Abstract

The heterodimeric cytokine Interleukin 12 (IL-12) is critical for T-helper 1 responses. The gene encoding the IL-12 p40 subunit is induced in macrophages following exposure to bacterial products. This study provides a functional analysis of the p40 promoter control elements in the murine macrophage cell lines RAW 264.7 and J774. Results show that two immune response associated transcription factors, C/EBPb and C/EBPd, are induced by lipopolysaccharide in these cell lines. The most critical cis-acting element is located between positions -96 and -88 relative to the transcription start site. Electrophoretic mobility shift assays reveal LPS-inducible functional binding of both in vitro and cell line produced C/EBPb and C/EBPd to the critical element. In vitro transcription/translation of the putative control elements suggest the heterodimer C/EBPb (LAP)/C/EBPb (LIP) to function in vivo. In addition, transient transfection assays show the C/EBPb isoform LAP to activate transcription of the IL-12 p40 promoter, while C/EBPb (LIP) shows transcriptional inhibition. The results suggest that C/EBP proteins play critical roles in the bacterial induction of the IL-12 p40 promoter.

Introduction

The cytokine Interleukin 12 (IL-12) is synthesized by macrophages upon exposure to bacterial products and intracellular pathogens. A main function of IL-12 is to mediate the signalling for differentiation of effecter T-helper 1 (Th1) cells and to secrete the Th1 cytokines, IL-2 and IFN-g (29). Th1 cells take part in cell-mediated immunity associated with intracellular microbes (1, 17). IL-12 has been shown to generate a Th1 response against human pathogens such as Mycobacterium tuberculosis (32), Mycobacterium leprae (25), and Leishmania species (19), and during the pathogenesis of the human immunodeficiency virus (5, 6, 7). Because of the role of IL-12 in the pathogen-induced immune response, its expression may be important for the development of vaccines against several intracellular pathogens (2). While the induction of IL-12 by intracellular pathogens instigates the protective host Th1 response, overexpression of Th1 cytokines and IL-12 may take part in the formation of chronic autoimmune and inflammatory diseases (24). Thus, an elucidation of macrophage expression of IL-12 may supply information concerning the pathogenesis of infectious and inflammatory diseases and may aid in the development of novel approaches for manipulation of the immune response.

Two covalently linked glycosylated chains, p40 and p35, constitute the IL-12 heterodimer. The subunits are encoded by distinct genes and together make up the biologically active p70 heterodimer (13, 21, 31). The p35 gene is induced in several tissues (13). p40 is strongly induced by intracellular bacteria and bacterial products (8) and its mRNA is found in macrophages and other cells that synthesize IL-12 (13).

This paper presents the results of a functional analysis of the murine IL-12 p40 promoter in the RAW 264.7 and J774 cell lines. The study implicates two main control elements involved in LPS-induced promoter activity. Both C/EBPb and C/EBPd show significant nuclear induction by lipopolysaccharide. The two transcription factors also show selective binding of the IL-12 p40 promoter in vitro. In vitro expression and electrophoretic mobiliy shift assays provide evidence that the precise heterodimeric element responsible for activation of p40 may be composed of C/EBPb (LAP) and C/EBPb (LIP) subunits. In addition, overexpression of C/EBPb (LAP) causes for increased transcription of the IL-12 p40 promoter in transient transfection assays, while overexpression of C/EBPb (LIP) results in decreased transcription. These findings aid in the exposition of the principle mechanisms involved in the regulation of this important cytokine.

Materials and Methods

Nuclear extracts and DNA binding assays

The method of Dignam et al. (10) as previously described (14) was used to prepare RAW 264.7 nuclear extracts with the following modifications: 1 µg of aprotinin (Sigma) per ml, 0.5 µg of leupeptin (Sigma) per ml, 1 µM pepstatin (Sigma), and 1mM phenylmethylsulfonyl fluoride (Sigma). Annealed single-stranded oligonucleotides with 5' GATC overhangs (Operon) that were gel purified were used to make electrophoretic mobility shift assay (EMSA) probes. The Klenow enzyme was used to fill in two hundred nanograms of probe with [a-³²P]dGTP and [a-³²P]dCTP. A NucTrap purification column (Stratagene) was used to purify the labeled probes. Wild-type IL-12 p40 EMSA probe sequence is displayed below (see Fig. 4A). Binding for C/EBP EMSAs was optimized at room temperature for 45 min, and EMSA products were separated on 5% acrylamide-1´ Tris-glycine-EDTA gel run at 4^oC for 3 h at 150 V. 2 µl of antibody was added to extracts for supershift experiments, in addition to poly (dI-dC) and binding buffer 30 minutes prior to addition of labeled probe at room temperature. Santa Cruz Biotechnology, Inc. supplied polyclonal antibodies. Kodak X-AR 5 film was used to expose dried gels at -80^oC with an intensifying screen.


