is recruited to the element by protein-protein interaction.

Transfected ICSBP can induce endogenous IL-12 p40 transcription

Lastly, we wished to determine whether exogenous ICSBP could stimulate transcription of the endogenous IL-12 p40 gene. RAW cells were transiently transfected with the ICSBP vector or control vector and expression of the murine IL-12 p40 transcripts was monitored by RT-PCR analysis. Remarkably, exogenous ICSBP alone led to induction of IL-12 p40 mRNA in the absence of stimuli, at a level comparable to that observed with control cells after IFN-{gamma}/LPS stimulation (Fig. 7Go, lower panel). In ICSBP-transfected cells, treatment with LPS or IFN-{gamma}/LPS did not significantly alter IL-12 p40 mRNA levels. As expected, transfection of the control vector (LK440 in Fig. 7Go) did not stimulate expression of IL-12 p40 transcripts in the absence of stimuli. Levels of hypoxanthine phosphoribosyltransferase, tested as a control, were comparable in both transfectants and with or without stimulation. To compare ICSBP induction of the endogenous IL-12 p40 with that of the exogenous IL-12 p40 promoter activity, the 0.243-kb human IL-12 p40 reporter was cotransfected and its activity measured (upper panel in Fig. 7Go). ICSBP transfection led to stimulation of IL-12 p40 promoter activity in the absence of IFN-{gamma}/LPS, while control vector did not, in agreement with results in Figs. 2–5GoGoGoGo. These results demonstrate that ICSBP is a bona fide activator of the IL-12 p40 gene, which once induced by IFN-{gamma}/LPS is capable of directly stimulating transcription in vivo.



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  		FIGURE 7. Stimulation of endogenous IL-12 p40 transcription by ICSBP. RAW cells (1.5 x 107) were transfected with 10 µg of 0.243-kb human IL-12 p40 luciferase reporter along with 20 µg of control empty vector (LK440) or ICSBP expression vector (ICSBP). Cells were plaved in media containing 1.2% DMSO for 6 h and primed overnight with IFN-{gamma}. LPS were added 6 h before harvesting. Reporter data represent the average of triplicate transfections ± SD. RT-PCR was performed on RNA extracted from transfected cells. cDNAs were analyzed by using oligonucleotides specific for murine IL-12 p40 or hypoxanthine phosphoribosyltransferase (HPRT).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study shows that ICSBP can activate both human and mouse IL-12 p40 promoter activity in macrophages, even in the absence of IFN-{gamma}/LPS stimulation. Our results indicate that IL-12 p40 promoter activation is critically dependent on ICSBP, a factor inducible by IFN-{gamma}/LPS, which can occur without requiring secondary cytokines that are induced following IFN-{gamma}/LPS stimulation, nor activation of other transcription factors controlled by IFN-{gamma} or LPS. Consistent with the critical role for ICSBP, ICSBP-/- CL-2 cells failed to stimulate IL-12 p40 mRNA and promoter activity.

Interestingly, some features associated with IL-12 p40 expression in vivo are shared with those of ICSBP, supporting the likelihood that ICSBP is indeed an activator of IL-12 p40 transcription. First, IL-12 p40 is not an immediate early gene, rather it is induced relatively late in IFN-{gamma}/LPS-stimulated macrophages and requires synthesis of a new protein (10, 42). ICSBP is an immune cell-specific transcription factor, induced in macrophages after IFN-{gamma}/LPS stimulation (19, 20, 43). Second, IL-12 p40 mRNA and promoter activation requires synergistic action of IFN-{gamma} and LPS ( Figs. 1–6GoGoGoGoGoGo) (2, 10). Likewise, ICSBP is synergistically induced by the two stimuli (Fig. 6GoB) (41). Third, IL-12 p40 transcription is selectively abrogated by the lack of ICSBP (Fig. 1Go). Similarly, studies of Leishmania infection indicate the presence of a pathway selectively directed to IL-12 activation (44). Infection with Leishmania stimulates induction of IL-1{alpha}, Il-1ß, as well as TNF-{alpha}, but it selectively impairs induction of IL-12 in macrophages, leading to compromised IFN-{gamma} production and host defense. This is reminiscent of the defect seen in ICSBP-/- macrophages, which fail to induce IL-12 p40, but not IL-1{alpha}, IL-1ß, and TNF-{alpha} (Fig. 1Go) (24). The demonstration that transfection of ICSBP alone can induce endogenous IL-12 p40 gene transcription provides conclusive evidence that ICSBP is a key transcriptional factor necessary for activating IL-12 p40 transcription in vivo.

Based on these observations, it might be surmised that ICSBP governs a late-acting transcriptional pathway that is activated subsequent to immediate IFN-{gamma}/LPS signaling, which then selectively targets IL-12 p40 transcription (see a model in Fig. 8Go). This pathway likely activates a subclass of IFN-{gamma}/LPS-inducible genes and may include additional target genes yet to be identified. In addition to IFN-{gamma}/LPS, various pathogens and CD40/CD40L interactions are shown to stimulate IL-12 p40 expression. Interestingly, these stimuli also fail to induce IL-12 p40 expression in ICSBP-/- macrophages (26, 27), indicating that the CD40/CD40L signals converge to the ICSBP-dependent pathway.



