Differential Expression and Distinct Functions of IFN Regulatory Factor

Differential Expression and Distinct Functions of IFN Regulatory Factor 4 and IFN Consensus Sequence Binding Protein in Macrophages This information is current as of October 1, 2021. Sylvia Marecki, Michael L. Atchison and Matthew J. Fenton J Immunol 1999; 163:2713-2722; ; http://www.jimmunol.org/content/163/5/2713 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Differential Expression and Distinct Functions of IFN Regulatory Factor 4 and IFN Consensus Sequence Binding Protein in Macrophages1

Sylvia Marecki,* Michael L. Atchison,† and Matthew J. Fenton2*

IFN regulatory factor 4 (IRF4) and IFN consensus sequence binding protein (ICSBP) are highly homologous members of the growing family of IRF proteins. ICSBP expression is restricted to lymphoid and myeloid cells, whereas IRF4 expression has been reported to be lymphoid-restricted. We present evidence that primary murine and human macrophages express IRF4, thereby extending its range of expression to myeloid cells. Here, we provide a comparative analysis of IRF4 and ICSBP expression and function in distinct cell types. These IRF proteins can form speciﬁc complexes with the Ets-like protein PU.1, and can activate transcription via binding to PU.1/IRF composite sequences. EMSA analysis revealed that murine macrophages contained both

IRF4/PU.1 and ICSBP/PU.1 complexes, analogous to B cells. Over-expression of ICSBP in these macrophages activated tran- Downloaded from scription of a PU.1/IRF-dependent promoter, whereas over-expression of IRF4 had no effect on this promoter. In contrast, over-expression of either IRF4 or ICSBP in both macrophages and NIH-3T3 ﬁbroblasts suppressed transcription of the PU.1- independent H-2Ld MHC class I promoter. In NIH-3T3 ﬁbroblasts, IRF4 and ICSBP also synergized with exogenous PU.1 to activate transcription of a PU.1/IRF-dependent promoter. Furthermore, both IRF4 and ICSBP activated transcription of the IL-1␤ promoter in both cell types. While this promoter is PU.1-dependent, it lacks any known PU.1/IRF composite binding sites.

Synergistic activation of the IL-1␤ promoter by these IRF proteins and PU.1 was found to require PU.1 serine 148. Together, these http://www.jimmunol.org/ data demonstrate that IRF4 and ICSBP are dichotomous regulators of transcription in macrophages. The Journal of Immunol- ogy, 1999, 163: 2713–2722.

nterferon responsiveness is a critical component of both in- Several reports have described the phenotype of mice with a null nate and adaptive immunity, with macrophages being respon- mutation of the ICSBP gene. Holtschke et al. (12) reported that I sive to both type I and type II IFNs. Signal transduction fol- ICSBP-deficient mice display increased susceptibility to infections lowing stimulation with IFN involves a variety of transcription with either vaccinia or lymphocytic choriomeningitis viruses. factors that bind specific DNA motifs known as IFN-stimulated These mice also spontaneously develop a syndrome similar to hu- 3 by guest on October 1, 2021 response elements (ISRE) and IFN-␥-activated sequences (GAS). man chronic myelogenous leukemia. Fehr et al. (13) subsequently These binding sites are found within the promoters of many IFN- reported that ICSBP-deficient mice were highly susceptible to in- responsive genes (reviewed in Ref. 1). The IFN regulatory factor fection with Listeria monocytogenes, which correlated with im- (IRF) family (reviewed in Ref. 2), which includes IRF1, IRF2, paired macrophage effector functions and IFN-␥ responsiveness. IRF3, IRF4 (previously termed Pip, LSIRF, and ICSAT), IFN con- Giese et al. (14) and Scharton-Kersten et al. (15) reported that ␥ sensus sequence binding protein (ICSBP), ISGF3 /p48, and sev- ICSBP-deficient mice were unable to control infection with Leish- eral viral homologues, mediates cellular responses to IFNs. The mania major and Toxoplasma gondii, respectively. This impaired family members IRF1 and ISGF3␥/p48 have been reported to ac- resistance to infection appears to be a consequence of the inability tivate some IFN-responsive genes (3, 4), whereas IRF2 and ICSBP of these mice to express the IL-12 p40 subunit, and thereby to have generally been reported to repress transcription (5, 6). Other mount an effective Th1-type immune response (15). Impaired family members, such as IRF3 and IRF4, have been reported to IL-12 p40 gene expression in the ICSBP-deficient mice implicated function as both activators and repressors of transcription, depend- ing upon the specific promoter (7–11). ICSBP as a transcription factor that directly or indirectly activates IL-12 p40 gene expression. This contrasts with earlier studies that concluded that ICSBP is a negative regulator of several IFN-responsive genes, including MHC class I, IFN-␤, ISG-54, ISG-15, *Pulmonary Center and Department of Pathology, Boston University School of Med- icine, Boston, MA 02118; and †Department of Animal Biology, School of Veterinary 2Ј-5Ј oligoadenylate synthetase, and the Ig ␭ enhancer (5, 7). Medicine, University of Pennsylvania, Philadelphia, PA 19104 The importance of IRF4 in vivo has been recently demonstrated Received for publication March 4, 1999. Accepted for publication June 25, 1999. in mice that lack a functional IRF4 gene. Lymphocyte develop- The costs of publication of this article were defrayed in part by the payment of page ment in IRF4-deficient mice was found to be blocked at a late charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. stage, leading to a severe accumulation of immature lymphocytes and to defective T cell cytokine production (16). The effects of 1 This work was supported by National Institutes of Health Grant GM57053. IRF4 deficiency on myeloid cell development and function have 2 Address correspondence and reprint requests to Dr. Matthew J. Fenton, Pulmonary Center, R-220, Boston University School of Medicine, 80 East Concord Street, Bos- yet to be reported. Recent analyses of ICSBP- and IRF4-deficient ton, MA 02118. E-mail address: [email protected] mice have revealed both similar and distinct phenotypes. For ex- 3 Abbreviations used in this paper: ISRE, IFN-stimulated response elements; GAS, ample, ICSBP-deficient mice spontaneously develop a chronic my- IFN-␥-activated sequence; IRF, IFN regulatory factors; ICSBP, IFN consensus sequence binding protein; PEC, peritoneal exudate macrophage; CAT, chloramphenicol elogenous leukemia-like syndrome, whereas IRF4-deficient mice acetyl transferase; ECL, enhanced chemiluminescence. do not (16). In contrast, both IRF4- and ICSBP-deficient mice

