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J. Biol. Chem., Vol. 279, Issue 53, 55609-55617, December 31, 2004
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¶
From the
Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York 10021 and Laboratory of Molecular Growth Regulation, NICHD, National Institutes of Health, Bethesda, Maryland 20892
Received for publication, June 11, 2004 , and in revised form, October 12, 2004.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-mediated signaling and in the development and function of NK, NK T cells, and cytotoxic T lymphocytes (2–7). IRF-1 also has direct anti-proliferative effects, thus acting as a tumor suppressor and tumor susceptibility gene. Interferon consensus sequence-binding protein (ICSBP)/IRF-8 is restricted in its expression to myeloid and lymphoid cell lineages. It can function both as a transcriptional repressor and an activator depending on the partners with which it interacts, and it plays crucial roles in myeloid differentiation, generation of plasmacytoid dendritic cells (8), macrophage activation, and tumor suppression (1, 9).
IL-12 is a heterodimeric cytokine produced primarily by macrophages and dendritic cells in both innate and adaptive immune responses. It is a key factor in the induction of T cell-dependent and independent activation of macrophages, NK cells, generation of T helper type 1 cells and CTL, induction of opsonic complement-fixing antibodies, and resistance to intracellular infections (10). The genes encoding the two heterologous chains of IL-12, p40 and p35, are located on different human and mouse chromosomes. Together, p40 and p35 form the biologically active IL-12 (also called p70). The highly coordinated expression of p40 and p35 genes to form IL-12 p70 in the same cell type at the same time is essential for the initiation of an effective immune response. The production of IL-12 p40 in activated professional antigen-presenting cells is generally in great excess over that of the p35 chain, making the latter molecule a limiting step in the formation of bioactive IL-12 (11).
Previous studies by us and others have revealed an intimate relationship between IRF-1, ICSBP, and IL-12 in that IRF-1 acts as a critical component of IFN- signaling in the selective activation of the IL-12 p35 transcription (12) in synergy with LPS-mediated events (13, 14) and that IRF-1 and ICSBP cooperatively regulate the transcription of IL-12 p40 gene in IFN--amplified IL-12 production (15, 16). In this work, we show that ICSBP and IRF-1, activated primarily by IFN-, also cooperate in their transcriptional activation of the IL-12 p35 gene by directly binding to a specific site in the human IL-12 p35 promoter. This study provides additional mechanistic information regarding how innate and adaptive immune activation jointly lead to greater IL-12 production and persistent inflammatory responses.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cells—The murine macrophage cell line RAW 264.7 (RAW cells hereafter) was obtained from American Type Culture Collection and maintained in RPMI 1640 medium supplemented with 2 mM glutamine, 100 units/ml penicillin and streptomycin, and 10% FBS (endotoxin <1 ng/ml; Hyclone). Mouse inflammatory peritoneal exudate macrophages were obtained by lavage 4 days after injection of sterile 3% thioglycolate broth (1 ml of intraperitoneal). Cells were washed and resuspended in RPMI 1640 medium containing 10% fetal calf serum and standard supplements. Macrophages were plated in 24-well tissue culture dishes (0.5 x 106 cells/well). After 2 h of incubation to allow for adherence of macrophages, monolayers were washed three times to remove nonadherent cells and incubated with RPMI 1640 medium containing 10% fetal calf serum and standard supplements. The next day, some of the wells were treated with 1 µg/ml LPS and 10 ng/ml IFN- in a final volume of 1 ml or 10 ng/ml IFN first for 16 h (priming) followed by LPS.
Human blood derived-monocytes were isolated from fresh blood by Ficoll/Hypaque gradient centrifugation. Mononuclear cells were incubated for 1 h in polystyrene tissue culture flasks (Falcon, BD Biosciences). After 1 h of incubation to allow for adherence of monocytes, monolayers were washed three times to remove nonadherent cells and incubated with RPMI 1640 medium containing 10% fetal calf serum and standard supplements. The treatment of human monocytes is the same as above unless otherwise stated.
