of August 31, 2018.This information is current as

Complexα1:STAT-1Functional Implications of the TNF ReceptorSignaling in Macrophages: Regulation and

α as Modulators of TNF-γ and IFN-αSTAT-1

Duane R. Wesemann and Etty N. Benveniste

http://www.jimmunol.org/content/171/10/5313doi: 10.4049/jimmunol.171.10.5313

2003; 171:5313-5319; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/171/10/5313.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2003 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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STAT-1� and IFN-� as Modulators of TNF-� Signaling inMacrophages: Regulation and Functional Implications of theTNF Receptor 1:STAT-1� Complex1

Duane R. Wesemann and Etty N. Benveniste2

TNF-� and IFN-� cooperate in the activation of macrophages. TNF-�-dependent activation of NF-�B is stronger in the presenceof IFN-�. STAT-1� associates with TNFR1 in TNF-�-treated cells, and this association attenuates TNF-�-mediated NF-�Bactivation. We hypothesized that nuclear localization of STAT-1� due to IFN-� signaling would preclude it from being recruitedto the TNFR1 and therefore enhance TNF-�-induced NF-�B activation. In the RAW264.7 macrophage cell line, TNF-� treatmentindeed recruits STAT-1� to the TNFR1, and this association is abrogated when cells are exposed to IFN-�. TNF-� treatmentinduces a more robust activation of NF-�B in STAT-1�-deficient cells, and restoration of STAT-1� inhibits TNF-�-dependentNF-�B activation. Our results suggest that a receptor-proximal level of cross-talk exists between these two cytokine pathways:IFN-� limits STAT-1� availability to the TNFR1 by depleting STAT-1� from the cytoplasm, thus allowing for optimal NF-�Bactivation upon TNF-� ligation. The Journal of Immunology, 2003, 171: 5313–5319.

T umor necrosis factor-� is a pleiotropic cytokine that elic-its a broad spectrum of cellular responses including mac-rophage activation, fever, acute phase response, as well as

cell proliferation, differentiation, and apoptosis (for review, seeRef. 1). Of the two types of TNFRs characterized (TNFR1 andTNFR2), TNFR1 is responsible for most of the biological proper-ties of TNF-� (1). The cytoplasmic domain of TNFR1, when ac-tivated by ligand binding, recruits the adaptor protein TNFR1-associated death domain protein (TRADD)3; the TNFR1-TRADDcomplex leads to at least two opposing downstream signaling cas-cades. The TNFR1-TRADD complex forms the death-initiated sig-naling complex by recruiting Fas-associated death domain protein(FADD), leading to caspase activation and apoptosis (2). Alterna-tively, the TNFR1-TRADD complex can recruit receptor interact-ing protein and/or TNFR-associated factor 2, leading to mitogen-activated protein kinase (MAPK) and NF-�B activation (3, 4).NF-�B activation is critical for the expression of proinflammatorycytokines, chemokines, adhesion molecules, acute phase proteins,and costimulatory molecules, as well as regulators of apoptosis andcell proliferation (for review, see Ref. 5).

The central biological actions described for IFN-� are macro-phage activation, antiviral activity, antiproliferative activity on tu-

mor cells, production of free radicals, and expression of adhesionmolecules as well as class I and II MHC Ags (for review, see Ref.6). Produced mainly by Th1 and NK cells (6), IFN-�, when boundto the IFN-� receptor, induces the transphosphorylation of Januskinase 1(JAK1) and JAK2, which are associated with the IFN-�receptor subunits. The subsequent phosphorylation of a tyrosineresidue on the IFN-� receptor by activated JAKs provides a dock-ing site for latent STAT-1�. Bound STAT-1� becomes tyrosinephosphorylated by activated JAKs, dissociates from the receptorcomplex, homodimerizes, then translocates to the nucleus to in-duce transcription via binding to the �-activation site in IFN-�-inducible promoters (for review, see Ref. 7). One of the STAT-1�-inducible genes is IFN-regulatory factor (IRF)-1 (8). IRF-1 isitself a transcription factor that recognizes the sequence termedIFN stimulation response element and plays, like STAT-1�, a cen-tral role in IFN-� inducible gene expression (9).

IFN-� and TNF-� synergize in the production of a large numberof proinflammatory cytokines, chemokines, and costimulatorymolecules (for review, see Ref. 10). Cooperativity between thesepathways occurs at multiple levels. Most, but not all, of the pro-moters induced synergistically by IFN-� and TNF-� contain bind-ing sites for STAT-1� or IRF-1 and NF-�B (10). For these genes,synergism occurs by the binding of TNF-�-activated NF-�B andIFN-�-activated STAT-1� or IRF-1 to their respective sites withinpromoters, thereby affecting gene expression at the transcriptionallevel (10). Another level of cross-talk exists at the level of NF-�Bactivation. Though not known to activate NF-�B directly, IFN-�enhances and prolongs TNF-�-dependent NF-�B activation (10);enhanced I�B� degradation upon costimulation is a likely molec-ular reason for the synergy (11). dsRNA-activated protein kinase Ris involved in the synergistic activation of NF-�B by TNF-� andIFN-� (12, 13).