were unable to mount an effective anti-viral response to lympho- that specifically recognize PU.1, IRF1, or IRF2, as well as polyclonal goat cytic choriomeningitis viruses, compared with wild-type mice (12, antisera that specifically recognize IRF4 or ICSBP, were purchased from 16), demonstrating the requirement of these factors in antiviral Santa Cruz Biotechnology (Santa Cruz, CA). Normal, preimmune goat and rabbit sera were purchased from Pierce (Rockford, IL). immunity. A better understanding of the functional roles of IRF4 and ICSBP in vivo will depend upon the identification of genes Cell lines and tissue culture conditions that are uniquely regulated by each of these transcription factors. The RAW264.7 murine macrophage, NIH-3T3 murine fibroblast, and IRF4 and ICSBP, by themselves, possess only weak DNA bind- Sup-T1 murine T cell lines were purchased from the American Type Cul- ing affinity (17, 18). However, the binding of ICSBP to DNA is ture Collection (ATCC; Manassas, VA). RAW264.7 and NIH-3T3 cells dramatically increased following interaction with IRF1, IRF2, or were maintained in DMEM culture medium (BioWhittaker, Walkersville, the Ets-like transcription factor PU.1 (18, 19). Previous studies MD) supplemented with 10% heat-inactivated FBS (HyClone Laborato- ries, Logan, UT), 10 mM HEPES, 2 mM L-glutamine, 100 U/ml penicillin, reported that IRF4 could bind to PU.1, and that these IRF4/PU.1 and 100 ␮g/ml streptomycin (BioWhittaker). Sup-T1 cells were cultured in complexes were essential for Ig light chain expression (19–21). RPMI 1640 culture medium (BioWhittaker) supplemented as described PU.1 stabilizes the binding of both IRF4 and ICSBP to a composite above. Murine peritoneal exudate macrophages (PEC) were elicited by PU.1/IRF motif found in both the Ig ␭ and ␬ light chain enhancers thioglycollate injection of BALB/c mice (The Jackson Laboratory, Bar in B cells. These interactions between PU.1 and IRF4 appear to be Harbor, ME). Following elicitation (72 h), peritoneal cells were harvested, and macrophages were obtained by adherence purification. Macrophages stabilized, at least in part, by interactions between DNA binding were used within 5 days of harvest. Murine splenic B cells were obtained domains (22). Recent work by Brass et al. (23) has also demon- from BALB/c mice using complement-mediated lysis to remove T cells strated the importance of direct physical interaction between PU.1 and adherence to deplete macrophages, as previously described (37). Hu- and IRF4 in transcriptional regulation. The Ig enhancer composite man monocytes were obtained by adherence purification from total PBMC, Ј Ј as we described previously (38). All cells were cultured at 37°C, 5% CO2, Downloaded from motif consists of a 5 PU.1 binding site and a 3 sequence that in a humidified incubator. Endotoxin levels in all medium components resembles an ISRE half site. Most recently, a highly similar PU.1/ were Ͻ10 pg/ml final concentration, as indicated by BioWhittaker or mea- IRF composite element was identified within the myeloid-specific sured by Limulus amebocyte lysate kit (BioWhittaker). Cells were stimu- gp91phox promoter (24). This gp91phox promoter element is bound lated with LPS or recombinant IFN-␥ at a final concentration of 100 ng/ml by a complex comprised of PU.1, ICSBP, and IRF1. for the times indicated in the text. PU.1 regulates the transcription of several myeloid-specific Nuclear extraction and cytosolic lysate preparation genes in the absence of interaction with either IRF4 or ICSBP. http://www.jimmunol.org/ Nuclear extracts were prepared essentially as described by Schreiber et al. These include the Ig heavy and light chains (19, 20), M-CSF re- ϩ (39). Approximately 1.0 ϫ 107 cells were harvested, washed with Ca2 ␤ phox ϩ ceptor (25), IL-1 (26), gp91 (27), and macrophage scavenger and Mg2 -free PBS (BioWhittaker), and pelleted by centrifugation at receptor (28) genes. A protein kinase CK2 phosphorylation site at 800 ϫ g for 10 min at 4°C. The resulting cell pellets were resuspended in ␮ serine 148 has been shown to be functionally important (21). Mu- 400 l of buffer I (10 mM KCl, 10 mM Tris-HCl (pH 7.8), 5 mM MgCl2, tation of this serine to an alanine resulted in a complete loss of 0.5 mM DTT, 0.3 M sucrose, 10 mM ␤-glycerol phosphate, 0.1 mM EGTA, 1 mM PMSF, and 5 ␮g/ml each of aprotinin, leupeptin, chymo- LPS-induced transcriptional activity. These data demonstrate that statin, and antipain), then incubated on ice for 10 min. Subsequently, 25 ␮l serine 148 phosphorylation is critical for inducible transcriptional of 10% Nonidet P-40 (Sigma) was added to each sample and vortexed. The activation by PU.1. Consistent with these findings, physical and nuclei were pelleted by centrifugation for 1 min at 5000 ϫ g. Supernatants by guest on October 1, 2021 maximal functional interaction between PU.1 and the IRF members were collected and stored for later use as cytosolic lysates. Nuclear pellets IRF4 and ICSBP also requires the presence of serine 148 (20, 21). were resuspended in a nuclear extraction buffer (buffer II) containing 320 mM KCl, 10 mM Tris-HCl (pH 7.8), 5 mM MgCl2, 0.5 mM DTT, 10 mM Unlike most other IRF family members, ICSBP expression is ␤-glycerol phosphate, 0.1 mM EGTA, 25% glycerol, 1 mM PMSF, and 5 mainly restricted to cells of the immune system, including lym- ␮g/ml each of aprotinin, leupeptin, antipain, and chymostatin. Samples phocytes and macrophages (5). However, recent work by Li et al. were extracted on ice for 15 min followed by centrifugation at 16,000 ϫ g (29, 30) demonstrated that ICSBP is expressed in the retinal epi- for 10 min at 4°C. Protein concentration was determined using the Bio-Rad (Hercules, CA) assay kit. All nuclear extracts were stored at Ϫ70°C, and thelia and ocular lens. ICSBP is constitutively expressed in mac- multiple freeze-thawing cycles were avoided. rophages, although its expression can be further up-regulated by IFN-␥, but not significantly by IFN␣␤ (31–33). Moreover, LPS EMSA and DNA probes ␥ and IFN- synergistically enhance ICSBP steady-state mRNA and A double-stranded oligonucleotide containing a single copy of the Ig ␬ 3Ј protein levels (34). IRF4 has been reported to only be expressed in enhancer (Ig ␬) composite PU.1/IRF sequence (5Ј-CTTTGAGGAACT- lymphocytes (7, 10, 16, 35, 36), and its expression was not altered GAAAACAGAACCT-3Ј; (40)) was utilized as an EMSA probe. Italicized by IFN-␥ treatment (10, 36). In contrast to these earlier reports, we sequences represent PU.1 (5Ј) and IRF (3Ј) binding sites. Mutant oligonu- demonstrate here that macrophages express both IRF4 mRNA and cleotides were also generated where either the PU.1 (GGAA to TTCA) or IRF (AAAC to ATCA) binding sites were previously described (20). An protein. The capacity of macrophages to express both IRF4 and unlabeled double-stranded oligonucleotide containing a single copy of the ICSBP raises the possibility that they perform distinct functions in human IL-1␤ cap site-proximal PU.1 binding site (GCAGAAGT; (26)) or these cells. To assess the function of these IRF proteins in mac- IL-2R ␣-chain NF-␬B site (GGGGAATTCC) were utilized as competitor 32 rophages, we examined their ability to regulate the transcription of DNA. DNA probes were labeled with [␣- P] deoxynucleotide triphos- phates (DuPont-NEN, Boston, MA) using E. coli DNA polymerase Kle- selected promoters. Here, we report that IRF4 and ICSBP can now fragment (United States Biochemicals, Cleveland, OH), as recom- function as both transcriptional activators and repressors of differ- mended by the manufacturer. Unincorporated nucleotides were removed ent promoters in a cell type- and promoter-specific manner. Our using Sephadex G-25 columns (5 Prime 3 3 Prime, Boulder, CO). Nuclear data suggest a complex molecular mechanism that regulates the extracts (typically 3 ␮g) were incubated with radiolabeled probe DNA (0.1 ␮ expression of genes by IRF4 and ICSBP in macrophages. ng, typically 10,000 cpm) in the presence of 2 g poly dI-dC (Pharmacia, Piscataway, NJ), 1.0 mM EDTA, 10 mM Tris-HCl (pH 7.9), 25 mM glycerol, and 0.5 mM DTT in a final volume of 20 ␮l, as previously described Materials and Methods (41, 42). Binding reactions were incubated at room temperature for 30 min. Reagents and Abs In competition experiments, unlabeled DNA was added at a 100-fold molar excess in addition to the binding reaction. For supershift experiments, 2 ␮g Trypsin, proteinase K, antipain, aprotinin, chymostatin, and leupeptin were of antiserum was added as indicated in the text in addition to the binding purchased from Sigma (St. Louis, MO). Recombinant murine IFN-␥ was reaction. Following incubation, a portion of each binding reaction (7 ␮l) purchased from R&D Systems (Minneapolis, MN). LPS, Escherichia coli was electrophoresed through a 7% nondenaturing low ionic strength poly- serotype 055:B5, was purchased from Sigma. Polyclonal rabbit antisera acrylamide gel, dried, and visualized by autoradiography. The Journal of Immunology 2715

