Triggering of the Human Interleukin-6 Gene by Interferon-g andTumor Necrosis Factor-a in Monocytic Cells Involves Cooperationbetween Interferon Regulatory Factor-1, NFkB, and Sp1Transcription Factors*

(Received for publication, May 22, 1995, and in revised form, September 1, 1995)

Josiane Sanceau‡§, Tsuneyasu Kaisho¶, Toshio Hirano¶, and Juana Wietzerbin‡

From ‡INSERM, U365, “Interferons et Cytokines,” Institut Curie, Section de Recherches, 26, rue d’Ulm, 75231 Paris,France and the ¶Biomedical Research Center, Osaka University Medical School, Division of Molecular Oncology, 2-2Yamada-oka, Suita, Osaka 565, Japan

We investigated the molecular basis of the synergisticinduction by interferon-g (IFN-g)/tumor necrosis fac-tor-a (TNF-a) of human interleukin-6 (IL-6) gene inTHP-1 monocytic cells, and compared it with the basis ofthis induction by lipopolysaccharide (LPS). Functionalstudies with IL-6 promoter demonstrated that three re-gions are the targets of the IFN-g and/or TNF-a action,whereas only one of these regions seemed to be impli-cated in LPS activation. The three regions concernedare: 1) a region between 273 and 236, which is the min-imal element inducible by LPS or TNF-a; 2) an elementlocated between 2181 and 273, which appeared to reg-ulate the response to IFN-g and TNF-a negatively; and 3)a distal element upstream of 2224, which was inducibleby IFN-g alone. LPS signaling was found to involveNFkB activation by the p50/p65 heterodimers. Synergis-tic induction of the IL-6 gene by IFN-g and TNF-a, inmonocytic cells, involved cooperation between theIRF-1 and NFkB p65 homodimers with concomitant re-moval of the negative effect of the retinoblastoma con-trol element present in the IL-6 promoter. This removaloccurred by activation of the constitutive Sp1 factor,whose increased binding activity and phosphorylationwere mediated by IFN-g.

IL-61 is a multifunctional cytokine involved in controllingmany cell functions, including antibody synthesis by B cells, Tcell cytotoxicity, stem cell differentiation, and induction ofacute phase proteins. IL-6 is produced in response to a varietyof noxious stimuli, including viral and bacterial infections (1–4). Deficient regulation of the IL-6 gene is involved in thepathogenesis of autoimmune diseases and affects normal andleukemic hematopoiesis (4–9). Recent studies demonstrated

the presence of a defect in IL-6 production in Fanconi’s anemia(10, 11), suggesting that is partly responsible for the alteredhemopoiesis in Fanconi’s anemia patients.The transcriptional regulatory elements present in the 59-

flanking region of the human IL-6 gene have been studied byseveral laboratories. Various elements responsible for IL-6gene induction have been identified, including phorbol 12-my-ristate 13-acetate, cAMP, NFkB, NF-IL-6, and multiple cyto-kine-responsive elements (3, 4, 12–16). Tumor suppressor geneproducts p53 and pRB have been reported to suppress IL-6promoter activity (17).Different laboratories, including ours, showed that there are

cell type- dependent differences in the mechanism and tran-scription factors involved in IL-6 gene induction. Thus, TNF-ainduced IL-6 in a variety of cell types (18–21), but it failed to doso in monocytes (2, 3, 20). We have shown that IFN-g is anessential co-signal for TNF-a in the induction of IL-6 in mono-cytic THP-1 cells (22).The role of IL-6 in the immune response, acute phase reac-

tion, and hematopoiesis, and the fact that monocytic cells ap-peared to be one of the major physiological sources of thiscytokine, prompted us to analyze the molecular mechanismresponsible for its induction in this cell type.Although the mechanism of transcriptional activation medi-

ated by different inducers in several cell types has been exten-sively studied, the mechanism responsible for synergistic IL-6gene induction by IFN-g and TNF-a in monocytic cells has stillnot been identified.We postulated that both IFN-g and TNF-a activate tran-

scription factors, which should act simultaneously to induceIL-6 gene expression. This hypothesis was supported by ourprevious finding that sequential stimulation with IFN-g, fol-lowed by TNF-a stimulation, did not lead to IL-6 mRNA induc-tion (22). This implied that IFN-g and TNF-a induced or acti-vated different transiently expressed components, which mustact together in cooperation to trigger IL-6 gene expression.It has been suggested that the induction of NFkB binding

activity by TNF-a contributes to the activation of the IL-6promoter in some cell lines, including monocytic cells (3, 18–20). The NFkB/Rel family of transcription factors consists of atleast five proteins, including p65 (Rel A), p50, c-Rel, and p52,which are related to each other through an N-terminal stretchof 300 amino acids called the Rel homology domain. DNAbinding occurs through dimerization of the family members,resulting in numerous homo- and heterodimeric combinationsof NFkB/Rel proteins. The C-terminal region of p65, c-Rel, andRel-B harbor a transcriptional activation domain, and the invivo transcriptional activity is attributed to the p65-, c-Rel-,

* This work was supported by grants from the Institut NationalScientifique et de la Recherche Medicale (INSERM) and the Associationpour le Recherche contre le Cancer (ARC). The costs of publication ofthis article were defrayed in part by the payment of page charges. Thisarticle must therefore be hereby marked “advertisement” in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.§ To whom correspondence should be addressed: INSERM U365,

“Interferons et Cytokines,” Institut Curie, Section de Recherches, 26,rue d’Ulm, 75231 Paris, France. Tel.: 33-1-4325-8267; Fax:33-1-4407-0785.

1 The abbreviations used are: IL-6, interleukin-6; TNF-a, tumor ne-crosis factor-a; EMSA, electrophoresis mobility shift assay; IRF-1, in-terferon-regulatory factor 1; RCE, retinoblastoma control element;FCS, fetal calf serum; PBS, phosphate-buffered saline; BSA, bovineserum albumin; IFN, interferon; MOPS, 4-morpholinepropanesulfonicacid; ISRE, IFN-a-stimulated response element; ISGF3, interferon-stimulated gene factor 3.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 270, No. 46, Issue of November 17, pp. 27920–27931, 1995© 1995 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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and Rel-B-containing dimers (23).The elements involved in the IFN-g-mediated induction of

IL-6 expression are not known. The binding of IFNa/b to theirspecific receptors rapidly activates a latent cytoplasmic tran-scription factor, ISGF3 (interferon-stimulated gene factor 3).ISGF3 is transiently activated and has been shown to stimu-late ISRE-dependent transcription (24–27). Other regulatoryfactors, including interferon-regulatory factor 1 (IRF-1) andIRF-2, have also been shown to be involved in the regulation ofthe IFN system. IRF-1 and IRF-2 bind to similar cis elementswithin type I IFN and IFN-inducible genes. IRF-1 functions asa transcriptional activator, while IRF-2 represses IRF-1 func-tion (28–33). Both factors appear also to bind specifically toISRE sequences (34, 35).We performed a functional analysis of the 59-flanking region

of IL-6 gene using transient transfection of CAT reporter genelinked to IL-6 promoter, in order to clarify the molecular mech-anism involved in the synergistic induction by IFN-g andTNF-a of the IL-6 gene in human monocytes. The monocyticTHP-1 cell model appears to be particularly suitable for molec-ular dissection of IFN-g/TNF-a-mediated synergistic inductionof IL-6 gene expression, because in these cells, the IL-6 gene isnot constitutively expressed (22).Analysis of IL-6 promoter constructs, electrophoresis mobil-

ity shift assay (EMSA), and immunoprecipitation analysis haveshown that the synergistic induction of IL-6 gene by IFN-g andTNF-a in human monocytic cells involved cooperation betweenIRF-1 and NFkB binding elements, as well as the removal ofthe negative effect of the retinoblastoma control element (RCE)present in the IL-6 promoter (IL-6-RCE). This IL-6-RCE con-tained, among other components, a core 59-CCGCC-39 sequencehomologous with the consensus binding motif, which is thetarget of the Sp1 factor. Sp1 is a constitutively expressed tran-scription factor present in a wide range of cell types and bindsa GC-rich consensus sequence present in many cellular andviral promoters. Sp1 contains three zinc fingers that mediateDNA binding and four domains that mediate transcriptionalactivation (36). Upon binding to the DNA containing the GCbox in the cell nucleus, Sp1 becomes phosphorylated on multi-ple sites by double-stranded DNA-dependent kinase (37). It hasbeen shown that specific interaction between DNA-binding do-mains of the p65 subunit-NFkB and Sp1 bound the DNA mod-ulates transcription of human immunodeficiency virus type 1in response to cellular activation (38).Our results show that the contribution of IFN-g to the trig-

gering of IL-6 gene expression in human monocytes involves achange in the amount and phosphorylated state of Sp1, to-gether with the induction and activation of IRF-1. These factorscooperate synergistically with homodimer p65-NFkB, which isactivated by TNF-a.