In the RAW264.7 macrophage cell line, IFN-� and TNF-� syn-ergize in the activation of NF-�B (14, 15). In these cells, IFN-�synergizes with endogenously produced TNF-� to give strongerand more prolonged NF-�B activation than does exogenousTNF-� treatment or IFN-� treatment alone (achieved by the use ofa neutralizing anti-TNF-� Ab to inhibit signaling by endogenouslyproduced TNF-�) (14, 15). These results prompted us to search for

Department of Cell Biology, University of Alabama, Birmingham, AL 35294

Received for publication March 24, 2003. Accepted for publication September2, 2003.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported in part by grants from the National Institutes of Health(NS-36765 and NS-39954) (to E.N.B). D.R.W was supported in part by the MedicalScientist Training Program at the University of Alabama (Birmingham, AL), waspreviously supported by National Institutes of Health Predoctoral Fellowship T32AI-07051-26, and is currently supported by National Institutes of Health PredoctoralFellowship F30 NS-47051.2 Address correspondence and reprint requests to Dr. Etty N. Benveniste, Departmentof Cell Biology, University of Alabama, 1530 3rd Avenue South, McCallum BasicHealth Sciences Building 395, Birmingham, AL 35294-0005. E-mail address:tika@uab.edu3 Abbreviations used in this paper: TRADD, TNFR1-associated death domain pro-tein; FADD, Fas-associated death domain protein; MAPK, mitogen-activated proteinkinase; JAK, Janus kinase; IRF, IFN-regulatory factor; RPA, RNase protection assay.

The Journal of Immunology

Copyright © 2003 by The American Association of Immunologists, Inc. 0022-1767/03/$02.00

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mechanisms by which these two signaling pathways could com-municate to enhance NF-�B activation. Recently, in an Ab arrayscreen for STAT-1� binding proteins, TNFR1 was shown to in-teract with STAT-1� in a TNF-�-dependent manner in Hela cellsand A549 lung epithelial cells (16). When associated with theTNFR1, STAT-1� inhibited TNF-�-mediated NF-�B activation(16). Because STAT-1� is a critical component of IFN-� signal-ing, we hypothesized that IFN-� may attenuate the ability ofSTAT-1� to interact with the TNFR1, thereby augmenting TNF-�-mediated NF-�B activation. We demonstrate via coimmunopre-cipitation experiments that TNF-� treatment leads to an associa-tion between STAT-1� and the TNFR1, and that this complex isattenuated in the presence of IFN-�. Immunofluorescence studiesshow that STAT-1� is located in the cytoplasm in both untreatedand TNF-�-treated cells, where it is found to grossly colocalizewith TNFR1. IFN-� leads to a pronounced STAT-1� nucleartranslocation, suggesting a potential mechanism for the ability ofIFN-� to inhibit the STAT-1�:TNFR1 complex. Coupled withthese findings, exposure of cells to IFN-� allows cells to respondmore vigorously to constitutively present endogenous TNF-� byenhanced activation of NF-�B and NF-�B responsive genes. Wealso demonstrate that TNF-� treatment leads to enhanced NF-�Bactivation in cells deficient in STAT-1�, and upon restoration ofSTAT-1�, TNF-�-dependent NF-�B activation and RANTES (anNF-�B-dependent gene) expression is inhibited. These data sug-gest that STAT-1� interacts with the TNFR1 and acts as a repres-sor of TNF-�-dependent NF-�B activation. Additionally, IFN-�-dependent control of STAT-1� availability to the TNFR1 maycontribute to the regulation of NF-�B activation through TNFR1.We propose that this represents a novel form of cross-talk thatcontributes to the synergistic proinflammatory response by thesetwo cytokines.

Materials and MethodsRecombinant proteins and reagents

Recombinant murine IFN-� was purchased from Genzyme (Boston, MA).Murine and human TNF-� were purchased from Endogen (Woburn, MA)and Calbiochem (La Jolla, CA), respectively, and neutralizing anti-murineTNF-� Ab was purchased from Endogen (Woburn, MA). Rabbit poly-clonal TNFR1 Ab, mouse monoclonal STAT-1� Ab and consensus NF-�Bbinding oligonucleotide were purchased from Santa Cruz Biotechnology(Santa Cruz, CA). Rabbit polyclonal anti-STAT-1� Ab was purchasedfrom Upstate Biotechnology (Lake Placid, NY). Mouse monoclonal anti-FADD Ab was purchased from BD PharMingen (San Diego, CA). Biotin-ylated anti-mouse TNFR1 was purchased from Caltag Laboratories (Bur-lingame, CA). Streptavidin-conjugated HRP was purchased from SouthernBiotechnology Associates (Birmingham, AL).