Western blot analysis Reporter gene assays Nuclear extracts and cytosolic lysates were prepared from cells as de- CAT reporter activity was assessed using a two-phase fluor diffusion CAT scribed above. Samples (50–150 ␮g total protein) were electrophoresed assay as previously described (46). Equal concentrations of cell lysates through either a 15 or 10% SDS-polyacrylamide gel (noted in figure leg- (15–35 ␮g total protein per sample) were used in the assay. Protein con- end), transferred to a nitrocellulose membrane (Bio-Rad), and blocked with centration was determined utilizing the Bio-Rad protein assay kit, accord- 5% nonfat dry milk (Carnation) in TBST containing 0.1% Tween 20 (Sig- ing to the manufacturer’s instructions. Data were calculated by plotting ma). PU.1 protein was detected using a 1:2000 dilution of anti-PU.1 anti- total cpm of acetylated chloramphenicol vs time, and the slopes for each sera and developed using a 1:4500 dilution of a protein A conjugated to reaction were calculated within a linear kinetic range. Luciferase activity HRP (Amersham, Arlington Heights, IL). IRF4 and ICSBP proteins were was measured using the luciferase assay system (Promega), according to visualized using a 1:3000 dilution of specific antisera and developed with manufacturer’s instructions and performed as previously described (47). a 1:1500 dilution of protein G-HRP (Pierce). Membranes were visualized Lysates were assayed for total protein using the Bio-Rad protein assay. using an enhanced chemiluminescence (ECL) reagent (CL-HRP substrate Luciferase activity was measured using 5–20 ␮g total protein as measured system; Pierce), according to the manufacturer’s instructions. by light emissions in a scintillation counter.