Cell lines and reagents

Dulbecco Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Gibco) (assayed for low endotoxin activity) was utilized to maintain the RAW 264.7 and J774 murine macrophage lines (American Type Culture Collection). LPS was obtained from Sigma Chemicals.


Plasmids

pBluescript KS+ (Stratagene) was used to isolate a 10kb murine IL-12 p40 genomic clone. DNA sequencing and restriction mapping was employed to determine the location of the promoter region and intron-exon structure. The sequence of one kilobase of the promoter region was determined (Sequenase, version 2) and was found to match that previously published (18). Chloramphenicol acetyltransferase (CAT) assay p40 promoters were cloned from pGL2B into pCAT basic (Promega) modified to contain a KpnI site and a BglII site in the polylinker. C/EBPb isoforms LIP (pSCT-LAP with a deleted NcoI fragment) and LAP (pSCT-LAP) expression plasmids containing a cytomegalovirus (CMV) promoter/enhancer were originally constructed by Patrick Descombes and Uelie Schibler (9) and were obtained from Andrew Henderson and Kathryn Calame. An endotoxin-free plasmid purification system (Qiagen) was used to purify all plasmids used in transient transfections.


Transfections

A previously described protocol (26) was employed with modifications to transiently transfect RAW 264.7 cells by electroporation. For CAT transfections, 7.5 x 10⁶ cells were suspended in 200 µl of DMEM with 10% FBS plus 20 µg of CAT plasmid and 1 µg of HSP-b -galactosidase reporter (provided by Bradley Cobb, UCLA). Cells were electroporated in a 0.4-mm-long cuvette (Bio-Rad) in a Bio-Rad Gene Pulser at 250 V and 960 µF. The cells were incubated for 10 min at room temperature and were washed with 5 ml of PBS and resuspended in 1 ml of DMEM. Each transfection product was divided into two 500-µl aliquots, and each was diluted to 2.5 ml with medium in a six-well plate. 50 µg of total protein from cell extracts were used to perform CAT assays, as per the Promega protocol. A PhosphorImager (Molecular Dynamics) was employed to quantitate the conversion of [¹⁴C]chloramphenicol to its acetylated forms.

Figure 1. C/EBPb and C/EBPd are induced by lipopolysaccharide in the RAW 264.7 and J774 murine macrophage cell lines. (A) An immunoblot assay was performed with cytoplasmic and nuclear extracts from unactivated RAW 264.7 cells and from cells activated with LPS (5 µg/ml) for 4 h. 6 µg of extract was hybridized at room temperature with anti-C/EBPb primary antibody (Santa Cruz Biotechnology) for 1 h, and then with secondary antibody for 1 h. The hybridization complexes were treated with ECL reaction for 1 min, and were exposed to film. (B) Similar immunoblot assay hybridizing with anti-C/EBPd primary antibody. (C) Similar mmunoblot assay using anti-C/EBPa antibody. (D-F) Immunoblot assays using the same respective methods as described in A-C, with J774 cells as the source of cytoplasmic and nuclear extract.

Figure 2. The transcription activator of the IL-12 p40 promoter may be a C/EBPb (LAP)/C/EBPb (LIP) heterodimer. (A) An immunoblot assay was performed with in vitro transcribed/translated presumptive IL-12 p40 activators. 6 µg of protein was incubated with C/EBPb and C/EBPd primary antibody (Santa Cruz Biotechnology) for 1 h, and then with secondary antibody for 1 h. An ECL reaction was performed and the complexes were exposed to film. (B) An EMSA was performed with nuclear extracts from unactivated RAW 264.7 cells (lane 1) and from cells activated with LPS (5 µg/ml) for 4 h (lane 2), and with in vitro transcribed/translated presumptive IL-12 p40 transcription activators (lanes 3-8). Labeled probe was incubated with 6 µg of protein and 2 µg of poly (dI-dC) at room temperature for 45 min, and electrophoresis followed.

Figure 3. Effects of overexpressed C/EBPb isoforms LIP and LAP on p40 promoter activity. 15 µg of the -355-to-+55 IL-12 p40 promoter-CAT reporter was cotransfected in transient assays into RAW 264.7 cells with expression plasmids for LAP and LIP (9) containing a CMV promoter/enhancer. The amounts of the LIP and LAP plasmids cotransfected are indicated below the graph. Addition of a CMV promoter/enhancer-luciferase plasmid normalized plasmid amounts to 20 µg per transfection. Twenty-four hours after transfection, cells were either not activated or were activated with 5 µg of LPS per ml for 24 h.