Cells

The murine macrophage cell line RAW264.7 was maintained in DMEMsupplemented with 10% FBS as previously described (17). The U3A cells(a generous gift from Dr. G. Stark, The Cleveland Clinic, Cleveland, OH)were maintained with 10% FBS in DMEM/F12 as previously described(18).

Stable transfection of U3A cells

STAT-1� and pcDNA3 stable transfectants were created by transfectingU3A cells with either STAT-1� (a generous gift of Dr. J. Darnell, Rock-efeller University, New York, NY) or the pcDNA3 backbone vector aspreviously described (18). STAT-1� expression was determined by immu-noblotting for STAT-1�.

Nuclear extracts and EMSA

EMSA was performed with 5–10 �g of nuclear extract as previously de-scribed (15). For supershift experiments, 1 �g of anti-p50, anti-p65 orisotype-matched control Abs (Santa Cruz Biotechnology, Santa Cruz, CA)were included in the reaction mixture. For competition experiments, 100�unlabeled NF-�B consensus probe or 100� of a probe (Santa Cruz Bio-

technology) containing a mutation in the NF-�B binding site were added tothe reaction mixture. Bound and free DNA were then resolved by electro-phoresis through a 6% polyacrylamide gel in 0.5� Tris-borate-EDTAbuffer at 250 V for 1 h.

Coimmunoprecipitation and Western blotting

RAW264.7 cells were incubated with medium, IFN-� (10 ng/ml), TNF-�(50 ng/ml), or a combination of IFN-� and TNF-� for various time periods,followed by lysis in cold radioimmunoprecipitation assay (RIPA) buffer(PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS). Lysateswere then cleared of debris by centrifugation (12,000 � g, 10 min) and 900�g of protein were precleared with 0.25 �g of rabbit IgG Ab and 30 �l ofprotein A/G bead slurry (Pierce, Rockford, IL) for 2.5 h. Cleared lysateswere then incubated overnight with 1.5 �g of rabbit anti-TNFR1 Ab and anadditional 3 h with 30 �l of protein A/G bead slurry. Beads were thenwashed five times with cold RIPA buffer, boiled in sample buffer, separatedon 10% SDS-PAGE, transferred to a nitrocellulose membrane, and probedwith a monoclonal anti-STAT-1� Ab for detection of endogenous TNFR1:STAT-1� association. For detection of immunoprecipitated TNFR1, mem-branes were stripped and reprobed with a biotinylated anti-TNFR1 Ab.Following which, membranes were incubated with a streptavidin-HRP con-jugate. For detection of components of the TNFR1 signaling pathway,RIPA lysates were subjected to SDS-PAGE, transferred to a nitrocellulosemembrane, and probed with Abs to TNFR1, TRADD, FADD, and NF-�Bp65 (Santa Cruz Biotechnology) as well as an anti-actin Ab (Sigma-Al-drich, St. Louis, MO) to control for loading. The ECL method was used forprotein detection.

Immunofluorescence

RAW264.7 cells were grown in two-chamber culture slides and were in-cubated in the absence or presence of IFN-� (10 ng/ml), TNF-� (50 ng/ml),or both for 30 min. Cells were permeabilized/fixed in a 1:1 mixture ofmethanol/acetone at �20°C for 10 min, air-dried, and blocked in 1.5% goatserum in PBS for 30 min. Cells were then stained with polyclonal TNFR1for 1 h, rinsed three times in PBS, and then stained with goat anti-rabbitAlexa Fluor 488 (Molecular Probes, Eugene, OR) for 45 min. Cells wereagain rinsed with PBS followed by staining with mouse monoclonal anti-STAT-1� Ab. Cells were then rinsed three times in PBS and stained withgoat anti-mouse Alexa Fluor 594 (Molecular Probes) for 45 min. Cellswere again rinsed three times and counterstained with Hoechst 33258 (Sig-ma-Aldrich) followed by a brief rinsing. Images were obtained with anOlympus IX70 digital camera (Melville, NY) and analyzed with IPLab 3.2software (Scanalytics, Fairfax, VA).

RNA isolation, riboprobes, and RNase protection assay (RPA)

Total cellular RNA was isolated from confluent monolayers of cells. Theriboprobes for Bcl-xL, Bcl-xS, RANTES, L32, and GAPDH were pur-chased from BD PharMingen and prepared following the manufacturer’sguidelines. The IRF-1 riboprobe was prepared as previously described(15). Twenty micrograms of total RNA were hybridized with Bcl-xL, Bcl-xS, RANTES, IRF-1, L32, and GAPDH riboprobes as described previously(15). Quantification of the protected RNA fragments was performed byscanning with the PhosphorImager (Molecular Dynamics, Sunnyvale, CA).Values for Bcl-xL, Bcl-xS, IRF-1, and RANTES mRNA expression werenormalized to GAPDH or L32 mRNA levels for each experimentalcondition.