RNA isolation and RT-PCR Results Total RNA from macrophages, fibroblasts, and murine B cells was purified IRF4 and ICSBP can form specific complexes with PU.1 in using RNA STAT-60 (Leedo Medical Laboratories, Houston, TX), as rec- macrophages ommended by the manufacturer. RNA was converted to cDNA using avian We performed a series of EMSA analyses to determine whether myeloblastosis virus RT (Promega, Madison, WI). PCR were performed using between 100 ng and 2 ␮g of cDNA, 0.5 ␮M oligonucleotide primers ICSBP/PU.1 complexes could be formed in RAW264.7 macro- ␮ (each), 1.5 mM MgCl2, 150 M dNTPs and 2.5U Taq polymerase in a final phages and primary B cells. The EMSA probe used in these ex- reaction volume of 75 ␮l. Thirty amplification cycles were performed periments is a PU.1/IRF composite binding site (see Materials and Downloaded from (95°C denaturation, 30 s; 55°C annealing, 1 min; 72°C extension, 1.5 min). Methods for sequence) that contains an ISRE half site located Intron-spanning IRF4, CD19, and ␤-actin PCR primers used in this study are listed below. The IRF4 primers correspond to sequences that do not downstream and adjacent to a consensus PU.1 binding site. Such share sequence similarity with ICSBP. Following amplification, a portion sites have been identified within the Ig light chain enhancers and of the PCR reactions were electrophoresed through a 1.2% agarose gel. The the gp91phox promoter (19, 20, 24). IRF proteins alone do not bind 554-bp IRF4, 747-bp CD19, and 285-bp ␤-actin products were visualized to this composite site, although concomitant binding of PU.1 to the using ethidium bromide. Sense-strand IRF4 primer: 5Ј-GCT GCA TAT site stabilizes IRF binding to DNA as a consequence of its inter- http://www.jimmunol.org/ CTG CCT GTA TTA CCG-3Ј; anti-sense strand IRF4 primer: 5Ј-GTG GTA ACG TGT TCA GGT AAC TCG TAG-3Ј; sense-strand CD19 prim- action with PU.1 (7). As shown in Fig. 1, PU.1 and IRF proteins er: 5Ј-CCC CAG AAG TCC TTA CTG-3Ј; anti-sense CD19 primer: 3Ј- that were present in unstimulated macrophage and B cell nuclear GCC TCT CGA TGG TCA GGT TT-3Ј; sense-strand ␤-actin primer: 5Ј- extracts specifically bound to the composite site. Abs against PU.1 Ј TCA TGA AGT GTG ACG TTG ACA TCC GT-3 ; anti-sense strand (lane 7) and ICSBP (lane 3) could supershift the DNA-protein ␤-actin primer: 5Ј-CCT AGA AGC ATT TGC GGT GCA CGA TG-3Ј. complexes, whereas Abs against IRF1 (lane 5) and IRF2 (lane 6) did not supershift the complexes. Abs that recognize PU.1 blocked Plasmids the binding of both PU.1 and ICSBP to the DNA, suggesting that Expression plasmids encoding the full-length, wild-type and S148A mutant ICSBP could not bind to DNA in the absence of PU.1. Unexpect- murine PU.1 proteins were previously described (21). An expression plas- edly, specific Abs for IRF4 (Fig. 1A, lane 4) could also supershift by guest on October 1, 2021 mid encoding the full-length murine ICSBP protein was provided by Dr. Keiko Ozato (National Institutes of Health, Bethesda, MD) and was pre- the DNA-protein complexes generated using macrophage extracts. viously described (43–45). Expression plasmids encoding the murine IRF4 Like ICSBP, Abs that recognize PU.1 blocked the binding of both proteins were previously described (35). The control vector pcDNA3.1 was PU.1 and IRF4 to the DNA, indicating that IRF4 could not bind to purchased from Invitrogen (Carlsbad, CA), and was used to maintain a DNA in the absence of PU.1. The IRF4 Ab was also able to su- constant quantity of plasmid DNA in each transfection. The (PU.1/IRF)4 pershift a complex when incubated with B cell nuclear extracts chloramphenicol acetyl transferase (CAT) (wild type), (mPU.1/IRF)4 CAT (PU.1 binding site mutant), and (PU.1/mIRF)4 CAT (IRF binding site mu- (Fig. 1B, lane 4). Furthermore, normal preimmune goat and rabbit tant) reporter constructs containing four copies of the composite PU.1/IRF sera used at similar concentrations as all other antisera did not binding site derived from the Ig ␬ 3Ј enhancer were previously described produce any supershifted complexes when incubated with either ␤ (20). The IL-1 luciferase reporter construct was generated from the pre- macrophage or B cell nuclear extracts (lanes 8 and 9). In addition, viously described 7 mXT-CAT reporter plasmid (26). Briefly, the XbaIto TaqI fragment (positions Ϫ3759 to ϩ11) from the human IL-1␤ promoter when EMSA analysis was performed upon primary murine and was subcloned into the promoterless pGL3 luciferase reporter plasmid human macrophage nuclear extracts, similar results were obtained (Promega). The H-2Ld MHC class I reporter plasmid was provided by Dr. (data not shown). Mutation of the IRF binding site abolished bind- Keiko Ozato, and was previously reported (5). ing of the PU.1/IRF complexes, but did not affect the binding of PU.1 alone (data not shown), while mutation of the PU.1 site Transient transfections blocked the binding of both PU.1 and IRF proteins to the oligo- Transient transfections were performed using SuperFect reagent (Promega) nucleotide (data not shown). Together, our data suggest that mac- as per the manufacturer’s instructions. Briefly, cells were plated on 6-well rophages express IRF4, or a related protein that is also recognized dishes and transfected when cells reached 80% confluence. Plasmid DNA by the Ab, and that this protein can bind to the PU.1/IRF com- ␮ was added to 100 l of Opti-Mem reduced serum media (Life Technolo- posite sites in the presence of PU.1. gies, Rockville, MD). All transfections utilized a total of 4 ␮g of plasmid DNA consisting of 2 ␮g of reporter plasmid, 1 ␮g of each expression vector, and the balance was made up with empty vector described above IRF4 mRNA and protein are expressed in macrophages ␮ unless otherwise noted in the text. A total of 10 l of SuperFect was added To confirm that IRF4 is expressed by macrophages, we used se- to the DNA-media mixture, incubated for 10 min, diluted with 600 ␮lof serum-containing media, and added to individual wells. Each reaction was quence-specific, intron-spanning oligonucleotide primers to am- prepared separately and in triplicate. Fresh media containing serum was plify cDNA synthesized from both primary and cell line murine added 2–3 h after transfection. All conditions were incubated for an addi- mRNA. These primers were selected from regions of the IRF4 tional 16–24 h. CAT and luciferase assays were performed as described mRNA that lack sequence similarity with ICSBP transcripts. Using below. All transfection experiments were repeated at least three times using different plasmid preparations, and a single representative experiment is RT-PCR, these primers generated a 554-bp product using cDNA shown. Each single experiment represents triplicate independent transfec- synthesized from RAW264.7 cells and primary murine PEC (Fig. tions, and data are expressed as average values Ϯ SD. 2, lanes 2 and 4). A PCR product of identical size was generated 2716 EXPRESSION AND FUNCTION OF IRF4 AND ICSBP IN MACROPHAGES

FIGURE 2. Detection of IRF4 transcripts in murine macrophages by RT-PCR. Total RNA was prepared from NIH-3T3 fibroblasts (NIH), RAW264.7 macrophages (RAW), primary murine B cells (B cells), and primary murine PEC, as described in the text. RT-PCR was performed using IRF4-, CD19-, and ␤-actin-specific oligonucleotide primers. PCR products were fractionated on a 1.2% agarose gel and stained with ethidium bromide. The expected 554-bp IRF4 product was observed in PCR reactions containing cDNA from the macrophages and B cells, but not the NIH-3T3 fibroblasts. The expected 747-bp product for CD19 was observed ␤

only in B cells. The expected 285-bp -actin product was observed in all Downloaded from PCR reactions. No PCR products were generated using RNA that had not been reverse transcribed.