MATERIALS AND METHODS

Cell Culture—THP-1 cells (strain TB202; American Type CultureCollection, Rockville, MD) were grown (7% CO2) in RPMI 1640 (LifeTechnologies, Inc.) supplemented with 10% heat-inactivated FCSshown to be endotoxin-free (,0.1 IU/ml; Myoclone, Life Technologies,Inc.).Reagents—rHuIFN-g (specific activity, 2 3 107 units/mg protein) was

a gift from Roussel-Uclaf (Romainville, France); rHuIFN-a2 was pro-vided by Shering (Kenilworth, NJ); rHuTNF-a (specific activity 6 3 107

units/mg protein) was produced by Genentech and provided by Boeh-ringer-Ingelheim (Dr. G. R. Adolf, Vienna, Austria); LPS (Escherichiacoli, serotype 0111:B4) was purchased from Sigma. Rabbit affinity-purified polyclonal antibodies against human Rb, Sp1, IRF-1, IRF-2,and NFkB family proteins were from Santa Cruz Biotechnology, Inc.(Tebu, France). Monoclonal anti-phosphoserine was from Sigma. Puri-fied Sp1 protein was from Promega. Enhanced chemiluminescence(ECL) Western blotting kit reagent and Rabbit reticulocyte lysate sys-tem were from Amersham (Les Ulis, France).

IL-6-CAT Construct and the 59-Deletion Mutants—A 1.2-kilobasepair BamHI-XhoI fragment which contained the IL-6 59-flanking regionof promoter (18) was deleted by using the Exo-mung deletion kit (Strat-agene), inserted into a pCAT™-Basic plasmid (Promega) at the SalIblunt end and HindIII sites, resulting in the following plasmids:del(2181) (2181 to 114), del(2108) (2108 to 114), del(273) (213 to114), and del(236) (236 to 114). A del(2224) (2224 to 114) plasmidwas generated by deleting the BamHI-NheI fragment (21160 to 2224).All resultant plasmids were verified by sequencing.Transient Transfections—2 3 107 THP-1 cells (logarithmic growth

phase, washed three times with PBS) suspended in 200 ml of DMEM(Dulbecco’s minimal essential medium, Life Technologies, Inc.) weremixed gently with 15 mg of indicated dried supercoiled IL-6 promoter-plasmid DNAs and 5 mg of pSVb-galactosidase control plasmid as aninternal reference (Promega) (purified by two cycles of CsCl gradient)and were electroporated at 220 V, 960 microfarads, for 42 ms using aGene Pulser wired to an electroporation chamber (Bio-Rad). Cells weremaintained at room temperature for 10 min before dilution in 10 ml ofprewarmed RPMI 1640 supplemented with 10% FCS. Cells were stim-ulated 1 h later with IFN-g (400 units/ml) and/or TNF-a (400 units/ml),or with LPS (1 mg/ml) in fresh RPMI 1640/10% FCS. After stimulationof 36 h, cells were analyzed for CAT activity, essentially as described byGorman et al. (39). Each CAT reaction was performed with 20 mgprotein extract and 2.3 nmol of 14C-labeled chloramphenicol (54 mCi/mmol) for 2 h at 37 °C. b-Galactosidase enzyme was measured in thesame cell extracts as described previously (40).Nuclear Extracts and EMSA—To prepare nuclear extracts, 40 3 106

cells were washed twice in chilled PBS and then resuspended in 500 mlof lysis buffer containing 10 mM HEPES (pH 7.9), 50 mM NaCl, 1 mM

EDTA, 5 mM MgCl2, 10 mM sodium orthovanadate, 10 mM sodiummolybdate, 0.2 mM phenylmethylsulfonyl fluoride, 10 mg/ml proteaseinhibitors (pepstatin, leupeptin, aprotinin), and 0.05% Nonidet P-40.After swelling for 20 min on ice, glycerol was added (final concentration5%, v/v) and nuclei were pelleted by centrifugation at 600 rpm for 10min, at 2 °C. After rapid washing with the same buffer (containing 140mM NaCl instead of 50 mM), the nuclei were gently resuspended in 100ml of storage buffer containing 10 mM HEPES (pH 7.9), 400 mM NaCl,0.1 mM EDTA, 0.5 mM dithiothreitol, 5% glycerol, 0.5 mM phenylmeth-ylsulfonyl fluoride, 10 mM sodium vanadate, 10 mM sodium molybdate,and 10 mg/ml protease inhibitors. After 60 min on ice with gentle (andoccasional) mixing, particulate matter was eliminated by centrifugationat 100,000 3 g for 10 min at 4 °C. Protein content in the supernatantwas determinated using Bradford’s method (81).For EMSA, nuclear protein (10 mg) was incubated with radiolabeled

probe (20,000 cpm) in buffer containing 50 mM NaCl, 10 mM HEPES(pH 7.9), 5 mM Tris-HCl (pH 7.9), 1 mM dithiothreitol, 15 mM EDTA,10% glycerol, 500 mg/ml BSA-FV, and 800 mg/ml denaturated salmonsperm DNA (in a final volume of 12.5 ml) (without EDTA in the case ofSP1 oligonucleotide probe). After a 30-min incubation on ice, the nucle-oprotein complexes were resolved by a nondenaturing electrophoresis ina 5% polyacrylamide gel for 3 h at 20 mA in 1 3 TGE buffer (50 mM

Tris-HCl (pH 8.8), 180 mM glycine, and 2.5 mM EDTA), in refrigeratedcondition. The gel was dried and exposed overnight to a PhosphorIm-ager screen (Molecular Dynamics, Sunnyvale, CA). For competitionexperiments, a 100-fold molar excess of the unlabeled oligonucleotideswas added 15 min before incubation of nuclear extracts with the end-labeled oligonucleotides, while antisera were mixed directly with nu-clear extracts and binding buffer (without salmon sperm DNA) 1 hbefore adding salmon sperm DNA and radiolabeled probe.Immunoprecipitation and Immunoblot Analysis—Nuclear protein

samples (150 mg) were precleared with rabbit IgG nonimmune antiseraand protein A-Sepharose (Pharmacia) 2 h at 4 °C. After centrifugation(5 min, 4 °C), the supernatants were incubated with specific antibodies(1:500eme dilution) overnight at 4 °C, and then 8 mg of protein G-Sepharose (Pharmacia) was added and gently rocked for 4 h at 4 °C.Protein A- and protein G-Sepharose were swollen in 50 mM Tris-HCl(pH 8.8), 500 mM KCl, 1% Triton X-100, 0.5 mM phenylmethylsulfonylfluoride, 10 mg/ml protease inhibitors, 10 mM sodium molybdate, 10 mM

sodium orthovanadate. The protein G-Sepharose immunocomplexeswere successively washed three times with 100 mM Tris-HCl (pH 8.8),500 mM KCl, 0.5% Triton X-100, once with 10 mM Tris-HCl (pH 7.6), 150mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1%SDS, 10% glycerol, and once with 10 mM Tris-HCl (pH 8.8). All washeswere performed with buffers containing the anti-proteases and anti-phosphatases mixture. The proteins were eluted in 50 ml of 3% SDS,30% glycerol, 150 mMKCl, 10 mM Tris-HCl (pH 6.7), 200 mM b-mercaptoethanol (10 min at 95 °C), and specific immunoprotein complexes wereseparated by SDS-polyacrylamide gel electrophoresis, transferred to a

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nitrocellulose membrane (0.45 mM, Schleicher & Schuell) in refrigeratedconditions (200 mA, 5 h). BSA-saturated membranes were incubatedfirst with a specific antibody (overnight at 4 °C, 1:500 dilution, in PBS5% BSA-FV, 0.1% Tween 20), washed with PBS, 0.1% Tween 20, andthen incubated with 125I-protein A (Amersham, 500,000 cpm/ml of PBScontaining 2% BSA-FV, 0.1% Tween 20) for 1 h at 20 °C. After extensivewashing, membranes were exposed to PhosphorImager (MolecularDynamics).Shift-Western Blotting—Protein-DNA complexes were analyzed by