ResultsTNF-� and IFN-� effects on TNFR1:STAT-1� complexformation

RAW264.7 cells were treated with medium, IFN-�, TNF-�, orIFN-� plus TNF-� for 30 min and then assayed for TNFR1:STAT-1� interaction via coimmunoprecipitation by immunopre-cipitating TNFR1 and probing for STAT-1�. As shown in Fig. 1A,treatment with IFN-� had no effect on complex formation, whiletreatment with exogenous TNF-� lead to an association of TNFR1and STAT-1�. Of most interest was the observation that inclusionof IFN-� inhibited TNF-�-mediated TNFR1:STAT-1� complexformation (Fig. 1A). Immunoprecipitation with an isotype-matchedcontrol Ab was without effect (Fig. 1A). To determine the tyrosinephosphorylation status of STAT-1� with these treatments, 10% ofthe input samples were subject to Western blot analysis and probedwith an Ab specific for STAT-1� that was phosphorylated on the

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Y701 residue. As shown in Fig. 1B, IFN-�, but not TNF-�, leadsto the tyrosine phosphorylation of STAT-1�. These results indicatethat TNF-� induces the association of STAT-1� and TNFR1 in atyrosine phosphorylation-independent manner, and that formationof the TNFR1:STAT-1� complex is inhibited upon inclusion ofIFN-�.

To determine the time course of TNFR1:STAT-1� complex for-mation, cells were treated with medium, TNF-�, or a combinationof TNF-� and IFN-� for 0.5–6 h (Fig. 1C), and then were assayedfor TNFR1:STAT-1� interaction via coimmunoprecipitation asdescribed above. The TNFR1:STAT-1� complex was induced by0.5 h and was stable for up to 6 h. Shown as well is that inclusionof IFN-� inhibits TNF-�-mediated TNFR1:STAT-1� complexformation at all time points tested (Fig. 1C). To determine the dosedependency of this effect, cells were left untreated, or were incu-bated with increasing doses of TNF-� (1–100 ng/ml), as well asincreasing doses of IFN-� (0.1–100 ng/ml) in the presence ofTNF-� (100 ng/ml). Complex formation was observed using 1ng/ml TNF-�, and increasing the dose of TNF-� did not result ina stronger signal (Fig. 1D). IFN-� treatment at all concentrationstested inhibited complex formation (Fig. 1D). We have also seeninhibition of complex formation with IFN-� at 0.01 ng/ml (data notshown). These results suggest that the TNF-�-induced TNFR1:STAT-1� complex is induced to form with a threshold amount ofTNF-�, and that IFN-� is a potent inhibitor of this process.

IFN-� depletes cytoplasmic availability of STAT-1� andattenuates TNFR1:STAT-1� colocalization

Immunofluorescence technology was used to confirm TNFR1 andSTAT-1� localization under the influence of TNF-� and IFN-�. Inthese experiments, cells were left untreated or given IFN-�,TNF-�, or both for 30 min, fixed, and then stained for TNFR1

(green) followed by staining for STAT-1� (red). Cells were alsocounterstained with the Hoechst DNA binding dye (blue). Asshown in Fig. 2, STAT-1� and TNFR1 appear to grossly colocal-ize in unstimulated cells, as manifested by the yellow coloration inthe red/green overlays (Fig. 2A). Upon TNF-� treatment,STAT-1� remains in the cytoplasm where it remains grossly co-localized with TNFR1 (Fig. 2C). IFN-� treatment induces nucleartranslocation of STAT-1�, and colocalization of STAT-1� andTNFR1 is attenuated (Fig. 2, B and D). Because there is such scantcytoplasm in monocytic cells, discriminating between cytoplasmicSTAT-1� and membrane-bound TNFR1 is not straightforward.These results qualitatively show a significant proportion of IFN-�mediated STAT-1� nuclear translocation, and suggest that theability of IFN-� to inhibit the formation of the TNFR1:STAT-1�complex may be related to its ability to localize STAT-1� to thenucleus, thereby depleting availability to the cytoplasmic domainsof the TNFR1.

IFN-� exposure allows for optimal TNF-� dependent NF-�Bactivation by constitutively produced endogenous TNF-�

The possibility of STAT-1� as a component of the TNF-� signal-ing pathway led to the speculation that STAT-1� may be a centralmediator of receptor proximal cross-talk between these two sig-naling pathways. The effect of IFN-� activation of STAT-1� onTNF-�-mediated NF-�B activation was examined. We have pre-viously shown that IFN-� synergizes with endogenously secretedTNF-� in RAW264.7 cells to mediate optimal NF-�B activation(14, 15). Initially, we hypothesized that IFN-� activated NF-�B byup-regulating the expression of TNF-�, which subsequently sig-nals to the cells in an autocrine fashion to activate NF-�B. How-ever, IFN-�-mediated activation of NF-�B, to some degree, pre-cedes IFN-� induction of TNF-� as measured by ELISA (15).