These analyses were performed using semiquantitative methods,

where logarithmic dilutions of cDNA were used as templates for http://www.jimmunol.org/ PCR and then analyzed by densitometry to assure that amplification was performed in the linear range. The amount of IRF4 PCR products generated using cDNA generated from the RAW264.7 FIGURE 1. EMSA analysis of PU.1/IRF complexes in nuclear extracts cells was consistently and substantially less that that generated from RAW264.7 macrophages. A, A radiolabeled double-stranded oligo- from the primary B cells or primary macrophages. When the levels nucleotide probe containing a single copy of the Ig ␬ chain 3Ј enhancer of IRF4 message were normalized to ␤-actin, both RAW264.7 PU.1/IRF composite site was incubated with nuclear extracts prepared macrophages and primary macrophages were found to contain from RAW264.7 macrophages, as described in the text (lane 1). Non-cross- similar amounts of IRF4 mRNA. These levels were 25–35% of by guest on October 1, 2021 reactive Abs against ICSBP, IRF4, IRF1, IRF2, PU.1, normal, preimmune that seen in primary B cells (data not shown). From these data, we goat or rabbit sera were included in some binding reactions, as indicated in conclude that macrophages do express IRF4 transcripts, although the figure. Unlabeled probe DNA, unlabeled double-stranded oligonucle- at levels lower than those found in B cells. ␤ otide probe containing a single copy of the IL-1 cap site-proximal PU.1 To determine whether this IRF4 mRNA was translated into pro- binding site or an unlabeled IL-2 NF-␬B binding site were included in tein, Western blot analysis was performed. Nuclear extracts were some binding reactions (100-fold molar excess) as indicated in the figure. B, A radiolabeled double-stranded oligonucleotide probe containing a sin- prepared from primary murine macrophages, RAW264.7 macro- gle copy of the Ig ␬ chain 3Ј enhancer PU.1/IRF composite site was in- phages, and primary human monocytes. These samples were frac- cubated with nuclear extracts prepared from primary B cells, as described tionated by SDS-PAGE and probed using non-cross-reactive Abs in the text. The EMSA was performed identically as described in A. The against either IRF4 or ICSBP. Nuclear extracts from primary mu- mobility of the radiolabeled DNA complexes containing PU.1 (PU.1), an rine B cells were also included in the figure, because they express IRF protein plus PU.1 (PU.1/IRF), and supershifted (SS) complexes are both IRF4 and ICSBP. As shown in Fig. 3A, immunoreactive IRF4 indicated by arrows. NE, nuclear extract; NGS, normal goat sera; NRS, and ICSBP protein were present in both the macrophage and B cell normal rabbit sera; CC, cold competitor. Unbound probe DNA migrates at lysates. To confirm that the Abs were non-cross-reactive and rec- the bottom of the gel. ognized proteins of the predicted molecular size, three duplicate lanes were transferred to nitrocellulose membranes and probed with the Abs singly, or in combination. As shown in Fig. 3B, the using cDNA synthesized from primary murine B cells (lane 3). IRF4 and ICSBP Abs each identified a single protein with the Meanwhile, no PCR product was generated from NIH-3T3 fibro- expected molecular size (42 and 45 kDa, respectively). Together, blast cDNA (lane 1), cells that do not express IRF4, ICSBP, or these data demonstrate that macrophages express both IRF PU.1. To confirm that the PCR product generated from primary proteins. murine macrophages was not due to B cell contamination, we performed PCR using primers to detect expression of the B cell-spe- Differential expression of IRF4 and ICSBP in macrophages cific gene CD19. A PCR product of the expected size (747 bp) was Yamagata et al. (10) reported that IRF4 expression and function in generated only from the B cell cDNA. These data confirmed that lymphocytes were not activated by IFN treatment. In contrast, the IRF4 PCR product generated using primary macrophage RNA ICSBP is expressed constitutively at low levels in macrophages, was not due to the presence of contaminating B cells. In all cases, and this expression can be further up-regulated by IFN-␥ treatment a 285-bp PCR product was generated using ␤-actin primers. The (32–34). We sought to determine whether IRF4 expression could PCR products generated from macrophage cDNA were subse- be regulated by IFN-␥ in macrophages. The capacity of IFN-␥ to quently sequenced and found to be identical to the sequence of up-regulate ICSBP gene expression in the RAW264.7 macro- IRF4 (data not shown). phages was confirmed by Western blot analysis of nuclear extracts The Journal of Immunology 2717 Downloaded from

FIGURE 3. Detection of IRF4 protein in macrophages. A, Nuclear extracts were prepared from NIH-3T3 ﬁbroblasts (NIH), primary murine B cells (B cells), primary murine PEC, RAW264.7 macrophages (RAW), and ␾ ␮

human monocytes (Hu M ), as described in the text. Extracts (150 g per http://www.jimmunol.org/ lane) were fractionated on 15% denaturing polyacrylamide gels, transferred to nitrocellulose, and probed with either the anti-IRF4 or anti-ICSBP Abs. The membranes were then developed using protein G-HRP, and visualized using ECL. The molecular sizes of the protein bands are indicated in the figure. B, To confirm that the IRF4 and ICSBP Abs were non-cross-reactive, three identical membranes were prepared from RAW264.7 cytosolic lysates (30 ␮g), fractionated on 10% SDS-PAGE gels, and probed with each Ab singly or together. The membranes were developed using protein FIGURE 5. LPS induces nuclear translocation of IRF4. A, RAW264.7 G-HRP, and visualized using ECL. The molecular sizes of the protein cells were stimulated with LPS (100 ng/ml) for various times, as indicated bands are indicated in the figure. in the figure. Cytosolic lysates and nuclear extracts were prepared from by guest on October 1, 2021 unstimulated and LPS-stimulated RAW264.7 cells, as described in the text. Aliquots of either nuclear or cytosolic fractions (150 ␮g/lane) were fractionated by SDS-PAGE. Protein was transferred to nitrocellulose, probed (Fig. 4), and densitometric quantification of the Western blots re- ␥ with an anti-IRF4 Ab, developed using protein G-HRP, and visualized vealed that6hofIFN- stimulation increased the nuclear levels of using ECL. The same blots were stripped and reprobed with an anti-PU.1 ϳ ICSBP by 26-fold. In contrast to ICSBP, levels of nuclear IRF4 Ab. The molecular sizes of the protein bands are indicated in the figure. B, protein in the macrophages was not altered by IFN-␥ treatment, Densitometric quantification of the Western blot data in A. Densitometric consistent with previous findings. Cytosolic levels of IRF4 and data are expressed as relative optical density values. Dark bars represent nuclear extracts, and light bars represent the cytosolic fraction.

ICSBP mirrored that observed in the nucleus (data not shown), consistent with previous ﬁndings in lymphocytes (13, 36). In addition, the levels of nuclear PU.1 protein were not altered by IFN-␥ treatment (Fig. 4).

LPS induces nuclear translocation of IRF4 in macrophages IRF4 activity has been reported to be induced in activated lymphocytes (10, 36). Therefore, we sought to determine whether LPS stimulation of the RAW264.7 cells could similarly activate IRF4. As shown in Fig. 5A, levels of nuclear IRF4 protein increased FIGURE 4. Differential induction of IRF4 and ICSBP expression in within 2 h following LPS stimulation, with a concomitant decrease IFN-␥-stimulated macrophages. RAW264.7 cells were treated with 100 in the cytosolic levels of this protein. In contrast, ICSBP protein ␥ ng/ml IFN- for various times as indicated (in hours) or remained unstimu- levels in both the nucleus and cytosol were not measurably altered lated. Cells were harvested, nuclear extracts were prepared, and a portion following LPS stimulation within the time period examined (data of the extracts (150 ␮g/lane) were fractionated by SDS-PAGE. Gels were then transferred to nitrocellulose, probed with an anti-ICSBP Ab, devel- not shown). As a control for cross-contamination of the samples, oped using protein G-HRP, and visualized using ECL. Blots were then we found that these nuclear extracts did not contain a protein found stripped and reprobed with an anti-IRF4 Ab, and subsequently, with an solely in the cytosol (p105 NF-␬B; data not shown). Densitometric anti-PU.1 Ab. The molecular sizes of the protein bands are indicated in the quantiﬁcation of the Western blots revealed that6hofLPSstim- ﬁgure. ulation increased the nuclear levels of IRF4 by ϳ3-fold (Fig. 5B). 2718 EXPRESSION AND FUNCTION OF IRF4 AND ICSBP IN MACROPHAGES