Reagents—All of the antibodies used in this study were purchased from Santa Cruz Biotechnology, Inc. Expression vector ICSBP and control LK440 were provided by Keiko Ozato. All of the plasmid DNA were prepared with Qiagen Endo-free Maxi-Prep kits. Recombinant human and murine IFN- was purchased from Genzyme (Boston, MA). LPS from Escherichia coli 0217:B8 and thioglycolate medium were purchased from Sigma.
ELISA—Supernatants from macrophage cultures were harvested at 6, 12, and 24 h after LPS stimulation and stored at –70 °C. Mouse IL-12 p40 and p70 were detected by using the OPT-EIA ELISA kit (BD Biosciences) according to the manufacturer's instructions. Concentrations were calculated by regression analysis of a standard curve.
RNase Protection Assay (RPA)—Mouse peritoneal macrophages were pretreated with IFN- for 16 h followed by treatment with LPS for an additional 4 h. 10 µg of total RNA for each determination was subjected to a multiprobe RNase protection kit (BD Biosciences), mCK2b, for mouse according to the manufacturer's instructions.
Quantitative real-time PCR (qRT-PCR)—To determine the level of IL-12 p35 mRNA by quantitative real time PCR, we used a modified protocol from Rajeevan et al. (17). cDNA converted from 1 µg of total RNA was diluted in a several concentrations. Diluted cDNA was mixed with a pair of primers (10 µM) derived from mouse IL-12 p35 or glyceraldehyde-3-phosphate dehydrogenase cDNA sequences and SYBR green PCR master mixture (Applied Biosystem) in a 15-µl volume. PCR cycling was performed as follows: 2 min at 50 °C; 10 min at 95 °C for 1 cycle followed by 40 cycles at 15 s at 95 °C; and 1 min at 60 °C. The PCR primers used were: forward primer, 5'-AAATGAAGCTCTGCATCCTGC-3', and reverse primer, 5'-TCACCCTGTTGATGGTCACG-3', for mouse IL-12 p35; forward primer, 5'-AACT TTGGCATTGTGGAAGG-3', and reverse primer, 5'-ACACATTGGGGGTAGGAACA-3', for mouse glyceraldehyde-3-phosphate dehydrogenase.
Nuclear Extract Preparation—Nuclear extracts for Western blot and DNA affinity binding assay were prepared according to the methods of Schreiber et al. (18).
Western Blotting—SDS-PAGE was performed according to Laemmli (19). Gels were electroblotted to polyvinylidene difluoride membranes and blocked in 5% milk in Tris buffer, pH 8.0. Primary antibody was added at the concentration of 1 µg/ml in Tris buffer containing 1% milk powder and left overnight at 4 °C. After extensive washing, secondary antibody conjugated to horseradish peroxidase was added at a 1:5000 dilution in 5% milk. After extensive washing, blots were subjected to enhanced chemiluminescence detection (PerkinElmer Life Sciences).
Transfection Assay—Transient transfections were performed by electroporation as described previously (20).
DNA Affinity Binding Assay—Complementary biotinylated oligonucleotides encompassing the human IL-12 p35 promoter ICSBP-RE (–235/–208) or the mutant ICSBP-RE were synthesized and annealed. 4 µg of biotinylated double-stranded DNA were conjugated to 200 µl of streptavidin-bound magnetic beads (Dynabeads, M280; Dynal) in binding/washing buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.1 M NaCl) for 30 min at room temperature. Unconjugated DNA was collected with a magnetic particle concentrator. DNA-conjugated beads were then blocked by 0.5% bovine serum albumin in TGEDN buffer (120 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.1 M NaCl, 1 mM dithiothreitol, 0.1% Triton X-100, 10% glycerol) at room temperature for 1 h. Beads were washed once in TGEDN buffer and resuspended in 100 µl of TGEDN. 20-µl beads conjugated to 4 µg of DNA were equilibrated with TGEDN buffer and incubated with 1000 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 and unconjugated protein were separated by 12% SDS-PAGE and detected by immunoblot analysis using rabbit anti-IRF-1 antibody with the enhanced chemiluminescence kit. Nuclear extracts were prepared from RAW cells treated with 10 ng/ml IFN- for 16 h followed by incubation with LPS alone or plus IFN- for an additional 4 h.