FIGURE 1. TNF-� and IFN-� effects on TNFR1:STAT-1� complex formation. RAW264.7 cells were incubated in the absence or presence of IFN-� (10ng/ml), TNF-� (50 ng/ml), or both for 30 min (A) or 0.5–6 h (C) and lysed with SDS-containing RIPA buffer. Following which, 1 mg of protein lysate wasimmunoprecipitated with rabbit anti-TNFR1 (1.5 �g) and captured with protein A/G beads. The beads were washed and the captured immune complex was subjectto reducing SDS-PAGE and transferred to nitrocellulose membranes. Blots were probed with mouse monoclonal anti-STAT-1� (4 �g/ml) (A) or rabbit polyclonalanti-STAT-1� (2 �g/ml) (C) and detected using the ECL method. Blots were also probed with biotinylated anti-TNFR1, and subsequently incubated withstreptavidin-HRP for detection of immunoprecipitated TNFR1 (A and C). B, Ten percent of the coimmunoprecipitation input samples from (A) were subject toSDS-PAGE analysis and probed with anti-phospho-Y-701 STAT-1� Ab, stripped, and reprobed with rabbit polyclonal STAT-1� Ab (C). D, RAW264.7 cells wereincubated with increasing concentrations of TNF-� (0–100 ng/ml) and increasing concentrations of IFN-� (0–100 ng/ml) in the presence of 100 ng/ml TNF-�for 30 min. Protein lysates were harvested and subjected to coimmunoprecipitation analysis for detection of the TNFR1:STAT-1� complex as described above.Blots were also probed with biotinylated anti-TNFR1 as described above (D). Representative of three experiments.

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These results indicate that although IFN-�-induced TNF-� secre-tion contributes to IFN-�-mediated activation of NF-�B, there maybe additional mechanisms responsible for the more rapid IFN-�-mediated NF-�B activation.

To examine the effect of IFN-� and TNF-� on NF-�B activa-tion, EMSA was performed on nuclear extracts from RAW264.7cells treated with TNF-�, IFN-�, TNF-� plus IFN-�, or IFN-� inthe presence of a neutralizing anti-TNF-� mAb. Using a consensusNF-�B binding sequence as a probe, a small level of constitutiveNF-�B activity was observed, which is due to the fact that these

cells constitutively secrete TNF-� (Fig. 3A, lane 1). Addition ofexogenous TNF-� slightly enhanced NF-�B activation (Fig. 3A,lane 2), while IFN-� treatment clearly enhanced NF-�B activation(Fig. 3A, lane 3). The combined treatment with IFN-� and TNF-�was additive with respect to NF-�B activation compared with ei-ther cytokine alone (Fig. 3A, lane 4). This result suggests thatexposure of RAW264.7 cells to IFN-� allows constitutively ex-pressed, endogenous TNF-� to more actively induce NF-�B acti-vation. The ability of IFN-� to induce NF-�B activation was com-pletely abrogated when endogenously produced TNF-� was

FIGURE 2. IFN-� depletes cytoplas-mic availability of STAT-1� and attenu-ates TNFR1:STAT-1� colocalization.RAW264.7 cells were left untreated (A),or treated with IFN-� (10 ng/ml) (B),TNF-� (50 ng/ml) (C), or both (D) for 30min. Cells were then fixed with methanol:acetone (1:1) and incubated with a rabbitpolyclonal anti-TNFR1 Ab (5 �g/ml) fol-lowed by an incubation with anti-rabbitAlexa Fluor 488 (4 �g/ml). Cells were sub-sequently incubated with mouse monoclo-nal anti-STAT-1� Ab (5 �g/ml) followedby an incubation with anti-mouse AlexaFluor 594 (4 �g/ml). Cells were washedwith PBS between incubations. Imageswere obtained via fluorescence microscopy.Shown are representative fields. Represen-tative of two experiments.

FIGURE 3. IFN-� exposure allows for optimalTNF-�-dependent NF-�B activation by constitutivelyproduced endogenous TNF-�. A, EMSA experimentswere performed using nuclear extracts (7 �g) preparedfrom RAW264.7 cells treated with TNF-� (50 ng/ml),IFN-� (10 ng/ml), TNF-� plus IFN-�, or IFN-� in thepresence of a neutralizing anti-TNF-� Ab or an iso-type-matched control Ab (10 �g/ml) for 3 h, and thenwere assayed using a radiolabeled oligonucleotidecontaining the consensus NF-�B binding sequence. Todetermine the composition of the NF-�B complex, 1�g of anti-p50, anti-p65, and isotype-matched controlAbs were included in the reaction mixture. For com-petition experiments, 100� unlabeled NF-�B consen-sus probe or 100� of a probe containing a mutation inthe NF-�B binding site were added to the reactionmixture. Representative of three experiments. B, RNAwas isolated from RAW264.7 cells treated for 8 h withthe same cytokine profile described in (A) and subjectedto RPA with a riboprobe mixture containing radiolabeledBcl-xL, Bcl-xS, and GAPDH. C, RAW264.7 cells wereincubated with medium, IFN-�, or IFN-� plus neutraliz-ing anti-TNF-� Ab for 8 h, RNA isolated, then examinedby RPA for IRF-1 and GAPDH mRNA expression. Rep-resentative of two experiments.