FIGURE 7. Synergistic activation of transcription by PU.1 and IRF proteins. The (PU.1/IRF)4 reporter plasmid was transiently cotransfected with FIGURE 6. PU.1/IRF composite elements are differentially regulated various combinations of PU.1, IRF4, and ICSBP expression plasmids into by IRF4 and ICSBP in macrophages. The (PU.1/IRF)4 reporter plasmid, NIH-3T3 cells. Untransfected cells do not express PU.1, IRF4, or ICSBP. ␬ Ј containing four tandem repeats of the Ig chain 3 enhancer composite site CAT activity was determined as described in the text. Transfections were ligated upstream of a minimal thymidine kinase promoter and the CAT performed in triplicate. Data are expressed as the mean rate of radiolabeled Downloaded from reporter gene, was transiently transfected into RAW264.7 macrophages, as chloramphenicol accumulation over time (slope) Ϯ SD. described in the text. RAW264.7 cells were also transfected with reporter plasmids in which the IRF binding site (PU.1/mIRF)4) or the Ets binding site (mPU.1/IRF)4) was mutated. In some cases, these cells were cotransfected with either an IRF4 expression vector, an ICSBP expression vector, or the empty expression vector. CAT activity was determined as described promoter. Of the IRF proteins examined to date, only IRF4 and in the text. Transfections were performed in triplicate. Data are expressed ICSBP have been shown to bind to PU.1 in vitro (35). Therefore, http://www.jimmunol.org/ as the mean rate of radiolabeled chloramphenicol accumulation over time we cotransfected RAW264.7 cells with the (PU.1/IRF)4 reporter Ϯ (slope) SD. plasmid and an expression plasmid encoding IRF4 or ICSBP. As shown in Fig. 6, over-expression of ICSBP resulted in a 4-fold While our data are only suggestive of nuclear translocation, they enhancement in basal promoter activity. Meanwhile, over-expres- are consistent with a previous study by Lin et al. (8). These in- sion of ICSBP had no effect upon the basal expression of either the vestigators reported that stimulation of various ﬁbroblast cell lines Rous sarcoma virus long terminal repeat, or the NF-␬B-dependent with Sendai virus resulted in the rapid nuclear translocation of E-selectin (ELAM-1) reporters. Reporter plasmids, in which either IRF3, a ubiquitously expressed IRF family member.

the IRF or PU.1 binding sites were mutated, lacked basal activity by guest on October 1, 2021 and could not be activated by over-expression of ICSBP. In con- A composite PU.1/IRF site is functional in macrophages trast, over-expression of IRF4 did not result in enhanced transcrip- One objective of these studies was to determine whether IRF pro- tion of the promoter, as was seen with ICSBP. We also found that teins could form functional associations with PU.1 in macro- transfecting the cells with increasing amounts of the ICSBP ex- phages. This physical and functional interaction has been previ- pression plasmid (100–2000 ng) resulted in dose-dependent acti- ously demonstrated for Ig light chain enhancer expression in B vation of the reporter, whereas increasing amounts of IRF4 had no cells (19, 20). We sought to determine whether such composite effect on reporter gene expression (data not shown). Moreover, sites were also functional in macrophages, cells that express PU.1 these data also demonstrate that IRF4 over-expression in macro- constitutively. We transfected RAW264.7 macrophage cells tran- phages does not activate the (PU.1/IRF)4 reporter plasmid, which siently with a reporter plasmid containing the CAT reporter gene contrasts with its ability to activate this reporter in fibroblast cell under the control of a HSV thymidine kinase promoter ligated lines (19, 20). downstream of four tandem copies of a PU.1/IRF composite element (plasmid designated (PU.1/IRF)4), or reporter constructs containing composite elements in which either the IRF (designated Functional synergy between PU.1 and IRF proteins (PU.1/mIRF)4) or the Ets (designated (mPU.1/IRF)4) binding sites were mutated. EMSA analysis was used to demonstrate that the Interactions between PU.1 and IRF4, or between PU.1 and ICSBP, (PU.1/mIRF)4 mutant was still capable of binding PU.1 in vitro cannot be easily examined in macrophages because these cells (data not shown). In the absence of PU.1 binding, as in the case of constitutively express all three transcription factors. To study these the (mPU.1/IRF)4 mutant, the affinity of IRF binding is markedly interactions in isolation, we expressed these factors in fibroblast reduced (20). As shown in Fig. 6, the (PU.1/IRF)4 reporter plas- lines that do not intrinsically express PU.1, IRF4, or ICSBP. We mid was constitutively expressed in RAW264.7 macrophages, cotransfected NIH-3T3 cells with the (PU.1/IRF)4 reporter plas- whereas mutation of the IRF binding site completely abolished this mid and various combinations of PU.1 and IRF expression plas- activity. Similarly, mutation of the PU.1 binding site substantially mids. As shown in Fig. 7, neither IRF4 nor ICSBP alone could reduced transcription. These data demonstrate that a PU.1/IRF activate the promoter, although PU.1 alone could activate a low composite element can activate transcription in macrophages, that level of transcription. Coexpression of PU.1 and either IRF4 or neither the PU.1 nor the IRF binding site alone is sufficient for full ICSBP synergized to induce a high level of promoter activity in the activation of transcription, and that both proteins appear to be re- NIH-3T3 cells. It is likely that this synergy only became apparent quired for this activation. in the fibroblasts because the basal level of reporter gene activity We subsequently sought to determine whether IRF4 and/or in these cells was extremely low due to the absence of endogenous ICSBP could function in conjunction with PU.1 to activate the PU.1, IRF4, and ICSBP. The Journal of Immunology 2719

FIGURE 9. Activation of the IL-1␤ promoter by IRF4, but not by ICSBP, is dose-dependent. RAW264.7 cells were transiently cotransfected with the IL-1␤ promoter and 100, 500, 1000, or 2000 ng of either IRF4 or ICSBP expression plasmid. Total plasmid DNA was maintained constant using empty pcDNA-neo vector. Transfections were performed in tripli- Downloaded from cate. Data are expressed as the mean fold activity over background Ϯ SD.