EMSA, as described above. After polyacrylamide gel electrophoresis,Western blots were done by semidry blotting using the Multiphor IINovaBlot electrophoretic transfer unit (Pharmacia) as described byDemczuk et al. (41). The first filter below the gel was nitrocellulose(BA85, 0.45 mm, Schleicher & Schuell) followed by a second anion-exchange filter DEAE membrane (Shleicher & Schuell). Radiolabeledcomponents were detected by autoradiography on DEAE membrane.For protein detection, BA85 membrane was first blocked as describedabove for immunoblot analysis; primary antibodies were applied at adilution of 1:1000, and ECL detection was performed according to themanufacturer’s procedures (Amersham).Nucleotide Sequences of Oligonucleotides Used in This Study—Puri-

fied synthetic oligonucleotides were provided by Eurogentec (Seraing,Belgium), and covered IL-6 promoter fragments between 273 and 254(A; 59-ctagaTGGGTTTTCCCATGAGTTCTt-39), between 2126 and2101 (B; 59-ctagaGCCCCACCCGCTCTGGCCCCACCCTCt-39), be-tween 2173 and 2145 (C; 59-ctagaATGCTAAAGGACGTAACATTGCA-CAATCTt-39), between 2207 and 2184 (D; 59-ctagaCTAAGCT-GAACTTTTCCCCCTAGTt-39), between 2283 and 2242 (E;59-ctagaTGAGTCACTAATAAAAGAAAAAAGAAAGTAAGGAAGAGTG-Gt-39). Oligonucleotides with mutated sequences were also used: 2283mt15, 59-ctagaTGGTTAGATAATAAAAGAAAAAAGAAAGTAAAGGAA-GAGTGGt-39, and 2283 mt31, 59-ctagaTGAGTCACTAATAAAA-GAAAAAAGAAAGTCGCATAAGAGTGGt-39. For competition experi-ments, we used synthetic oligonucleotides covering: (i) the NFkBbinding site from the human immunoglobulin k light chain enhancer(42) 59-ctagaCAGAGGGGATTTCCGAGAGGTt-39, (ii) the IFN-a-stim-ulated response element (ISRE) of 29,59-oligo(A) synthetase (43) 59-ctagaGATCCATGCCTCGGGAAAGGGAAACCGAAACTGAAGCCt-39,(iii) the C3-IRF (44) 59-ctagaAAGGGAAAGGGAAAGGGAAAGGGt-39,(iv) the AP-1 consensus motif 59-ctagaTGAGTCACTGAGTCACTGAGT-CACt-39, and (v) the SP1 consensus motif 59-ctagaGATGGGCGGAGT-TAGGGGCGGGACTATCt-39 (45). The lowercase letters represent thebases included for creating restriction sites. The underlined lettersindicate the mutated bases. After annealing, the synthetic double-stranded oligonucleotides were end-labeled using the Klenow-DNA po-lymerase and [a-32P]dCTP (DuPont NEN) and were purified onElutip™ columns (Schleicher & Schuell).RNA Extraction and Northern Blot Analysis—Total cellular RNA

was prepared by denaturation in guanidinium thiocyanate, followed bypelleting through a cesium chloride cushion (40). For Northern blotanalysis, 15 mg of total RNAs were loaded on a 1% agarose gel in MOPSbuffer, containing 0,7% formaldehyde and transferred onto nylon Hy-bond N1 membrane (Amersham). Probe hybridizations (106 cpm/ml)were carried out overnight at 65 °C in a Rapid Hybridization Buffer(Amersham).cDNA Probes—The cDNA probe for human IRF-1 was a 0.9-kilobase

KpnI fragment excised from pUCIRF-1 vector. The human IRF-2 cDNAwas a 1.2-kilobase XbaI fragment excised from pHIRF4S-51 vector.These cDNAs were a generous gift from Dr. T. Taniguchi (Osaka,Japan). The cDNA probes were labeled using the Redi-prime randomprimer labeling kit (Amersham), using [a-32P]dCTP (DuPont NEN).DNA Extraction—Plasmid DNA were prepared with the Qiagen plas-

mid kit (Qiagen, Coger, France), followed by two cycles of purification ofclosed circular DNA by equilibrium centrifugation in CsCl-ethidiumbromide gradients (40).

RESULTS

Functional Analysis of 59 Cis-regulatory Elements of the Hu-man IL-6 Gene—Functional analysis of the 59-flanking regionof the human IL-6 gene was carried out using the 21200 to 114fragment as well as a series of 59-deletion mutants of IL-6promoter, linked to a reporter pCAT-basic plasmid, which lacksboth enhancer and promoter sequences (Fig. 1A). These plas-mids were used to transfect THP-1 cells by electroporation, and1 h later, the cells were stimulated by either IFN-g and/orTNF-a, or by LPS, and tested for CAT activity after 36 h. When

THP-1 cells were transfected with the construct containing1200 base pairs of the IL-6 59-flanking region, they exhibited a5-fold increase in CAT activity in response to combined treat-ment with IFN-g 1 TNF-a, and a 2.5-fold increase after IFN-gtreatment, but displayed no significant response to TNF-aalone. As expected, LPS induced a 7-fold increase in CATactivity (Fig. 1B), in agreement with the observations reportedby other groups for other cell systems (3, 4, 17–20). Deletions ofthe regions between 21200 and 2224 and between 21200 and2181 resulted in constructs whose respective CAT activitiesrose 4-fold and 2.5-fold in response to IFN-g 1 TNF-a treat-ment, and 5-fold and 4-fold in response to LPS. Removal of theregion spanning nucleotides 21200 to 2224 abolished the sen-sitivity to IFN-g. Deletion of the IL-6 promoter fragment up tonucleotide 2108 resulted in a loss of the synergistic increase inCAT activity in response to IFN-g 1 TNF-a stimulation,whereas 2-fold CAT activity was still observed after LPS.Although successive deletions at the 59 end of the IL-6 pro-

moter fragment resulted in a progressive reduction of the in-ducibility of CAT activity by LPS, the construct containing theregion from 273 to 114 retained its sensitivity to LPS andcontinued to display a 3-fold increase in CAT activity. It wasinteresting to observe that this fragment became inducible byTNF-a and exhibited a 2-fold increase in CAT activity. How-ever, no synergistic effect was induced by IFN-g 1 TNF-a.Removal of the region from 21200 to 236 resulted in thecomplete loss of inducibility by IFN-g and/or TNF-a, and byLPS (Fig. 1B).The results of the deletion analysis described above sug-

gested the presence of at least three regions differentially in-volved in IL-6 gene regulation by IFN-g and/or TNF-a, and byLPS. The first region is a distal fragment between 21200 and2224 that positively regulates the response to IFN-g alone.Although deletion of this fragment does not eliminate the syn-ergistic IFN-g 1 TNF-a response, a slide reduction in themagnitude of the induction of CAT activity is observed. Com-puter-based observation showed that this fragment contains anAP-1 site between 2283 and 2277, and a copy of the IFNenhancer core sequence 59-AAAGGA-39 (2253/2248) (13). Thesecond region is a repressor element, i.e. the sequence between2181 and 273, which would negatively affect the synergisticresponse to IFN-g and TNF-a without affecting sensitivity toLPS. This IL-6 DNA sequence contains, from 2126 to 2101, adirect repeat which is strikingly similar to the c-fos basaltranscription element, with 21/26 nucleotides matching theRB-repressible RCE (the RB control element) identified in thec-fos gene (17, 46). In this connection, Santhanam et al. (17)showed in functional assays that the overexpression of wildtype RB in Hela cells strongly repressed the activity of IL-6promoter constructs. In addition to these two elements, a thirdregion located between 273 and 236, probably correspondingto the minimal element necessary for the LPS response, alsoallowed TNF-a sensitivity. Within this fragment were a puta-tive AP-1 motif (261 to 255) and the sequence 59-GGGATTT-TCC-39 (272 to 263), which is highly homologous to the im-munoglobulin k light-chain enhancer sequence 59-GGGG-ACTTTCC-39 (42).Activation of Nuclear Protein(s) Binding to the IL-6 Promoter