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blocked by addition of a neutralizing anti-TNF-� Ab (Fig. 3A, lane5) (an isotype-matched control Ab has no effect, Fig. 3A, lane 6).Anti-p50 and anti-p65 Abs were used to determine the composi-tion of the NF-�B complex. These Abs acted as competitors ofnucleotide binding compared with an isotype-matched control Ab(Fig. 3A, lanes 7–10), and suggest that the complex studied con-sists of the p65 and p50 NF-�B isoforms. Abs that act as compet-itors of binding instead of inducing a supershift have been ob-served previously (14, 15). Competition analysis using coldcompetitor DNA and mutant DNA was performed as well to dem-onstrate the specificity of this assay for the NF-�B binding se-quence (Fig. 3A, lanes 11 and 12).

Another parameter of NF-�B activation was assessed by exam-ining expression of the NF-�B regulated Bcl-x gene via RPA ofboth Bcl-xL and Bcl-xS mRNA splice variants. As shown in Fig.3B, mRNA for Bcl-xS was constitutively expressed in these cells(Fig. 3B, lane 1), slightly augmented by TNF-� treatment (Fig. 3B,lane 2), and highly up-regulated by IFN-� treatment (Fig. 3B, lane3: 8.3� induction). A simultaneous treatment with TNF-� plusIFN-� (Fig. 3B, lane 4) did not increase expression (8.5�) overthat seen with IFN-� alone (Fig. 3B, lane 3). The enhancement ofBcl-xS by IFN-� was due to the presence of endogenous TNF-�,

because the response was inhibited in the presence of a neutraliz-ing anti-TNF-� Ab (Fig. 3B, lane 5), while the isotype-matchedcontrol Ab was without effect (Fig. 3B, lane 6). Comparable resultswere observed for the expression of Bcl-xL mRNA (Fig. 3B).These results collectively indicate that the ability of IFN-� treat-ment to activate Bcl-x gene expression, which is a NF-�B-depen-dent response (19, 20), is actually IFN-� working in concert withconstitutively expressed TNF-�. It should be noted that inclusionof neutralizing anti-TNF-� Ab does not interfere with IFN-�-in-duced, STAT-1�-dependent expression of IRF-1 mRNA (Fig. 3C).

TNF-�-mediated NF-�B activation is inhibited by STAT-1�

To directly test the influence of STAT-1� on NF-�B activation, weused a cell line deficient in STAT-1� (U3A cells) and stably trans-fected them with either STAT-1� (U3A-STAT-1�) or thepcDNA3 vector (U3A-pcDNA3). These cells were then used todetermine whether TNF-�-induced NF-�B activation was affectedwhen STAT-1� was absent (U3A-pcDNA3) and then restored(U3A-STAT-1�). Using EMSA, we observed a strong activationof NF-�B by TNF-� in the U3A-pcDNA3 cells (Fig. 4A, lanes2–6), however, in the U3A-STAT-1� cells, TNF-�-mediatedNF-�B activation was partially attenuated (Fig. 4A, lanes 8–12).To validate this phenomenon, we investigated whether the pres-ence of STAT-1� affected the ability of TNF-� to regulate aNF-�B responsive gene. Using an RPA, TNF-� treatment led to a5.0- to 6.1-fold induction of RANTES mRNA at 6–12 h, respec-tively, in the U3A-pcDNA3 cells (Fig. 4B, lanes 1–3), whereasonly a 3.0-fold induction of TNF-�-induced RANTES mRNA lev-els was observed in the U3A-STAT-1� cells (Fig. 4B, lanes 5–8).Western blotting confirmed that protein levels of components ofthe TNFR1 signaling pathway (TNFR1, TRADD, FADD, andNF-�B p65) remain constant in the absence or presence ofSTAT-1� (Fig. 4C). These results demonstrate that STAT-1� in-deed inhibits TNF-�-mediated activation of NF-�B.

DiscussionIFN-� and TNF-� synergize in the activation of NF-�B (for re-view, see Ref. 10). Activated NF-�B is a key part of the promotionof autoimmune and chronic inflammatory disorders mediated bythe otherwise helpful immune system (for review, see Ref. 5). Thesynergistic activity of IFN-� and TNF-� is well-documented andoccurs on many different levels within the cell. Herein, we eluci-date a novel mechanism of cross-talk between these two signalingpathways occurring at the receptor level that may contribute to theobserved enhancement of TNF-�-dependent NF-�B activation inthe presence of IFN-�.