gest that repression by these two IRF family members is independent of their ability to interact with PU.1. http://www.jimmunol.org/ Effects of IRF proteins on a natural PU.1-dependent promoter The studies described above assessed the ability of IRF4 and ICSBP to interact with PU.1 and to activate a synthetic promoter containing tandem copies of a PU.1/IRF composite site. We subsequently sought to determine whether IRF4 and ICSBP could activate transcription of a natural PU.1-dependent promoter. We FIGURE 8. Both IRF4 and ICSBP repress transcription of an MHC selected IL-1␤ as a potential gene that could be regulated by both class I promoter in distinct cell types. An H-2Ld MHC class I reporter PU.1 and IRF proteins in macrophages. The IL-1␤ promoter has plasmid was cotransfected with either an IRF4 or ICSBP expression plas- by guest on October 1, 2021 ␥ mid, or with empty vector alone, into RAW264.7 macrophages (A)or been previously shown to be regulated by both LPS and IFN- , NIH-3T3 fibroblasts (B). CAT activity was determined as described in the and to require a functional PU.1 binding site within the cap site- text. Transfections were performed in triplicate. Data are expressed as the proximal promoter (46). We cotransfected RAW264.7 cells with mean rate of radiolabeled chloramphenicol accumulation over time an IL-1␤ reporter plasmid and increasing amounts of expression (slope) Ϯ SD. plasmids encoding either IRF4 or ICSBP (100–2000 ng). As shown in Fig. 9, we found that over-expression of ICSBP resulted in a consistent 40–50% increase in basal transcriptional activity, IRF4 and ICSBP can either activate or repress transcription in which was not further enhanced by increasing the quantities of a promoter-specific manner ICSBP expression plasmid used. In contrast, over-expression of Our data demonstrate that IRF4 and ICSBP can activate transcrip- IRF4 activated the IL-1␤ reporter in a dose-dependent fashion, tion of a promoter controlled by PU.1/IRF composite elements. with more than 7-fold activation at the highest amount of IRF4 Previous studies have reported that IRF4 and ICSBP function as a expression plasmid used. These results are qualitatively distinct transcriptional repressor in various cell types (5, 10, 18). There- from those obtained using the (PU.1/IRF)4 reporter plasmid (Fig. fore, it was important to confirm that IRF4 and ICSBP could also 6), suggesting that the two promoters are regulated differently by suppress transcription in our model system. The PU.1-independent these IRF proteins. H-2Ld MHC class I promoter had been previously reported to be IRF-dependent activation of PU.1/IRF composite elements de- negatively regulated by both IRF4 and ICSBP (5). We cotrans- pends on phosphorylation of PU.1 at serine 148 (7, 19–21). We fected RAW264.7 cells with a CAT reporter plasmid under the subsequently sought to determine whether synergistic activation of control of the H-2Ld promoter and an expression plasmid encoding the PU.1-dependent IL-1␤ promoter by PU.1 and IRF proteins also either IRF4 or ICSBP. As shown in Fig. 8, over-expression of required serine 148. We cotransfected NIH-3T3 cells with the either IRF4 or ICSBP resulted in reduced basal promoter activity, IL-1␤ reporter plasmid, the IRF4 or ICSBP expression plasmids, compared with cells transfected with the reporter alone. Similar and the wild-type or S148A mutant PU.1 expression plasmids, as results were obtained using transfected RAW264.7 cells (Fig. 8A) described above. As shown in Fig. 10A, IRF4, PU.1, and S148A and NIH-3T3 cells (Fig. 8B). Together, these findings demonstrate alone were incapable of activating expression of the reporter plas- that IRF4 and ICSBP can repress transcription of the PU.1-inde- mid. Synergistic activation of the IL-1␤ promoter was observed pendent H-2Ld promoter in both cell types, whereas only ICSBP when cells were cotransfected with IRF4 and PU.1 expression can activate transcription of the PU.1-dependent (PU.1/IRF)4 re- plasmids. Unlike wild-type PU.1, the S148A mutant failed to syn- porter in both cell lines. Furthermore, repression of the H-2Ld ergize with IRF4 to activate the reporter plasmid. Similar results promoter in RAW264.7 cells (that constitutively express PU.1) were obtained using this promoter in cells that over-expressed and NIH-3T3 cells (that lack PU.1) by both IRF4 and ICSBP sug- ICSBP (Fig. 10B). However, cotransfection of ICSBP with the 2720 EXPRESSION AND FUNCTION OF IRF4 AND ICSBP IN MACROPHAGES

genes, although the contribution of ICSBP to macrophage functions remains largely unknown. Our finding that IRF4 is expressed in macrophages raises the possibility that these two IRF proteins differentially regulate the same target genes. Thus, we compared the capacity of IRF4 and ICSBP to regulate transcription in RAW264.7 murine macrophages. We also tested the hypothesis that these two IRF proteins could form functional complexes with the Ets-like protein PU.1. The function of these IRF proteins in different cell lines, both with and without PU.1, are summarized in Table I. Together, these data suggest a complex molecular mechanism that regulates the expression of IFN-responsive genes by IRF4 and ICSBP. Previous studies had failed to detect IRF4 mRNA in macrophage cell lines using Northern blot analysis (7, 10). Our success in detecting these transcripts presumably resulted from the use of the more sensitive semiquantitative RT-PCR approach. Consistent with this possibility is our finding that the RAW264.7 cell line expressed markedly lower levels of IRF4 transcripts than primary B cells (Fig. 2). When normalized to ␤-actin expression, it appears Downloaded from that macrophages express 25–35% of the IRF4 message expressed in B cells. The lack of CD19 transcripts in RNA prepared from primary thioglycollate-elicited macrophages demonstrates the lack of B cell contamination (Fig. 2). These data support the conclusion that macrophages produce IRF4 message. In summary, confirma- tion that macrophages express IRF4 came from: 1) the sequence of http://www.jimmunol.org/ the RT-PCR product (data not shown), 2) specific binding of a 45-kDa protein by non-cross-reactive anti-IRF4 Abs (Fig. 3B), and 3) supershifting of a specific DNA-protein complex by both anti- PU.1 and anti-IRF4 Abs (Fig. 1). Therefore, we cannot fully ex- plain why previous studies failed to identify macrophages as cells that can produce IRF4, although the paucity of IRF4 message in FIGURE 10. The IL-1␤ promoter can be synergistically activated by macrophages may have been below the level of detection of de- PU.1 and IRF proteins. NIH-3T3 fibroblasts were transiently cotransfected tection techniques used in previous studies. with the IL-1␤ reporter plasmid, wild-type, or S148A mutant PU.1 expres- Several features indicate that IRF4 and ICSBP are differentially by guest on October 1, 2021 sion plasmid, and either the IRF4 (A) or ICSBP expression plasmid (B). regulated in macrophages. First, only ICSBP expression was in- Luciferase activity was determined as described in the text. Transfections creased following IFN-␥ treatment. This is consistent with data were performed in triplicate. Data are expressed as mean fold activity over obtained using lymphocytes where ICSBP, but not IRF4, expres- Ϯ background SD. sion was up-regulated by IFN-␥ (10, 33). Second, LPS stimulation appeared to induce nuclear translocation of IRF4, but not of ISCBP S148A mutant resulted in a complete loss of synergy, as compared (Fig. 5). Third, over-expression of ICSBP activated transcription with the incomplete loss of activity seen with coexpression of of the (PU.1/IRF)4 reporter plasmid (Fig. 6) and weakly activated IRF4 with S148A. These data demonstrate that a natural PU.1- transcription of the IL-1␤ reporter plasmid (Fig. 9). In contrast, dependent promoter can be synergistically activated by these IRF over-expression of IRF4 only enhanced transcription of the IL-1␤ proteins and PU.1. This synergistic activation with ICSBP requires reporter plasmid. However, both IRF4 and ICSBP were capable of a critical serine residue on PU.1, whereas IRF4 may be less de- repressing the PU.1-independent H-2Ld promoter (Fig. 8). To- pendent upon this serine. gether, these data suggest that the expression and function of IRF4 and ICSBP are regulated by different mechanisms in macrophages. Discussion Furthermore, such differences suggest that these IRF proteins serve ICSBP is expressed in both lymphoid and myeloid cells, and has nonredundant functions in macrophages which may, in part, be been reported to repress the expression of several IFN-responsive related to their ability to interact with PU.1.