Co-immunoprecipitation—RAW 264.7 cells were activated with 10 ng/ml IFN- for 5 h. The cells then were washed with cold phosphate-buffered saline, and nuclear extract was harvested. The nuclear extract was precleared by adding 20 µl of protein G-agarose and incubating at 4 °C for 1 h on a rocker. Precleared nuclear extract (500 µg) was incubated with 2 µg of IRF-1 or ICSBP antibody (Santa Cruz Biotechnology, Inc.) in the presence of 20 µl of 50% (v/v) protein G-agarose overnight at 4 °C with gentle rocking. After three washings with phosphate-buffered saline, precipitated complexes were solubilized by boiling in SDS buffer, fractionated by 12% SDS-PAGE, and transferred to polyvinylidene difluoride membrane. Western blotting was performed using an ICSBP or IRF-1 antibody.
Chromatin Immunoprecipitation (ChIP) Assay—The ChIP procedure was performed using an assay kit according to the manufacturer's instructions (Upstate Biotechnology). 107 human peripheral blood monocytes were stimulated with 10 ng/ml IFN- priming and 1 µg/ml LPS or alone and cross-linked by 1% formaldehyde for 10 min at 37 °C. Nuclei were prepared and subjected to sonication to obtain DNA fragments ranging from 200 to 1000 bp. Chromatin fractions were precleared with protein G-agarose beads followed by immunoprecipitation overnight at 4 °C with 2 µg of anti-IRF-1 antibody or anti-ICSBP antibody and its control antibody. Cross-linking was reversed for4hat 65 °C and was followed by proteinase K digestion. DNA was purified and subjected to PCR. The input DNA was diluted 200 times before PCR. The input and precipitated DNA were PCR-amplified by primers encompassing the ICSBP site in the human IL-12 p35 promoter (5' primer, 5'-GCGAACATTTCGCTTTCATT-3', and 3' primer, 5'-ACTTTCCCGGGACTCTGGT-3') in a buffer containing 2 mM MgCl2. The samples were amplified for 34 cycles by PCR and analyzed by electrophoresis on a 1.2% agarose gel or analyzed by quantitative real-time PCR as described above.
Statistical Analysis—Student's t test was performed wherever applicable. Mean ± S.D. is shown unless otherwise indicated.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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followed by LPS stimulation. IL-12 p40 and p70 production at various times over a period of 24 h was measured by specific ELISA. Because of the lack of a p35-speicifc ELISA due to the fact that it is secreted only as a heterodimer, the measurement of p70 production is a generally accepted indicator of IL-12 p35 production due to the one to one ratio of the p40-p35 interaction. As shown in Fig. 1, IL-12 p40 production stimulated by LPS alone (A) or IFN- plus LPS (B) was strongly impaired in ICSBP–/– macrophages compared with wild type or ICSBP+/– cells. Similarly, IL-12 p70 production induced by LPS (C) or IFN- plus LPS (D) was virtually absent in ICSBP–/– cells. These results indicate that ICSBP is an essential regulator of IL-12 p40 and p70 production.
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plus LPS was comparable between wild type and ICSBP+/– peritoneal macrophages (lane 3 versus lane 7 and lane 4 versus lane 8), whereas its level was significantly reduced in ICSBP–/– cells activated by IFN- and LPS (lanes 4 and 8 versus lane 12), a result consistent with our previous finding (16). IL-12 p35 mRNA levels were reduced in ICSBP–/– cells activated with LPS alone (lane 11) or IFN- plus LPS (lane 12). The qRT-PCR analysis (Fig. 2B) shows more definitively that both LPS- and IFN-/LPS-induced IL-12 p35 mRNA levels were drastically reduced in ICSBP–/– macrophages. These data strongly indicate that ICSBP regulates both IL-12 p40 and p35 mRNA expression.