DNA Fragments after Stimulation of THP-1 Cells—To deter-mine whether IFN-g and/or TNF-a induced the binding offactors that specifically recognize DNA sequences in the IL-6promoter, we prepared double-stranded synthetic oligonucleo-tides corresponding to IL-6 promoter DNA motif targets ofspecific transacting factors reported to be involved in the IL-6gene induction in other cell types (3, 12–16).Nuclear protein extracts, prepared from both untreated cells

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and cells stimulated for 1 h with either IFN-g and/or TNF-a orwith LPS, were submitted to EMSA with several radiolabeleddouble-stranded synthetic oligonucleotides. This experimentwas an initial experiment, which was designed to screen theearly induction of specific protein-DNA complexes.Fig. 2 shows an EMSA using five different oligonucleotides

corresponding to (i) NFkB-like and AP-1 binding motifs (A/273), (ii) IL-6-RCE (RB control element) target motif (B/2126),(iii) the typical cAMP/phorbol ester-responsive motif, and theIL-1/TNF-a responsive elements (C/2173), (iv) the negativeregulatory domain-like sequence (and two putative copies ofGGAAAmotifs considered to be responsible for IFN inducibility(D/2207), and (v) the consensus AP-1 motif and a copy of theIFN enhancer core sequence 59-AAAGGA-39 (E/2283) (13).Formation of protein-DNA complexes was apparent with the

three regions A, B, and E described above, which were involvedin the functional response of the 59-flanking regions of the IL-6gene (Fig. 2). Surprisingly, no specific complexes were observedwith regions C and D, previously reported to be involved inIL-6 gene regulation by various inducers in other cells systems(3, 4, 12–16).Induction of NF-kB Binding Activity by TNF-a in THP-1

Cells without Concomitant IL-6 Production—We investigatedthe kinetics of NFkB activation in IFN-g- and/or TNF-a-treatedTHP-1 cells, and LPS-treated cells. Cells were stimulated by

the various inducers for 0.5, 1, or 2 h and nuclear extractsanalyzed for binding to the NFkB motif, using the radiolabeledsynthetic oligonucleotide A/273 (Fig. 3). Activated complexeswere detectable within 1 h after stimulation with TNF-a alone,as well as with LPS (Fig. 3A). The binding activity shown aftercombined treatment with IFN-g and TNF-a was similar to thatobtained after treatment with TNF-a alone. The abundance ofthe complexes induced by TNF-a alone or combined with IFN-gand by LPS increased throughout 2 h of stimulation withoutcomplex mobility change (Fig. 3A). After LPS treatment, theamount of the complexes increased throughout 4 h of stimula-tion; in contrast, TNF-a either alone or combined with IFN-g,led to a transient increase for 2 h and dramatically decreasedthereafter (data not shown). The specificity of the protein-DNAcomplexes was confirmed by complete competition of the bind-ing with unlabeled oligonucleotide A/273 as well as with theimmunoglobulin NFkB consensus motif, and the absence ofcompetition with AP-1 or SP1 consensus oligomers (Fig. 3B).We did not observe any specific binding complexes in untreatedcells or in cells stimulated with either IFN-g or IFN-a.Polyclonal rabbit antibodies against p50 and p65 subunits of

NFkB were used to probe the nuclear IL-6-NFkB binding com-plexes for the presence of corresponding proteins (Fig. 3B).Addition of antisera against the p65-NFkB subunit super-shifted the protein-DNA complexes induced by TNF-a but im-

FIG. 1. Functional analysis of the 5*-cis-regulatory elements of the IL-6gene: relative CAT activity. A, sche-matic extended map of the IL-6 promoter,with the locations of DNA motifs knownto be implicated in IL-6 gene induction byvarious inducers, and deletion mutants ofthe human IL-6 promoter fused to thepromoterless CAT gene. B, to assess basalpromoter activity and its responsivenessto IFN-g and/or TNF-a, and to LPS,THP-1 cells were tranfected as describedunder “Materials and Methods.” CAT ac-tivity was determined after 36 h. The IL-6promoter response (-fold induction) is theratio of CAT activity in stimulated cells tothat in unstimulated cells, which is de-fined as 1.0. Values are means of eightindependent experiments with standarddeviation less than 10%.

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paired the formation of the complexes induced by LPS. Addi-tion of antisera against the NFkB subunit p50 did not modifythe TNF-a-induced complexes, but those induced by LPS werepartially supershifted. Antibodies against the p52 and c-RelNFkB subunits did not interact with any of these protein-DNAcomplexes. Nevertheless, combined treatment with antibodiesagainst p65 1 p52 1 c-Rel NFkB subunits appeared to result inthe disappearance of the minor residual complex left in TNF-a-treated cell extracts (data not shown). Results similar tothose shown for the TNF-a-induced complexes were obtainedusing nuclear extracts of cells stimulated by IFN-g 1 TNF-a.These results suggest that p65 is contained in the complexes,whatever the inducers used, whereas p50 only seems to be aconstituent of the protein-DNA complexes induced by LPS.To further investigate the subunit composition of the nuclear

IL-6-NFkB binding complexes, nuclear extracts were immuno-precipitated with specific antibodies, and analyzed by Westernblot (Fig. 3C). In agreement with the results shown in Fig. 3B,p65 subunit-NFkB was found after stimulation with TNF-aalone, with IFN-g 1 TNF-a and LPS, whereas antibody againstp50 only revealed a major protein of 50 kDa after LPSstimulation (Fig. 3C).We previously showed that in THP-1 cells, stimulation by

combined treatment with IFN-g and TNF-a was required forendogenous IL-6 gene expression and protein secretion, andthat TNF-a treatment alone was ineffective (22). Here, how-ever, TNF-a was effective in activating nuclear protein bindingto the NFkB-like element and in driving CAT expression aftertransfection with the del(273) construct containing the NFkB-

like sequence. These observations suggested the presence of arepressive element(s) that might negatively affect TNF-a-me-diated transcription of the endogenous IL-6 gene.Involvement of RCE (Retinoblastoma Control Element) in

IL-6 Gene Repression in THP-1 Cells: Role of SP1 Protein—Itwas reported that Rb protein but not its mutants, repress IL-6promoter transcriptional activity in NIH 3T3 cells (17). Thisrepression was mediated through a cis-acting element, firstdescribed in the c-fos promoter (46). The IL-6 promoter con-tains a G1C-rich sequence between 2126 and 2104 that in-cludes two CCACC motifs (2123 to 2118 and 2109 to 2103)previously identified as the RB response element (RCE), andone CCGCC motif similar to the Sp1-binding site (2119 to2115) (12, 13).The results shown in Fig. 1 suggest that a cis-DNA element,

which is located between 2181 and 273 and contains, amongothers, the RCE motifs, might be involved in repression of IL-6gene expression in THP-1 cells. These observations promptedus to investigate the interactions between proteins and theIL-6-RCE sequence in nuclear extracts of THP-1 cells stimu-lated with various inducers. For this investigation, we used anoligonucleotide spanning both sites (B/2126). EMSA revealedthe presence of a major constitutive complex in untreated cells(Fig. 4A, left section). No major modification was observed afterthis stimulation. Complex formation was specific, since it com-peted with the non-radiolabeled specific oligonucleotide but notwith the unrelated oligonucleotide AP-1 (Fig. 4B). Note thatprotein-DNA interactions appeared to involve the Sp1 ele-ments, since binding displayed complete competition with a

FIG. 2. IFN-g and/or TNF-a, and LPS induce binding of specific nuclear proteins to the 5*-flanking region of the human IL-6 gene.A representative EMSA analysis showed the specific binding to oligonucleotide probes corresponding to IL-6 promoter fragments: A, 273 to 254(lanes 1–6); B, 2126 to 2101 (lanes 7–13); C, 2173 to 2145 (lanes 14–19); D, 2207 to 2184 (lanes 20–25); E, 2283 to 2242 (lanes 26–32). Todetermine the binding specificity, nuclear extracts from untreated THP-1 cells, or cells treated for 1 h with IFN-g and/or TNF-a or with LPS, werecompared by EMSA: lanes 1, 7, 14, 20, and 26, untreated nuclear extracts; lanes 2, 8, 15, 21, and 27, IFN-g-treated nuclear extracts; lanes 3, 9,16, 22, and 28, TNF-a-treated nuclear extracts; lanes 4, 10, 17, 23, and 29, IFN-g 1 TNF-a-treated nuclear extracts; lanes 5, 11, 18, 24, and 30,LPS-treated nuclear extracts. a* indicates competition with specific unlabeled double-stranded oligonucleotides (lanes 7, 12, 18, 25, and 31), andb* indicates competition with unlabeled unrelated double-stranded oligonucleotide (lanes 13 and 32).