Table I. Summary of transfection data

Cell Line Reporter Overexpression of IRF4/Pip Overexpression of ICSBP

RAW264.7 (PU.1/IRF)4 No activation Activation NIH-3T3 (PU.1/IRF)4 Synergy with PU.1 Synergy with PU.1 NIH-3T3 (PU.1/IRF)4 No synergy with S148A No synergy with S148A

RAW264.7 IL-1␤ Activation Weak activation NIH-3T3 IL-1␤ Synergy with PU.1 Synergy with PU.1 NIH-3T3 IL-1␤ Partial synergy with S148A No synergy with S148A

RAW264.7 H-2Ld Repression Repression NIH-3T3 H-2Ld Repression Repression The Journal of Immunology 2721

Our findings also extend several previous reports on the func- contain little phosphorylated PU.1), and 2) that only IRF4 was tions of both ICSBP and PU.1/IRF complexes. Nelson et al. (5) capable of synergizing with the S148A PU.1 mutant (albeit at demonstrated that ICSBP repressed several ISRE-containing pro- lower levels than wild-type PU.1). moters in macrophages. In contrast, Eklund et al. (24) recently A role for ICSBP in the regulation of the gp91phox and IL-12 reported that the PU.1/IRF composite site within the gp91phox pro- p40 promoters has been reported, whereas the specific role of IRF4 moter was positively regulated by ICSBP in U937 cells. These in macrophages remains to be determined. However, mice lacking investigators found that PU.1, ICSBP, and IRF1 functioned to- either a functional IRF4 or ICSBP gene do exhibit impaired resis- gether to activate transcription of a reporter plasmid containing tance to intracellular infection (13–16). Mice deficient in IRF4 do tandem copies of this composite element. Our data agree with not generate mature, functional lymphocytes (16), although the these results, and support a model in which ICSBP functions to effects of IRF4 deficiency on macrophage function have not yet repress transcription when bound to ISRE sequences, but instead been reported. Future studies will be needed to determine precisely activates transcription when bound to PU.1. Thus, the capacity of how these transcription factors contribute to the phenotype ob- ICSBP to activate or repress transcription appears to be determined served in these knockout mice. in a promoter-specific context, defined by the proteins with which it interacts. Acknowledgments These previous studies used reporter plasmids containing tan- We thank Shuyan Wang and Carrie Riendeau for their excellent technical phox dem copies of the Ig light chain enhancer or gp91 PU.1/IRF assistance, Dr. Keiko Ozato for the ICSBP expression and MHC-I reporter composite elements to drive transcription (19, 20, 24). Here, we plasmids, and Dr. Stefanie Vogel for critical review of this manuscript. showed that a natural promoter (i.e., IL-1␤) could be activated by IRF4, and weakly by ICSBP, in RAW264.7 macrophages (Fig. 9). References Downloaded from Furthermore, this promoter is activated synergistically by both 1. Stark, G. R., I. M. Kerr, B. R. G. Williams, R. H. Silverman, and R. D. Schreiber. IRF4 and ICSBP with PU.1 in NIH-3T3 fibroblasts (Fig. 10). The 1998. How cells respond to interferons. Annu. Rev. Biochem. 67:227. ␤ 2. Nguyen, H., J. Hiscott, and P. M. Pitha. 1997. The growing family of interferon IL-1 promoter is not known to contain functional PU.1/IRF com- regulatory factors. Cytokine Growth Factor Rev. 8:293. posite sites, and future studies will be needed to identify the ele- 3. Drew, P. D., G. Franzoso, K. G. Becker, V. Bours, L. M. Carlson, U. Siebenlist, ments within this 3.7-kb IL-1␤ promoter fragment that mediates and K. Ozato. 1995. NF-␬B and interferon regulatory factor 1 physically interact and synergistically induce major histocompatibility class 1 gene expression. J. In- http://www.jimmunol.org/ this synergy. The function of ICSBP/PU.1 complexes has been terferon Cytokine Res. 15:1037. previously examined in NIH-3T3 fibroblasts (35). One study re- 4. Martin, E., N. C., and Q. Xie. 1994. Role of interferon regulatory factor 1 in ported that ICSBP/PU.1 complexes failed to activate the same induction of nitric oxide synthase. J. Exp. Med. 180:977. 5. Nelson, N., M. S. Marks, P. H. Driggers, and K. Ozato. 1993. Interferon con- (PU.1/IRF)4 reporter plasmid used also in our study. This finding sensus sequence-binding protein, a member of the interferon regulatory factor contrasts with our data in these same cells, but the differences may family, suppresses interferon-induced gene transcription. Mol. Cell. Biol. 13:588. 6. Yamamoto, H., M. S. Lamphier, T. Fujita, T. Tanaguchi, and H. Harada. 1994. be due to the IRF expression plasmids used in each study. Brass et The oncogenic transcription factor IRF-2 possesses a transcriptional repression al. (35) used an epitope-tagged ICSBP expression plasmid, and a latent activation domain. Oncogene 9:1423. whereas the experiments reported here were performed using a 7. Eisenbeis, C. F., H. Singh, and U. Storb. 1995. Pip, a novel IRF family member, is a lymphoid-specific, PU.1-dependent transcriptional activator. Genes Dev. wild-type ICSBP cDNA that lacked an epitope tag.

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