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/LPS-activated cells (filled diamonds). Note that, although difficult to discern in this graph because of the relatively low scale, the IL-12 p35 promoter was highly inducible by IFN- and LPS (10–20-fold) without exogenous ICSBP, as is the case here and as we have shown previously (12). An analysis of endogenous ICSBP protein expression revealed that ICSBP was primarily induced by IFN- in the nucleus, not in the cytoplasm, and that LPS treatment alone had little effect (Fig. 3B). These data clearly demonstrate the ability of ICSBP, primarily as an IFN--inducible transcription factor, to activate IL-12 p35 gene transcription.
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regulates IL-12 p35 transcription through ICSBP, we dissected the human IL-12 p35 promoter by sequentially deleting the full-length promoter, a 1143-bp genomic fragment (12), from the 5' end. The transcriptional start site of the human IL-12 p35 gene in macrophages has been mapped previously (21). Because ICSBP is primarily induced by IFN-, not LPS treatment, in macrophages (Fig. 3B), we stimulated RAW 264.7 cells with LPS in all of our transfection studies to provide the other essential set of transcription factors for maximally induced IL-12 p35 gene expression (12). As shown in Fig. 3C, the full-length IL-12 p35 promoter (–1082 to +61) was highly responsive to stimulation by ICSBP in resting cells (
8-fold compared with control vector-transfected cells) and in LPS-activated cells (27-fold). Sequential deletion of the full-length promoter down to –231 had no adverse impact on the inducibility of the p35 promoter by ICSBP. Note that the –231/+61 construct has lost a previously identified element, IRF-E-(–245 to –236), that is critical for response to IRF-1 (12). The loss of the IRF-E apparently had no effect on the response to ICSBP. A further deletion of 17 bp (to –214) resulted in a moderate loss of the ICSBP response in resting cells and a dramatic loss of response in LPS-stimulated cells, indicating the presence of a critical cis-element(s) in this region involved in response to ICSBP. By this approach, we identified the 17-bp region between –231 and –214 as a site containing a ICSBP-RE. Within this region, the GeneToolTM program (Double Twist, now defunct) predicted an IFN--regulated element consensus sequence (-IRE_CS), 5'-CTGGAGGC-3', from –225 to –219. Localization of ICSBP-RE—To assess whether the predicted sequence 5'-CTGGAGGC-3' located at –226 to –219 was critical for ICSBP response, we introduced 2-bp substitutions into this site in the context of the –231/+61 of the IL-12 p35 promoter and measured their response to ICSBP by transfection into RAW 264.7 cells treated with LPS. One of the substitution mutants (Fig. 4A) showed a different response from the wild type control in that the mutation reduced the ability by >70% of the –231/+61 IL-12 p35 promoter to stimulation by the co-transfected ICSBP without LPS (Fig. 4B, compare column 3 with column 1), similarly to the 5'-deletion construct of –214/+61 that has lost the entire 17-bp region including the ICSBP-RE (column 5). Secondary to that, the LPS-induced promoter activity of the mutant was also reduced proportionally (compare column 4 with column 2).
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and LPS in a sequence-specific manner since the mutant probe failed to recognize the binding (Fig. 5A). This result was corroborated by co-immunoprecipitation, which showed that an anti-IRF-1 IgG was able to pull down ICSBP in IFN--stimulated RAW 264.7 cells (Fig. 5B, top panel) or by reciprocal co-immunoprecipitation with anti-ICSBP (Fig. 5B, bottom), demonstrating their co-presence in the same complex that interacts with the ICSBP-RE (not necessarily directly interacting), as is the case in IL-12 p40 gene transcription (16).