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Sp1 consensus motif (Fig. 4B).Antibodies against purified Sp1 protein partially super-

shifted the B/2126 complex, whereas antibodies against Rbprotein had no effect on complex formation (Fig. 4B). Further-more, addition of purified Sp1 protein to the nuclear extractsled to a higher site occupancy without changing mobility (Fig.4B, right section).Taken together, these results suggest that Sp1 is part of the

protein-DNA complex formed with B/2126 oligonucleotide.This oligonucleotide, which encompasses the RCE-IL-6 pro-moter element, includes the core sequence 59-CCGCC-39 (2119to 2115), which covers part of the RCE motif (2123 to 2118);this motif appears to be homologous with the 59-CCGCCC-39core sequence, reported to be the consensus binding motif in-

volved in the interaction with the Sp1 factor. Furthermore,when we used an Sp1 consensus oligonucleotide as the radio-labeled DNA probe in EMSA experiments, a major constitutivecomplex was revealed (Fig. 4A, right section) with a mobilitysimilar to that obtained with the B/2126 probe (Fig. 4A, leftsection). The amount of the protein-Sp1 oligonucleotide com-plex rose slightly after stimulation by IFN-g, TNF-a, LPS, orIFN-a, and rose more markedly after combined stimulationwith IFN-g 1 TNF-a. Complex formation was impaired bycompetition with the specific unlabeled Sp1 oligonucleotide,and partial competition was observed with the B/2126 oligo-nucleotide (Fig. 4C). As expected, antibodies against the Sp1protein led to a supershift of the protein-DNA complex,whereas no effect was observed with pRb antibodies. In thesame way, as for the B/2126 probe complex, addition of Sp1protein greatly increased the amount of protein-DNA complexrevealed with the Sp1 oligonucleotide probe.To demonstrate the presence of Sp1 protein in the B/2126

DNA protein complex, we used shift-Western blot analysis,with either the B/2126 probe or the Sp1 consensus probe.When a complex was formed with the B/2126 probe (Fig. 5,A1), we could not reveal the presence of Rb protein (A2),whereas the antibodies against Sp1 protein recognized a spe-cific protein whose abundance increased slightly after IFN-g 1TNF-a treatment (A3). The antibodies against Rb protein iden-tified a faint lower band, probably corresponding to free Rbprotein (Fig. 5, A2).As expected, in a similar experiment using a radiolabeled

Sp1 probe (Fig. 5B), antibodies against Sp1 protein revealed aspecific protein contained in the protein-DNA complex (B3); theamount of Sp1 protein correlated with the amount of DNAprobe bound to this complex (B1), which increased after IFN-g1 TNF-a treatment (B3). A faint lower band was also seen,probably corresponding to free Sp1 protein. With pRb anti-bodies, no protein was revealed in the protein-DNA complex,and as in the experiment using the B/2126 probe, a lower bandwas detected, corresponding to free Rb protein (Fig. 5, B2).Immunoprecipitation of crude nuclear extracts of THP-1

cells stimulated with IFN-g alone or combined to TNF-a, usingantibodies against Sp1 protein, dramatically raised the level ofthe 95-kDa native Sp1 protein as shown by Western blot anal-ysis (Fig. 4D, upper section). To demonstrate that the effect ofIFN-g or IFN-g 1 TNF-a correlated with the activation of Sp1protein, the same immuno-Western blot was hybridized with amonoclonal anti-phosphoserine antibody. Fig. 4D (lower sec-tion) shows an increase in phosphorylated Sp1 protein, previ-ously reported to be the active form of this nuclear factor (36).These results correlate well with the increase in binding activ-ity shown by EMSA performed with the Sp1 probe after THP-1cell treatment with IFN-g 1 TNF-a (Fig. 4A, right section).Taken together, these results suggest the involvement of the

Sp1 protein in the protein-DNA interaction of the RCE presentin the IL-6 promoter. Although we cannot exclude the possibil-ity that other factors interact with the RCE-IL-6 promoter,shift-Western blot and antibody-supershift experiments never-theless suggest that Sp1 is part of the activity that interactswith this region.Regulation of IRF-1 and IRF-2 in the Synergistic Induction of

IL-6 Gene Expression by IFN-g 1 TNF-a—Functional analysis(Fig. 1) showed that a distal element between 21200 and 2224regulates positively the response to IFN-g alone, or combinedwith TNF-a. EMSA using probe E/2283, and encompassing acopy of the IFN enhancer core sequence, revealed the formationof 2 protein-DNA complexes, one of which was specificallyinduced by IFN-g treatment (Fig. 2). We investigated the ki-netics of complex activation in THP-1 cells treated with IFN-g

FIG. 3.Kinetics of induction of NFkB-binding protein in THP-1cells. A, THP-1 cells were stimulated with IFN-g and/or TNF-a, withIFN-a, or with LPS. Nuclear extracts were prepared after cell stimula-tion for 0.5, 1, or 2 h. EMSA was performed using radiolabeled oligo-nucleotide A/273. B, specificity of protein-DNA complexes. EMSA wasperformed with nuclear extracts from untreated cells (Cont) or cellsstimulated for 2 h with TNF-a or LPS. Competition studies were carriedout with specific oligonucleotides A/273 (273) and consensus NFkB, orwith unrelated oligonucleotides AP-1, B/2126 (2126), or Sp1. In addi-tion, binding studies including antibodies were carried out with twopurified rabbit antisera, specifically reactive with p50 or p65 NFkBsubunits (Ab p50, p65). NI, nonimmune antiserum added for nonspe-cific reactions. C, nuclear extracts from THP-1 cells, either untreated(Cont) or treated with various inducers for 2 h, were submitted toimmunoprecipitation using either specific antibodies against p50 or p65NFkB subunits, or nonimmune serum. After Western blot analysis,specific proteins were revealed with the corresponding antibodies, asdescribed under “Materials and Methods.” Arrows point to specific p65(left) or p50 (right) NFkB subunits. The relative mass of protein molec-ular markers is shown in the middle (Rainbow™, Amersham).

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and/or TNF-a, LPS, and IFN-a. The same nuclear extracts asthose already used for the NFkB experiments were analyzedfor binding activity to the IFN-enhancer motif, using probeE/2283 (Fig. 6A). In untreated cells, EMSA revealed the pres-

ence of a constitutive DNA-protein complex, C1 whose amountincreased in a time-dependent manner after stimulation byIFN-g and IFN-g 1 TNF-a. The amount of this C1 complex wasnot modified by TNF-a, LPS, or IFN-a. Note that, IFN-g alone

FIG. 4. Specificity of protein binding to RB control elements. A, EMSA was performed with nuclear extracts of THP-1 cells, eitheruntreated or stimulated for 0.5 h with IFN-g and/or TNF-a, LPS, or IFN-a, using radiolabeled double-stranded oligonucleotide B/2126 (left section),or radiolabeled consensus Sp1 oligonucleotide (right section). B, nuclear extracts of cells left untreated for 0.5 h (Cont) were submitted to EMSAusing the B/2126 radiolabeled probe to reveal specific competition between RCE-binding proteins and either unlabeled double-stranded oligonu-cleotide B/2126 or Sp1 consensus oligonucleotide. AP-1 unlabeled oligonucleotide was used as unrelated competitor. Nuclear extracts of untreatedcells (Cont) or IFN-g-treated cells were preincubated with antibodies (Ab) against Rb protein (Rb) or against Sp1 protein (Sp1), and radiolabeledprobe B/2126 was then added. Recombinant Sp1 protein (1.0 footprinting units) was mixed with nuclear extract of untreated cells (Cont) or cellstreated with IFN-g, and radiolabeled B/2126 probe was then added (right section). C, specifity of the EMSA complexes revealed with radiolabeledprobe Sp1. Before the addition of radiolabeled Sp1 oligonucleotide, nuclear extracts of untreated cells or cells treated with IFN-g, TNF-a, or IFN-g1 TNF-a were mixed either with unlabeled oligonucleotide (2126, Sp1), or with antibodies (Ab) against Sp1 protein or Rb protein;NI correspondedto nonimmune rabbit antiserum. Sp1 protein (1.0 footprinting unit) was added to the same nuclear extracts. D, immunoprecipitation of nuclearextracts of untreated THP-1 cells (Cont) or cells stimulated with IFN-g alone or IFN-g 1 TNF-a, using antibodies against Sp1 protein, followedby Western blot analysis. Upper part shows blot hybridization with Sp1 antibodies. Lower part, the same blot was dehybridized and reprobed withantibodies against phosphoserine protein. Left arrows point to the specific Sp1 protein. Right arrows (mass in kDa) point to the relative mass ofprotein molecular markers.