Consistent with results from other cell types (16), we demon-strate that TNF-� treatment leads to the physical association ofSTAT-1� and TNFR1 (Fig. 1). Immunofluorescence staining re-veals that STAT-1� is localized to the cytoplasm and grossly co-localizes with the TNFR1 in resting cells, and that STAT-1� re-mains cytoplasmic upon treatment with exogenous TNF-� wheregross colocalization with TNFR1 remains intact (Fig. 2). Notably,lysates for the coimmunoprecipitation studies were prepared usingSDS-containing RIPA buffer and the interaction of endogenousSTAT-1� and TNFR1 was detectable under these stringent con-ditions (Fig. 1). A previous report has demonstrated a direct asso-ciation of JAK1 and JAK2 with the TNFR1 upon TNF-� stimu-lation (21). In contrast, we were unable to detect any TNFR1:JAK1 or TNFR1:JAK2 association via coimmunoprecipitation inthe absence or presence of either TNF-� or IFN-� (data notshown). However, this does not rule out the possibility that JAKsmay still be involved in this process. Sorting out the molecularcomplexes formed with TNF-� and IFN-� stimulation will no

FIGURE 4. TNF-� mediated NF-�B activation is inhibited by STAT-1�. A, Nuclear extracts (7 �g) were prepared from the STAT-1�-deficientU3A cell line stably transfected with either STAT-1� (U3A-STAT-1�) orthe pcDNA3 backbone vector (U3A-pcDNA3) that were treated withTNF-� (50 ng/ml) for 0–90 min. EMSA experiments were then performedusing a radiolabeled oligonucleotide containing the consensus NF-�B bind-ing sequence. B, U3A-pcDNA3 and U3A-STAT-1� cells were treated withTNF-� (50 ng/ml) for 0–24 h. RNA was isolated and analyzed by RPA forRANTES (an NF-�B-inducible gene), and L32 mRNA and quantified us-ing IQMac software. Representative of three experiments. C, U3A-pcDNA3 and U3A-STAT-1� cell lysates were subject to SDS-PAGE,transferred to a nitrocellulose membrane, and probed with Abs to TNFR1,TRADD, NF-�B p65, STAT-1�, actin, and FADD.

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doubt shed light on the integration of these signaling pathways,and is the topic of ongoing research.

Interestingly, when cells are exposed to IFN-�, whether or notTNF-� is provided, gross colocalization of STAT-1� and theTNFR1 can no longer be demonstrated, either by coimmunopre-cipitation (Fig. 1), or by immunofluorescence (Fig. 2, B and D).STAT-1� has been shown to interact with the TNFR1 complexand has been implicated as a negative regulator of TNF-�-medi-ated NF-�B activation while in this configuration (16). Consistentwith the hypothesis that IFN-�-dependent removal of STAT-1�from the TNFR1 complex may sensitize cells to TNF-�-mediatedNF-�B activation, exposure of cells to IFN-� allows endogenous,constitutively present TNF-� to activate NF-�B to a greater degreethan the addition of exogenous TNF-� in RAW264.7 cells (Fig.3A). To rule out the possibility that IFN-� may be merely up-regulating TNF-� expression that would in turn signal in an auto-crine fashion to give rise to NF-�B activation, ELISA experimentswere done to show that TNF-� protein levels were unchanged forat least 3 h after IFN-� exposure in RAW264.7 cells (15). Addi-tionally, IFN-� enhancement of TNF-�-dependent NF-�B activa-tion was still observed in the presence of the protein synthesisinhibitor puromycin (data not shown), suggesting that protein syn-thesis was not required for the effect of IFN-� on TNF-�-mediatedNF-�B activation.

To further investigate the role of STAT-1� as a negative regu-lator of TNF-�-mediated NF-�B activation, we sought to measureTNF-�-mediated NF-�B activation in the absence and presence ofSTAT-1� using a STAT-1�-deficient cell line that was reconsti-

tuted with either STAT-1� or the pcDNA3 vector. TNF-� wasfound to induce a stronger NF-�B activation in cells lackingSTAT-1� than in the STAT-1� -reconstituted cells as measured byEMSA (Fig. 4A). As well, TNF-�-induced mRNA levels of RAN-TES, an NF-�B responsive gene, were also inhibited in the pres-ence of STAT-1� (Fig. 4B).