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ICSBP and IRF-1 Both Bind to the ICSBP-RE in Vivo in Activated Primary Monocytes—To determine whether ICSBP and IRF-1 could bind to the endogenous IL-12 p35 promoter at the ICSBP-RE in vivo in primary human monocytes, we performed ChIP using a pair of primers flanking the ICSBP-RE for PCR amplification (Fig. 6A). Fig. 6B shows that both IRF-1 and ICSBP binding to the ICSBP-RE were readily detectable in monocytes stimulated with IFN- but not with LPS (the faint band seen in lanes 1 and 2 of the anti-ICSBP panel were likely artifacts because of their different size). The combination of IFN- and LPS stimulation further enhanced ICSBP binding (lane 4). These data was further confirmed by qRT-PCR (Fig. 5C) in that both IFN- and IFN- plus LPS strongly induced ICSBP and IRF-1 binding to the ICSBP-RE. The pattern of the endogenous ICSBP nuclear expression was consistent with its binding activity in vivo (Fig. 5D). These results clearly demonstrate that ICSBP and IRF-1 could bind to the endogenous IL-12 p35 ICSBP-RE in primary monocytes in response principally to IFN- stimulation.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-mediated enhancement of IL-12 production represents the convergence of signals derived from innate and adaptive immune responses. IFN- is produced mainly by NK cells during innate responses and by activated T lymphocytes during adaptive immunity. The exposure of professional antigen-presenting cells to IFN- activates IRFs including IRF-1 and ICSBP that are critical for maximal IL-12 synthesis when coupled with microbial stimuli such as LPS. This work builds on our previous studies and further elucidates the mechanism whereby the two signals initiated by innate and adaptive stimuli, respectively, converge to induce high levels of IL-12 production and ensure highly active and persistent NK and T helper type 1 responses.
The critical role of ICSBP in IL-12 production is illustrated in the observation that IL-12 p35 and p40 mRNA expression is impaired in ICSBP-deficient macrophages (Fig. 2) and that IL-12 p40 synthesis is severely reduced (Fig. 1, A and B), whereas IL-12 p70 production is virtually absent (Fig. 1, C and D). There is a quantitative discrepancy between reduced IL-12 p35 and p40 mRNA expression in ICSBP–/– macrophages on the one hand and the total lack of IL-12 p70 synthesis by these cells on the other. This may be explained by the possibility that the reduced levels of IL-12 p40 and p35 proteins in ICSBP–/– cells fall below a certain "threshold" for the formation of IL-12 heterodimers. Alternatively, although less likely, the ICSBP deficiency causes an indirect effect on the assembly or secretion of the IL-12 p70 heterodimer. Post-translational regulation of IL-12 production has been reported previously (22–24). That in our transfection system, LPS coupled with forced ICSBP expression failed to induce significant activation of the IL-12 p35 promoter compared with unstimulated cells (Fig. 3A) is consistent with the fact that IL-12 p35 transcriptional activation also requires IRF-1, which is induced by IFN- but not by LPS (12). It should be stressed that the transcriptional activation of IL-12 p35 requires two signals, one typically provided by LPS and the other by IFN-. The LPS-initiated signal induces the activation of such transcription factors as NF
B and Sp-1 that are essential for IL-12 p35 transcription (12–14). The IFN--initiated signal induces IRF-1 and ICSBP that are critical for IL-12 p35 transcription and p70 production (12, 14). Both signals are necessary (either signal alone is not sufficient) to activate IL-12 p35 transcription. The fact that the ICSBP response of the IL-12 p35 ICSBP-RE mutant promoter was reduced by 70% regardless of the absence or presence of LPS (Fig. 4B) suggests that the LPS response of the promoter is not dependent on the integrity of the ICSBP-RE. In other words, the ICSBP and LPS responses of the IL-12 p35 promoter are separate.