FIG. 5. Sp1 protein is part of IL-6-RCE-protein complex. EMSA was per-formed using either the radiolabeledB/2126 oligonucleotide (A) or the radiola-beled Sp1 consensus oligonucleotide (B).Shift-Western blot analysis was then per-formed as described under “Materials andMethods.” Radiolabeled DNA was visual-ized on DEAE membrane (A1 and B1)whereas either Rb protein (A2 and B2) orSp1 protein (A3 and B3) was revealed onnitrocellulose by Western blot analysisusing the respective specific antibodiesagainst Rb protein and Sp1 protein.

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induced a second protein-DNA complex (C2), which was notobserved after TNF-a or LPS stimulation. EMSA carried outwith nuclear extracts of cells treated with IFN-a for 2 h re-vealed a faint C2 complex.To define the sequence specificity of the C1 and C2 com-

plexes, competition experiments were performed with syn-thetic oligonucleotides representing several mutations of the2283 to 2242 region either in the putative interferon enhancercore sequence 59-AAAGG-39 (2283/mt31) or in the AP1 motif(2283/mt15), and also with the ISRE of the 29,59-oligo(A) syn-thetase gene promoter, known to be implicated in IFN re-sponses (43), and with the C3 oligonucleotide defined by itsIRF-1-binding activity (44).Oligonucleotide E/2283 and the mutant 2283/mt15 com-

peted for the formation of both complexes (Fig. 6B, mt 15),whereas the mutant 2283/mt31 lost this capacity (Fig. 6B, mt31), indicating that the IFN enhancer core sequence 59-AAAGG-39 was involved in the DNA-protein interaction. Com-plete competition for the C1 and C2 complexes was observedwith the ISRE and C3 oligonucleotides. A trimer AP-1 motifwas not able to compete for either complex, in agreement withthe behavior of mutant 2283/mt15. No competition was ob-served neither with the Sp1 nor with B/2126 oligonucleotides(Fig. 6B).To investigate the possible relationship between the C1 and

C2 complexes and ISGF3 (25–27, 47), nuclear extracts ofTHP-1 cells treated for 2 h with IFN-a were tested for theirbinding activity, using the E/2283, ISRE, or C3 oligonucleo-tides as DNA radiolabeled probes (Fig. 7). The pattern of theprotein-DNA complexes was very similar with the E/2283 andC3 oligonucleotide probes, suggesting that they bind the IRF-1-related factor. For ISRE-DNA-protein binding, only IFN-atreatment resulted in a double upper-induced binding activity

(Fig. 7); several constitutive complexes showed no quantitativeor qualitative changes, whatever the inducers used. These re-sults suggest that C1 and C2 complexes did not involve ISGF3,but rather IRF-1/IRF-2, unlike the IFN-a-induced complexesrevealed with the ISRE probe.As shown in Fig. 6C, the amount of constitutive DNA-bind-

ing C1 complex was markedly reduced by the IRF-2 antibodies,while the inducible complex C2 was supershifted by the IRF-1antibodies. The IRF-1 antibodies also abolished the time-de-pendent increase in the C1 complex, which remained at itsconstitutive level. These results demonstrate that IRF-2 isinvolved in the formation of the constitutive C1 complex, andthat IRF-1 is involved both in the formation of the inducible C2complex and in the increase in the amount of C1 complex.To establish whether this typical IRF recognition sequence is

sufficient for IFN-g activation and for the synergistic action ofIFN-g with TNF-a, we constructed mutated IL-6 promoter CATexpression plasmids. For this purpose, we linked oligonucleo-tide E/2283, or oligonucleotide 2283/mt31, which carried amutation in the putative IFN enhancer core sequenceAAAGGA (2253 to 2248), to del(2224)-CAT and called theresulting constructs del(2224)/E and del(2224)/Emt31, respec-tively (Fig. 1A). These constructs were then transfected intoTHP-1 cells. As shown in Fig. 1B, the relative induction of CATactivity after THP-1 transfection with construct del(2224)/Edemonstrated that the IFN enhancer core sequence is sufficientto confer responsiveness to IFN-g, because the mutation of theAAAGGA motif (see del(2224)/Emt31) abolished IFN-g sensi-tivity; the IFN-g 1 TNF-a synergistic induction was preserved,although to a much smaller extent (2-fold induction). Theseresults suggest that in addition to the IFN enhancer core se-quence, surrounding sequences might contribute to the syner-gistic effect of IFN-g with TNF-a.Transfection of either of the constructs del(2224)/E or

del(2224)/mt31 resulted in a similar LPS-induced increase inCAT activity (7- and 6-fold, respectively), showing that the IFNenhancer core sequence did not play an essential role in LPSinduction (Fig. 1). However, LPS inducibility required the co-operation of other(s) factor(s), as demonstrated by the progres-sive reduction of CAT activity after the transfection of IL-6promoter 59 end successive deletions.These results showed that IFN-g 1 TNF-a and LPS induced

the IL-6 gene by different pathways.

FIG. 6. Induction and specificity of IFN-g regulated IRF-1/IRF-2 complexes. Radiolabeled double-stranded oligonucleotideE/2283 was used for EMSA. A, binding reactions were tested withnuclear extracts prepared from untreated THP-1 cells or cells treatedwith various inducers for 0.5, 1, or 2 h. Arrows indicate C1- andC2-specific protein-DNA complexes. B, nuclear extracts from THP-1cells treated for 2 h with IFN-g 1 TNF-a were used to determine thespecificity of the two complexes by means of the following unlabeleddouble-stranded oligonucleotides: E/2283 without mutation (2283), orwith mutation (mt) in the AP-1 motif (mt 15) or in the IFN enhancercore sequence (mt 31), ISRE, and C3. Oligonucleotides AP-1, B/2126(2126) and Sp1 were used as nonspecific competitors. C, before additionof radiolabeled probe E/2283, nuclear extracts from THP-1 cells treatedwith IFN-g 1 TNF-a for 2 h were preincubated with antibodies againstIRF-2, IRF-1, or nonimmune antiserum (NI).

FIG. 7. E/2283, C3-radiolabeled oligonucleotides, and ISRE-radiolabeled oligonucleotide gave different patterns of the pro-tein-DNA complexes. Nuclear extracts of THP-1 cells treated for 2 hwith IFN-g, TNF-a, IFN-g 1 TNF-a, LPS, or IFN-a were tested for theirbinding activity, using oligonucleotides radiolabeled with E/2283 (leftsection), C3 (middle section), or ISRE (right section). Double arrowsindicate specific protein-DNA complexes.

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Differential Regulation of IRF-1 and IRF-2 Expression byIFN-g or LPS—Previous reports have shown that the IRF-1and IRF-2 factors display antagonist activities, and that theup-regulation of the binding activity of one factor results in thedown-regulation of the binding activity of the other (28–32).Our demonstration that the binding activities of IRF-1 andIRF-2 correlated with the synergistic induction of IL-6 pro-moter by IFN-g 1 TNF-a (Fig. 6) prompted us to compare theregulation of IRF-1/IRF-2 by IFN-g, TNF-a, or IFN-g 1 TNF-awith their regulation by IFN-a.For this purpose immuno-Western blot analysis of nuclear

extracts of THP-1 cells after 2 h of stimulation was performedusing antibodies against IRF-1 or IRF-2. A polypeptide of 42kDa was revealed by the IRF-1 antibodies, but only after stim-ulation by IFN-g or IFN-g 1 TNF-a (Fig. 8A, right section). TheIRF-2 antibodies detected a constitutive IRF-2 protein whoseabundance was not modified after cell treatment with IFN-g orTNF-a either alone or combined for 2 h. In contrast, treatmentwith LPS or IFN-a reduced the amount of IRF-2 protein (Fig.8A, left section). Since formation of the constitutive C1 complexinvolved IRF-2 protein binding (see Fig. 6C), we attempted toestablish whether a transient modulation of this protein oc-curred soon after stimulation. As shown in Fig. 8A (left section),treatment with IFN-g 1 TNF-a, LPS, or IFN-a reduced thelevel of IRF-2 protein in nuclear extracts of cells stimulated for30 min. The effect of IFN-g1TNF-a was transient, because thereduction in the amount of IRF-2 protein was not seen innuclear extracts of cells that had been stimulated for 2 h. Incontrast, the inhibition by IFN-a or LPS of the IRF-2 proteinlevel lasted throughout the 2 h of stimulation.Since the amount of IRF-2 protein diminished after cell

stimulation by IFN-g 1 TNF-a, LPS, or IFN-a, whereas IRF-1protein was only induced by IFN-g, we explored this effect tosee if it was related to an increase in IRF-1 and (or) a decreasein IRF-2 gene expressions. For this purpose, total RNAs wereextracted after 0.5, 1, or 2 h from cells stimulated with variousinducers and then analyzed by Northern blot. As shown in Fig.