It has been known for some time that synergism between theIFN-� and TNF-� signaling pathways occurs by the binding ofTNF-�-activated NF-�B and IFN-�-activated STAT-1� or IRF-1to their respective sites within promoters, thereby affecting geneexpression at the promoter level (10) (Fig. 5). The observation thatSTAT-1� binds to TNFR1 and inhibits TNF-�-mediated NF-�Bactivation, together with the finding that IFN-� inhibits the TNF-�-induced TNFR1:STAT-1� complex, prompted us to propose amodel that is consistent with the observed enhancement of TNF-�-dependent NF-�B activation seen in the presence of IFN-�. Inthis model (Fig. 5), resting macrophages have cytoplasmicSTAT-1� available to interact with TNFR1 to serve as a negativeregulator of TNF-�-dependent NF-�B activation. When TNF-� isavailable in the absence of IFN-�, STAT-1� associates withTNFR1 to aid in receptor-proximal negative regulation of NF-�Bactivation. However, in the presence of IFN-�, STAT-1� is ty-rosine phosphorylated and subsequently localizes to the nucleuswhere it is no longer able to interact with TNFR1, thus allowingfor stronger TNF-�-dependent NF-�B activation. Consistent withthe hypothesis that STAT-1� tyrosine phosphorylation and nuclearlocalization is important for IFN-�-mediated inhibition of the

FIGURE 5. IFN-�-mediated inhibition of TNFR1:STAT-1� complex formation correlates with a greaterTNF-�-dependent NF-�B activation. In this model,macrophages have cytoplasmic STAT-1� available tointeract with TNFR1 that serves as a negative regulatorof TNF-�-dependent NF-�B activation. When TNF-�is available in the absence of IFN-�, this associationbecomes stronger, so that tight regulation of NF-�Bactivation is maintained. However, in the presence ofIFN-�, STAT-1� is tyrosine phosphorylated and sub-sequently localizes to the nucleus where it is not able tointeract with TNFR1, thus allowing for stronger TNF-�-dependent NF-�B activation. STAT-1� has alsobeen shown to sensitize cells to TNF-�-mediated ap-optosis by binding to the TNFR1 complex. When inthe nucleus, IFN-�-activated STAT-1� cooperateswith TNF-�-activated NF-�B at the promoter level toenhance gene expression. RIP, receptor interactingprotein; TRAF2, TNFR-associated factor 2.

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TNFR1:STAT-1� complex for up to 6 h (Fig. 1C), we have pre-viously shown that IFN-�-mediated STAT-1� tyrosine phosphor-ylation was detectable for up to 6 h in these cells (15). Addition-ally, STAT-1� was still detected in the nucleus through this 6 htime point (data not shown).

It is worthy of mention that although this model is consistentwith our data, it is by no means a comprehensive explanation of allthe effects that IFN-� has on the TNF-� signaling pathway. Ourresults may represent a contributing factor to the general phenom-enon of IFN-� sensitization of macrophages to TNF-� effects. Inthis regard, work from another laboratory has provided evidencethat the effect of STAT-1� at the TNFR1 complex is not limited toNF-�B modulation. It was shown that when STAT-1� interactedwith the TNFR1, TNF-� was a more potent inducer of apoptosis(16) (see Fig. 5). In light of these results, we tested the effect ofIFN-� on TNF-�-dependent apoptosis in RAW264.7 cells. Theinterpretation of the results from these experiments was problem-atic as IFN-� induces a degree of apoptosis in these cells indepen-dent of TNF-� treatment (data not shown). Additional experimentsdemonstrated that, unlike NF-�B activation, IFN-� had no effecton TNF-�-mediated MAPK activation (data not shown).

An intriguing question that remains is how TNF-� ligationcauses STAT-1� to associate with TNFR1. A previous study dem-onstrated that in Hela cells, TNF-� induces tyrosine phosphoryla-tion of STAT-1� and this is necessary for TNF-�-induced forma-tion of a complex of STAT-1� and the adapter protein TRADD(16). However, although STAT-1� tyrosine phosphorylation wasimplied to be necessary, the authors of this study did not directlytest whether STAT-1� tyrosine phosphorylation was a requirementfor the TNFR1:STAT-1� complex formation (16). We have shownthat TNF-� does not induce tyrosine phosphorylation inRAW264.7 cells (Fig. 1), but still induces TNFR1:STAT-1� com-plex formation/colocalization (Figs. 1 and 2). Moreover, theSTAT-1� that is complexed to TNFR1 in the coimmunoprecipi-tation studies does not react with Abs to tyrosine phosphorylatednor serine phosphorylated forms of STAT-1� (data not shown).Thus, an important difference between our results and those re-ported previously (16), is regarding the role of STAT-1� tyrosinephosphorylation. The results presented herein clearly demonstratethat tyrosine phosphorylation is not required for TNF-�-mediatedTNFR1:STAT-1� complex formation as TNF-� does not inducetyrosine phosphorylation in RAW264.7 cells. At this point, themechanism of TNF-�-dependent TNFR1:STAT-1� complex for-mation remains unknown, and is under study.

The studies represented herein define a novel level of cross-talkbetween the IFN-� and TNF-� signaling pathways, and advancethe concept that control of STAT-1� availability to TNFR1, orperhaps other TNFR signaling components by IFN-� may in partmediate the balance and/or control the threshold of TNF-�-medi-ated processes.

AcknowledgmentsWe thank Dr. George Stark (Cleveland Clinic, Cleveland, OH) for the U3Acells and Dr. James Darnell (Rockefeller University, New York, NY) forthe STAT-1� expression construct.

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