Figure 4. EMSA shows LPS-inducible binding of C/EBPb, and to a smaller degree C/EBPd, to the p40 promoter. (A) Oligonucleotide probe location within the p40 promoter. The thick line represents the probe spanning the putative C/EBP site (-110/-69). The TATA box is indicated by boldfaced letters and the transcription start site is indicated by the arrow. (B) An EMSA was performed with 5 mg of nuclear extract from unactivated (lanes 1-3), LPS-activated (lanes 4-6) cells, and in vitro expressed C/EBPb (lanes 7-9) and C/EBPd (lanes 10-12). 2 mg of poly(dI-dC) and extract were incubated for 30 min at room temperature without (lanes 1, 3, 6, 9) or with 2 ml of the following polyclonal antibodies (Santa Cruz Biotechnology): anti-C/EBPb (lanes 2, 5, 8, and 11), and anti-C/EBPd (lanes 3, 6, 9, and 12). Subsequent to this incubation, labeled probe -110/-69 was added to the mixture and incubated for 45 min at room temperature prior to electrophoresis.

Figure 5. EMSAs demonstrate the double specific activity of the Santa Cruz Biotechnology anti-C/EBPb antibody, and the single specificity of the laboratory-prepared anti-C/EBPb antibody. (A) An EMSA was performed with 5 µg of nuclear extract from LPS-activated cells (lanes 1-5) and in vitro expressed C/EBPb (lanes 6-10) and C/EBPd (lanes 11-15). Extract and 2 µg of poly(dI-dC) were incubated for 30 min at room temperature without (lanes 1, 6, and 11) or with (lanes 2-5, 7-10, and 12-15) increasing concentrations of anti-C/EBPb antibody (Santa Cruz Biotechnology). Following incubation, labeled probe -110/-69 was added, and incubation proceeded for 45 min at room temperature prior to electrophoresis. (B) A similar EMSA was performed using laboratory-prepared anti-C/EBPb antibody in place of the Santa Cruz Biotechnology stock.

Laboratory-prepared anti-C/EBPb antibody shows specificity only for C/EBPb

An EMSA supershift experiment was performed using LPS-activated J774 cell line nuclear extracts and in vitro expressed C/EBPb and C/EBPd. The lanes consisting of nuclear extract and in vitro expressed C/EBPb (Fig. 5B, lanes 1-10) serve as controls and show expected binding of the putative antibody to proteins present in the incubation. The lanes consisting of in vitro expressed C/EBPd (lanes 11-15) serve as the functional assay, the results of which show quite conclusively the absence of antibody binding at levels as high as 2.0 µl. Its specificity only for C/EBPb confirmed, the laboratory- prepared anti-C/EBPb antibody was used in subsequent experiments in place of the non-specific commercial version.


C/EBPd binds to the IL-12 p40 promoter

An EMSA supershift experiment using J774 nuclear extracts was performed to determine the relative amount of C/EBPd which binds to the p40 probe. The lane consisting of LPS-activated nuclear extract incubated with the specially prepared anti-C/EBPb antibody shows the presence of a light-intensity double band at the same vertical position on the gel as the active bands in the LPS-activated nuclear extract (Fig. 6, compare lanes 8 and 6). This band represents residual levels of C/EBPd bound to the radiolabeled probe. Thus, the specially prepared anti-C/EBPb antibody is able to bind to and shift only the C/EBPb protein and any C/EBPd present in the extract remains at its original position. These results show quite clearly the relative amounts of bound C/EBPb and C/EBPd, with C/EBPb present to a great degree, and C/EBPd present at a smaller, yet recognizable level. The lanes consisting of LPS-activated cell line nuclear extracts with antibodies for either C/EBPa (lane 7) or C/EBP (lane 10) show no decrease in active band intensity relative to extracts incubated without antibody (lane 6). This data suggests that C/EBPa and C/EBP may not take part in the trans-activation of the p40 promoter.

Figure 6. EMSA reveals more precisely the extent of binding of both C/EBPb and C/EBPd to the IL-12 p40 promoter. C/EBPa and C/EBP show no binding to the p40 promoter. An EMSA was performed with 5 µg of nuclear extract from unactivated (lanes 1-5) and LPS-activated (lanes 6-10) cells. Extract and 2 µg of poly(dI-dC) were incubated for 30 min at room temperature without (lanes 1 and 6) or with 2 µl of the following polyclonal antibodies: anti-C/EBPa (lanes 2 and 7), laboratory-prepared anti-C/EBPb (lanes 3 and 8), anti-C/EBPd (lanes 4 and 9), and anti-C/EBP (lanes 5 and 10). After incubation, labeled probe -110/-69 was added and incubation continued for 45 min at room temperature prior to electrophoresis.

Figure 7. Immunoblot assays demonstrate the LPS-induced upregulation of C/EBPb in both the J774 murine macrophage cell line and in primary cells. (A) An immunoblot assay was performed with nuclear extracts from J774 cells activated for increasing periods of time with lipopolysaccharide (5 µg/ml). 5 µg of extract was hybridized at room temperature with anti-C/EBPb antibody for 1 h, and then with secondary antibody for 1 h. The hybridization complexes were treated with ECL reaction for 1 min, and were exposed to film. (B) A similar immunblot assay using primary cells as the source of nuclear extract.