9, IFN-g already induced IRF-1 mRNA after 30 min, in anamount which increased for up to 2 h of cell stimulation. Nosynergistic effect was observed after combined treatment withIFN-g and TNF-a. Stimulation by IFN-a also triggered IRF-1gene expression, but with different kinetics of accumulationsince IRF-1 mRNA was barely detected after 1 h of IFN-astimulation but its amount increased thereafter. Two IRF-1mRNAs of 2.1 and 3 kilobases were revealed, with a similaraccumulation kinetic. Neither TNF-a nor LPS induced IRF-1gene expression in THP-1 cells.In contrast, IRF-2 mRNA was constitutively expressed (Fig.

9). No significant modification of the IRF-2 mRNA level wasobserved whatever the period of incubation and the inducersused. The reduction in the level of this protein after stimula-tions with various inducers, without concomitant decrease in

FIG. 9. Kinetics of IRF-1 and IRF-2 gene expression in THP-1cells. Total RNAs (15 mg) of THP-1 cells treated with the indicatedinducers for 0.5, 1, or 2 h were analyzed by Northern blot hybridizationwith the IRF-1-radiolabeled probe. After dehybridization, the samemembrane transfer was successively hybridized with IRF-2 and actinprobes. Arrows indicate specific mRNA.

FIG. 8. Modulation of IRF-1 or IRF-2 proteins. A, nuclear extracts of untreated THP-1 cells (CONT) or cells treated with various inducersfor 30 min (upper section) or for 2 h (lower section) were submitted to immunoprecipitation using specific antibodies against IRF-1 or IRF-2,followed by Western blot analysis. IRF-2 protein was analyzed after treatment of THP-1 cells for 30 min or 2 h by various inducers, whereas IRF-1protein was analyzed after cell stimulation for 2 h. Middle arrows indicate the relative mass of protein molecular weight markers. Left or rightexternal arrows indicate specific IRF-2 or IRF-1 proteins. B, in vitro translation: total RNAs were extracted from cells left untreated for 2 h (Cont)or stimulated for 2 h, and were translated in vitro at a concentration of 480 mg total RNA/ml, using reticulocyte lysate and [35S]methionine underdefined optimal conditions (97 mM KCl and 1.8 mM Mg(CH3COO)2). After incubation of 1 h at 32 °C, volume aliquots corresponding to 5 3 106 cpmof labeled polypeptides were submitted to immunoprecipitation followed by Western blot analysis, as described under “Materials and Methods.”

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the IRF-2 mRNA level, prompted us to make sure that IRF-2mRNA was functionally active. Accordingly, total RNAs wereextracted from cells left untreated from 2 h or stimulated for 2h and translated in vitro, using reticulocyte lysate and [35S]me-thionine. Identical number of counts/min (5 3 106 cpm) wereanalyzed by immuno-Western blot, using specific IRF-2 anti-bodies. Similar amounts of IRF-2 protein were detected what-ever the inducers used (Fig. 8B).These results indicate that the differential regulation of

IRF-2 protein by IFN-g, LPS, and IFN-a takes place at post-translational level.

DISCUSSION

We investigated the molecular basis for the IFN-g/TNF-a-mediated synergistic induction of the human IL-6 gene in theTHP-1 monocytic cell line by functional analysis of this gene’spromoter. The results provided evidence that three regionsinside this promoter are the targets of the IFN-g/TNF-a action:(i) a region between 273 and 236, which constitutes the min-imal element inducible by LPS or TNF-a; (ii) an element lo-cated between 2181 and 273, which appears to regulate neg-atively the response to IFN-g and TNF-a; and (iii) a distalelement upstream of 2224, which is the only one inducible byIFN-g alone.Each of the DNA elements corresponding to the three func-

tional regions inducible by IFN-g and/or TNF-a and by LPS ledto the formation of specific DNA-protein complexes.The IL-6 promoter DNA fragment between 273 and 236 was

found to contain an NFkB-like element near a putative AP-1binding motif. In THP-1 cells, TNF-a or LPS induced binding tothis DNA motif, whereas IFN-g neither induced NFkB bindingactivity nor amplified the effect of TNF-a when added togetherwith it. The EMSA-DNA competition experiments with consen-sus immunoglobulin-kB oligonucleotide allowed us to concludethat the formation of the complexes induced by TNF-a and LPSis due to NFkB binding factors. EMSA including antibodies andimmunoprecipitation with anti-p50 and anti-p65 NFkB sub-unit antibodies revealed only the nuclear accumulation of p50and p65 after stimulation by LPS. The predominance of the p65subunit was shown to be the major factor induced by TNF-a orIFN-g 1 TNF-a. Various dimer combinations of induced factorshave been reported to be stimulus-dependent and to displaydistinct DNA binding specificities by activating distinct sets ottarget genes (23). Thus, homodimer p65 was previously shownto be selectively activated in other systems by TNF-a (48, 49).In THP-1 cells, the induction of NFkB binding activity by

TNF-a did not correlate with concomitant IL-6 production (22),but required the addition of IFN-g, unlike activation by LPS.This indicated that the mechanism involved in the induction ofendogenous IL-6 gene in THP-1 cells is inducer-specific.Although NFkB binding factors are certainly involved in IL-6

gene induction, the functional analysis of IL-6-CAT constructdeletions reported here shows that significantly greater stim-ulation of the CAT reporter gene is obtained for constructs thatcontain the 59 distal sequence of IL-6 promoter (pr-1200). Suc-cessive deletions of the 59 end of the IL-6 promoter fragment(21200 to 2108) resulted in the gradual reduction of synergis-tic CAT activity by IFN-g 1 TNF-a.EMSA using the oligonucleotide E/2283, which contains a

copy of IFN-enhancer-core sequence, showed that IFN-g, butnot TNF-a or LPS, induced specific binding activity. Similarresults were obtained with C3 oligonucleotide, suggesting thatan IRF-1-related factor was a component of the C1- and C2-specific protein-DNA complexes. EMSA experiments using an-tibodies against IRF-1 or IRF-2 allowed us to conclude that theformation of the constitutive C1 complex involved IRF-2 bind-ing and that IRF-1 was involved, both in the binding activity

of the inducible C2 complex and in the increased abundance ofthe C1 complex due to stimulation by IFN-g.In agreement with these findings, transfection of construct

del(2224)/Emt31 into THP-1 cells confirmed that the IFN-enhancer-core sequence AAAGG, which is located between2253 and 2248 (13), is sufficient to induce the response toIFN-g alone.The IRF-1 binding site identified in the IL-6 promoter was

shown to be the central motif of the IFN-stimulated responseelement (ISRE) found in the promoter of IFN-a-stimulatedgenes such as the 29,59-oligo(A)-synthetase gene (43). In EMSAusing the E/2283 probe, the IRF-related complexes C1 and C2were impaired by the ISRE oligonucleotide. However, afterIFN-a treatment, no C1 or C2 binding activity was observed.When EMSA was performed with the ISRE oligonucleotide forpurposes of comparison, it only revealed ISGF3-related com-plexes after IFN-a treatment, as expected (24–27).Induction of IRF-1 protein-DNA binding by IFN-g correlated

with the induction of IRF-1 mRNA and with the concomitantIRF-1 protein synthesis. On the other hand, IRF-2 mRNAconstitutive expression was not modified whatever the induc-ers, whereas the level of constitutive IRF-2 protein decreasedtransiently after IFN-g 1 TNF-a treatment. LPS or IFN-atreatment led to the disappearance of the constitutive IRF-2protein.The different effects exerted on IRF-2 protein down-regula-

tion by IFN-g 1 TNF-a on the one hand and by LPS on theother might partly account for the difference between the mag-nitude of IL-6 induction by each of these inducers in monocyticTHP-1 cells.LPS was recently shown to induce or activate proteins that

recognize the 39-end instability sequence of an mRNA thatprevents the latter’s translation (50–52). Such a mechanismmight account for the down-regulation of IRF-2 protein in vivo,since here we were unable to show by Northern blot analysisthat this inducer modulates either the expression of mRNAIRF-2 or its translation in vitro. Alternatively, the constitutiveIRF-2 protein might be rapidly degraded after its activation byIFN-g or LPS, as shown in other systems (53), thus allowingthe binding of another activator which in the case of IFN-gwould be IRF-1.As LPS did not, under our experimental conditions, induce