We were unable to detect a direct interaction of ICSBP with the ICSBP-RE in vitro but able to do so in vivo by ChIP (Fig. 6B). This could be due to a low abundance of ICSBP in the complex or due to the shielding of ICSBP from antibody contact by IRF-1 in vitro. This "hindrance" by IRF-1 may not occur in vivo on the chromatin-wrapped IL-12 p35 promoter because, presumably, the conformation in which IRF-1 and ICSBP bind to the ICSBP-RE in the presence of the surrounding chromatin structures does not obstruct access by their respective antibodies during the ChIP procedure (Fig. 6B). Furthermore, other lines of evidence suggest that ICSBP functionally interact with IRF-1. They can synergize to stimulate IL-12 p35 transcription through the ICSBP-RE (Fig. 5C) by binding together to the ICSBP-RE in vivo (Fig. 6B). It should be pointed out that the ICSBP-RE we identified in the human IL-12 p35 promoter does not look like a conventional IRF-E (GAAA(G/C)(T/C)GAAA(G/C)(T/C)) (25) or ISRE ((A/G)NGAAANNGAAACT) (26). Thus, it is possible that ICSBP may interact with this element through an unidentified factor in addition to IRF-1. A third possibility is that ICSBP could enhance IRF-1 binding to the ICSBP-RE through an intermediary but itself does not come into direct contact with DNA.
Based on several studies by us as well as other groups, a model is emerging that schematizes the molecular events that lead to IL-12 production by antigen-presenting cells (Fig. 7 for the IL-12 p35 subunit gene). During the innate phase of an immune response, microbial antigens activate the NFB pathway via Toll-like receptors (Toll-like receptor 4 for LPS) in a MyD88-dependent manner (27, 28). Activated NFB alone stimulates moderate transcription of IL-12 p35 and p40 genes primarily through p50 and c-Rel (13, 29 –31). The initial small amount of IL-12 is able to stimulate NK cells to produce IFN-. IFN- can also be produced by activated T cells. IFN- then activates IRF-1 and ICSBP, which can bind two sites in the IL-12 p35 promoter, either alone to the IRF-E by IRF-1 (12) or together (ICSBP-RE, Figs. 5 and 6), resulting in much greater levels of transcription of IL-12 p40 (16) and p35 genes (12) and the production of IL-12. The combined activity of IFN- and LPS also leads to the activation of Sp1 and its binding to a site in the p35 promoter as part of selective remodeling of a single nucleosome within the –310 to –160 region (14). The combination of signals derived from innate and adaptive immune events stimulate synergistically the production of IL-12 and sustain the inflammatory response and cell-mediated immunity against pathogens.
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FOOTNOTES |
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¶ To whom correspondence should be addressed: Dept. of Microbiology and Immunology, Weill Medical College of Cornell University, 1300 York Ave., New York, NY 10021. Tel.: 212-746-4404; Fax: 212-746-4427; E-mail: xim2002{at}med.cornell.edu.
1 The abbreviations used are: IRF-1, interferon regulatory factor-1; ICSBP, interferon consensus sequence-binding protein; IL-12, interleukin-12; ICSBP-RE, ICSBP-response element; IFN-, interferon-; LPS, lipopolysaccharide; ELISA, enzyme-linked immunosorbent assay; RPA, RNase protection assay; qRT-PCR, quantitative real-time PCR; ChIP, chromatin immunoprecipitation; NK, natural kill; IRF-E, IRF-1 response element.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-actin (cytoplasm) and anti-PU.1 (nucleus) antibodies, respectively, to ensure equivalent protein loading. C, various truncation constructs of the human IL-12 p35 promoter-luciferase reporter were transiently transfected into RAW cells by electroporation together with an ICSBP expression vector or its control vector LK440. Cells were left alone or treated with LPS (7 h). ICSBP-stimulated luciferase activity from each construct was measured from cell lysates and normalized to the activity obtained with the control vector, respectively. Results shown are the mean ± S.E. of four independent experiments. The promoter coordinates all refer to the monocyte-specific transcription initiation site located at +1 (