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  		FIGURE 8. A model for an ICSBP pathway. IFN-{gamma} activates STAT1 via the GAS element. ICSBP is one of the STAT1 targets, whose expression is synergistically augmented by LPS, possibly via NF-{kappa}B. ICSBP is assembled onto a regulatory element (Ets in the human IL-12 p40 promoter) and activates a specific downstream pathway required for IL-12 p40 transcription. This model is compatible with the requirement of 1) new protein synthesis, 2) IFN-{gamma}/LPS synergy, and 3) selectivity of IL-12 p40 induction.

 
Transfection analysis revealed that only ICSBP, but neither IRF-1 nor IRF-2, can enhance IL-12 p40 promoter activity (Fig. 4Go). However, despite the inability to stimulate promoter activity on its own, IRF-1, when cotransfected with ICSBP, gave cooperative activation of IL-12 p40 promoter activity (Fig. 5Go), probably by direct interaction with ICSBP through the heterodimerization domain (45). Our data indicate that while ICSBP is an obligatory activator, IRF-1 plays an auxiliary role in IL-12 p40 transcription. This auxiliary role may partly explain the defect reported for IRF-1-/- mice in developing Th1 responses (28, 29). However, the deficiency in T cell responses and NK cell development may weigh more heavily than the defect in IL-12 induction to produce impaired development of Th1 cells in IRF-1-/- mice (46).

Deletion analysis in Fig. 6Go found that the Ets site contributes to ICSBP activation of human IL-12 p40 promoter activity. This site has previously been shown to be essential for IFN-{gamma}/LPS induction of IL-12 p40 promoter activity (10, 17). In our assays, neither the Rel/NF-{kappa}B nor the C/EBP site, previously reported to be involved in stimulating IL-12 p40 promoter activity (15, 16), appeared to have a dominant role in mediating ICSBP activation (Fig. 6Go). However, the slight stimulation of luciferase reporters lacking the Ets site by ICSBP suggests that an element(s) downstream from the Ets site may also play a role in ICSBP activation, albeit to a lesser extent. Supporting the possible involvement of the NF-{kappa}B site, the Ets site was reported to synergize with the site in B cells and that c-Rel proteins are present in the complexes that bind to the Ets site (47). It is of note that in the present study deletion analyses were performed with the human IL-12 p40 promoter. Although the structure of the mouse promoter is very similar to that of the human promoter, relative importance of each regulatory element for ICSBP-mediated activation may differ in the two promoters. To address this question, it will be necessary to do deletion/mutation analyses for the mouse IL-12 p40 promoter.

Consistent with a role for ICSBP as an IL-12 p40 activator, we found that the endogenous ICSBP induced by IFN-{gamma}/LPS in RAW cells was recruited to the Ets DNA (Fig. 7Go). The importance of the Ets element for ICSBP recruitment was supported by the lack of ICSBP recruitment to the Ets mutant (Fig. 7GoC). However, ICSBP is not likely to directly contact Ets DNA, but rather likely to be recruited to the Ets element through protein-protein interaction, because recombinant ICSBP alone failed to bind to the Ets DNA (Fig. 7Go, D and E). At present, the factor(s) that contacts the Ets site is not known. It is possible that the element interfaces multiple factors, rather than contacting a single protein (17). In this regard, it may be anticipated that ICSBP interacts with multiple factors through multiple domains (48, 49), because we have obtained evidence that the full-length protein is necessary for full activation of IL-12 p40 promoter activity (not shown).

The finding that ICSBP activates IL-12 p40 promoter activity is contrary to its repressive function noted for a number of IFN-{alpha}ß-inducible promoters (18, 38). However, the fact that ICSBP has both positive and negative transcriptional activities may not be surprising, because other IRF members that repress ISRE-mediated transcription, such as IRF-2 and Pip/IRF-4, are shown to be capable of enhancing transcription from other promoters (50, 51). What directs ICSBP and IRF-4 to act as an activator or a repressor is unclear at present, but presumably depends on the context of promoter sequences and proteins with which they interact. ICSBP is recently shown to activate the gp91phox promoter in cooperation with PU.1 (52). In addition, it has been reported that not only ICSBP but Pip/IRF-4 can form complexes with PU.1 in macrophages and regulate transcription from PU.1/IRF-dependent promoters (53). It would be interesting to assess the role of Pip/IRF-4 and PU.1 for the IL-12 p40 transcription.

In summary, this work identifies ICSBP to be an IFN-{gamma}/LPS-inducible IRF member that selectively activates IL-12 p40 transcription.


   Acknowledgments
 
We thank D. Stephany for flow cytometry analysis, J. Lou, H. Fukazawa, G. Gusella for help in ICSBP-/- bone marrow cell transformation, T. Taniguchi and G. Adolf for plasmids and IFNs, and K. Holmes, A. Sher, and S. Nedosposov for discussions in the early stage of this work. We also thank Y. Yun and N. Desai for technical assistance.


   Footnotes
 
1 Current address: Preclinical Department, Genetics Institute, Andover, MA 01810. Back

2 Current address: National Institute of Infectious Diseases, Musashimurayama, Tokyo, 208 Japan. Back

3 Address correspondence and reprint requests to Dr. Keiko Ozato, Laboratory of Molecular Growth Regulation, Building 6, Room 2A01, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-2753. Back

4 Abbreviations used in this paper: IRF, IFN regulatory factor; ICSBP, IFN consensus sequence binding protein; ISRE, IFN-stimulated response element; GAS, IFN-{gamma} activation site; GBP, IFN-{gamma} binding protein. Back

Received for publication October 22, 1999. Accepted for publication April 11, 2000.


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