IRF-1, we might be justified in assuming that one or severalother factors activated by LPS could act as a transcriptionalfactor, as suggested by our functional CAT analysis (Fig. 1B).Our results suggest that IRF-1 may be a critical downstream

signaling factor involved in IFN-g signal transduction in mono-cytes/macrophages, particularly for genes whose maximal ex-pression is triggered by combined treatment with IFN-g 1TNF-a (22, 54–56).The fact that TNF-a induced NFkB binding activity, which

correlated with the inducibility of the del(273)-CAT constructbut failed to induce reporter constructs containing additionalupstream sequences, suggests that a silencer of NFkB activitymust be present in the IL-6 promoter. Functional analysis ofdeletion constructs indicated that this negative element mightbe located between 2181 and 273. This region contains, amongothers, a retinoblastoma control element known to be involvedin pRb-mediated repression of the c-fos promoter (46). Thepresence, as reported by Santhanam et al., of a region analo-gous to the RCE in the IL-6 promoter between positions 2126/2101, exhibiting similar patterns of c-fos and IL-6 promotersregulation, suggests that RCE may be involved in IL-6 generepression in monocytic cells. Evidence that RCE has a repres-sor role was obtained, in NIH 3T3 cells, by deletion experi-ments and cotransfections of IL-6 promoter with pRB express-

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ing vector (17).It is tempting to suggest that alteration(s) in the interaction

between the putative suppressor factor(s) and RCE allowed usto obtain a synergistic effect of IFN-g with TNF-a in THP-1cells. Synergistic CAT induction was observed for del(2181)but not for del(2108). The del(2181)-CAT construct containsthe intact IL-6-RCE motif, whereas a truncated RCE motif ispresent in the del(2108)-CAT construct.EMSA experiments performed with oligonucleotide B/2126,

which contains the IL-6-RCE motif, revealed one major consti-tutive specific RCE-protein complex in nuclear extracts ofTHP-1 cells. This complex was completely inhibited by consen-sus Sp1 oligonucleotide and was supershifted by Sp1 antibod-ies. In addition, recombinant Sp1 protein increased this siteoccupancy. Furthermore, we showed by shift-Western blotanalysis that Sp1 protein is part of the IL-6-RCE-protein com-plex, whereas Rb protein, undetectable in this complex, wasnot.Our observations are in agreement with the hypothesis pro-

posed by Udvadia et al. that Rb regulates transcription partlyby virtue of its ability to interact functionally with RB controlproteins, including Sp1 (45, 57–60).EMSA performed with the Sp1 consensus probe showed that

stimulation of THP-1 cells by IFN-g 1 TNF-a enhanced theamount of the protein-DNA complex. This result correlateswith a marked increase by IFN-g in the amount of serine-phosphorylated Sp1 protein.It has been shown that Sp1 is selectively phosphorylated

upon binding to its cognate recognition elements on DNA,suggesting that phosphorylation represents an early event inthe processes leading to transcriptional activation by Sp1.Phosphorylation of Sp1 is catalyzed by a double-stranded DNA-dependent kinase and requires binding to DNA containing theGC box (34, 61).It is tempting to postulate that the effect of IFN-g was partly

mediated by increased binding of Sp1 to its target sequence inthe IL-6 promoter, with concomitant Sp1 factor activation byspecific phosphorylation. This alteration might impair the neg-ative effect mediated by the IL-6-RCE.On the other hand, NFkB has been shown to synergize with

a number of transcription factors, including Sp1 (62, 63). It hasbeen reported that Sp1 specifically interacts with the N-termi-nal region of Rel A (p65); similarly, Rel A (p65) bound directlyto the zinc finger region of Sp1 factor. This interaction isspecific, because Rel A did not associate with several othertranscription factors known to be zinc finger proteins (36–38).Since the p65 homodimer induced by TNF-a in THP-1 cells isnot, by itself, sufficient to induce IL-6 gene expression, we canpostulate that functional interaction between p65 NFkB andthe activated-Sp1 bound to the adjacent target Sp1 site con-tributed to the induction of the IL-6 gene.Our observations allowed us to conclude that IL-6 gene ex-

pression in THP-1 cells by IFN-g 1 TNF-a, or by LPS treat-ment is mediated through different signaling pathways.LPS signaling was shown to involve interaction with CD14

(64, 65), which triggered the mitogen-activated protein kinaseRas-Raf1-dependent cascade, leading to IkB phosphorylationand subsequent translocation allowing accumulation of thep50/p65 NFkB heterodimer in the nucleus (23, 66–68). Sincethis p50/p65 complex was also identified in our cell system, asimilar pathway might be responsible for the IL-6 gene expres-sion induced by LPS treatment in THP-1 cells.In contrast, the synergistic effect of IFN-g 1 TNF-a on IL-6

gene induction in THP-1 cells might involve three simultane-ous processes. The first process is the activation of NFkB byTNF-a by a different pathway from that of activation by LPS,

leading to the activation of the p65 homodimer. This possibilityis supported by a recent report showing that, in murine macro-phages, TNF-a activates the mitogen-activated protein kinasecascade in a mitogen-activated protein kinase kinase kinase-dependent but c-Raf-1-independent fashion (69). The secondprocess is the induction of IRF-1 by IFN-g through a signalingpathway involving the activation of Jak1/Jak2 and Stat 1 (70–75). The third process is a concomitant change by IFN-g, in thestate of phosphorylation and abundance of the constitutive Sp1nuclear factor interacting with IL-6-RCE. The phosphoserinekinase implicated in the signaling pathway leading to phospho-rylation of Sp1, presumably activated by IFN-g, remains to beinvestigated. Transcriptional induction of the IL-6 gene mightresult from a coordinated effect exerted by factor Sp1 togetherwith IRF-1 and p65 homodimer-NFkB.The fact that regulation by IFN-g of the IL-6 gene in human

monocytes involved IRF-1 may be more generally related to thetumor suppressor/differentiation properties of IFN-g (32, 76).Deletion of a chromosomal segment that contains the IRF-1gene, mapped in chromosome 5q31.1, is very often observed inhuman leukemia (77). It is therefore possible that an alterationin the balance of IRF-1/IRF-2 may impair optimal physiologicalinduction of IL-6 by IFN-g/TNF-a in the monocytic cell com-partment, leading to abnormal cell maturation and prolifera-tion in certain hematological disorders and to neoplasia.Alterations in IL-6 production such as overexpression in

multiple myeloma and other type of cancers (2–9, 78, 79) orinhibition in Fanconi’s anemia (10, 80) may be suspected toplay a role in pathological cell growth and to affect hematopoi-etic cell differentiation. In this regard, the fact that the trig-gering of IL-6 gene expression by IFN-g in monocytic cellsrequired the induction of IRF-1 and involved phosphorylatedSp1 protein may be of physiological relevance, and one of theimportant homeostatic properties of IFN-g within the cytokinenetwork.

Acknowledgments—We thank Dr. T. Taniguchi (Osaka, Japan) forthe generous gift of IRF-1 and IRF-2 cDNAs, Dr. G. R. Adolf (Boeh-ringer-Ingelheim, Vienna, Austria) for the gift of human TNF-a, andDr. Lando (Roussel-Uclaf, Romainville, France) for the gift of humanIFN-g. We are grateful to A. Birot for expert secretarial assistance andto C. Sylvestri for valuable technical assistance for plasmid-DNA puri-fications and cell cultures.

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Josiane Sancéau, Tsuneyasu Kaisho, Toshio Hirano and Juana WietzerbinB, and Sp1 Transcription FactorsκFactor-1, NF

in Monocytic Cells Involves Cooperation between Interferon RegulatoryαFactor- and Tumor NecrosisγTriggering of the Human Interleukin-6 Gene by Interferon-

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