IFN-{gamma} and IL-4 differently regulate inducible NO synthase gene expression through IRF-1 modulation

Eliana M. Coccia1,2, Emilia Stellacci1, Giovanna Marziali1, Günter Weiss3 and Angela Battistini1

1 Laboratory of Virology and
2 Immunology, Istituto Superiore di Sanità, Rome 00161, Italy
3 Department of Internal Medicine, University of Innsbruck, A-6020 Innsbruck, Austria

Correspondence to: E. M. Coccia, Email: e.coccia{at}iss.it


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
NO is a labile radical involved in several immunological, antimicrobial and inflammatory processes. In macrophages, NO formation is catalyzed by the cytokine-inducible enzyme inducible NO synthase (iNOS). The importance of IFN regulatory factor (IRF)-1 and of the signal transducers and activators of transcription (STAT)-1 for the induction of iNOS gene expression in response to IFN-{gamma} has been well defined. Here, we investigated the molecular events responsible for the inhibition of iNOS gene expression by IL-4 in the murine macrophage cell line RAW264.7. Unidirectional deletion analysis on iNOS promoter demonstrated that an IFN-stimulated responsive element (ISRE), contained in the –980 to –765 bp region of the iNOS promoter, may be involved in the IL-4-mediated inhibition of IFN-{gamma}-inducible iNOS transcription. Accordingly, the IFN-{gamma}-induced binding activity of IRF-1 to the ISRE sequence was reduced in cells pre-treated with IL-4, while the binding activity of STAT-1 to the STAT-binding element (SBE) within the same region of the iNOS promoter remained unaffected. Moreover, IL-4 even down-regulated IFN-{gamma}-inducible expression of IRF-1 mRNA. This could be related to a transcriptional mechanism by which IL-4 and IFN-{gamma} differentially influence the trans-acting activity of the STAT factors binding to SBE within the IRF-1 promoter. SBE is targeted by IFN-{gamma}-inducible STAT-1 and by IL-4-inducible STAT-6. Although STAT-6 has no trans-acting function on iNOS gene expression, it is able to inhibit the IFN-{gamma}-induced expression of IRF-1. Thus, IL-4 may down-regulate IFN-{gamma}-inducible iNOS transcription by activation of STAT-6 which in turn inhibits IRF-1 expression.

Keywords: cytokine, macrophage, monocyte, nitric oxide, signal transduction, transcription factors


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The control of inflammatory responses is mainly mediated by interplays in the cytokine network (1,2). Through complex interactions, cytokines not only cooperate, but also antagonize each other's functions. IL-4 and IFN-{gamma} represent a well-known example of a pair of mutually counteracting cytokines. In particular, the induction of gene expression in mononuclear phagocytes mediated by pro-inflammatory cytokines such as IFN-{gamma} and IL-2 is counteracted by anti-inflammatory lymphokines such as IL-4 and IL-10 (35). Moreover, IL-4 produced by Th2 induces Th2 proliferation and differentiation and inhibits Th1 proliferation. On the contrary, IFN-{gamma} acts on these cell populations in an opposite way by promoting Th1 differentiation and inhibiting Th2 proliferation (6). Therefore, IL-4 and IFN-{gamma} may both directly deactivate/activate macrophages as well as indirectly inhibit/promote the proliferation of IFN-{gamma}-producing Th1 cells. Most IFN-{gamma}-stimulated functions in monocytes and macrophages are antagonized by IL-4 via inhibition of IFN-{gamma}-induced gene transcription (5). Recent studies on intracellular cytokine signaling have provided specific evidence for the transcriptional modulation of target genes. An essential pathway for this transcriptional control involves members of cytoplasmic protein tyrosine kinases, termed the Janus kinases (Jaks). The activated Jaks phosphorylate the signal transducers and activators of transcription (STAT) proteins. After undergoing tyrosine phosphorylation, dimerization and translocation from the cytoplasm to the nucleus, STATs bind to specific cis-acting nucleotide sequences termed STAT-binding elements (SBE) (7). STAT-1 phosphorylation is mainly induced by IFN-{gamma}, whereas IL-4 induces the phosphorylation of STAT-6 (79). Both STAT-6 and STAT-1 recognize similar SBE motifs, providing a possible molecular explanation of the mutual effects exerted by these two transcription factors on IFN-{gamma}-induced gene expression (10,11). Among the genes that are differentially regulated by IFN-{gamma} and IL-4, inducible NO synthase (iNOS) represents a key gene for the antitumor, antimicrobial and immunosuppressive activity of macrophages (12). The murine iNOS gene promoter contains nearly 30 consensus binding sites for known transcription factors (13,14). Deletion and mutational analysis of the mouse iNOS promoter has identified several transcription factors which are of pivotal importance for the transcriptional regulation of this gene by IFN-{gamma} and lipopolysaccharide. These include NF-{kappa}B (15), IFN regulatory factor-1 (IRF-1) (16,17), nuclear factor IL-6 (NF IL-6) (18) and STAT-1 (19).

In the present report, we investigated the ability of IL-4 and IFN-{gamma} to regulate iNOS expression in macrophage cell line RAW264.7 focusing on the differential activation of critical transcription factors binding to the iNOS promoter. This has been explored by analysis of IRF-1 and SBE present within the iNOS promoter. The results show that IL-4 could inhibit IFN-{gamma}-stimulated iNOS transcription through repression of IRF-1 expression since STAT-1 binding activity was not affected. Thus, the anti-inflammatory effect of IL-4 occurs in part by inhibiting IFN-{gamma} induction of iNOS through the convergence of signaling by these cytokines on the IRF-1 promoter.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Cell culture and reagents
The macrophage-like cell line RAW264.7 was obtained from ATCC (Rockville, MD). The cells were grown in RPMI 1640 supplemented with 10% FCS endotoxin-free, penicillin (100 IU/ml) and streptomycin (100 µg/ml) (Whittaker Bioproducts, Walkersville, MD). Recombinant IFN-{gamma} (107 U/mg of protein) produced by Genentech (San Francisco, CA) was used at 10 and/or 100 U/ml. Mouse IL-4 was purchased from R & D System (Minneapolis, MN) and used at 20 ng/ml.

DNA electrophoretic mobility shift assay (EMSA)
Synthetic double-stranded oligonucleotides were end-labeled with [{gamma}-32P]ATP by T4 polynucleotide kinase. Nuclear cell extracts were prepared as described (20). Binding reaction mixture (20 µl final volume) contained labeled oligonucleotide probes (20,000 c.p.m.) in binding buffer [4% (v/v) glycerol, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 50 mM NaCl, 10 mM Tris–HCl, pH 7.5, 2 µg poly(dI)–poly(dC)]. Nuclear cell lysates (10 µg) were added and the reaction mixture was incubated for 20 min at room temperature. Then 13% (v/v) glycerol was added and samples were electrophoresed in 5% polyacrylamide gel in 0.5xTBE buffer for 1.30 h at 200 V at 18°C. Where indicated, cell extracts were incubated with 1 µg of anti-STAT-1 or anti-IRF-1 (Santa Cruz Biotechnology, Santa Cruz, CA) antibodies for 20 min at 4°C, before the addition of binding buffer containing labeled oligonucleotide. Competition studies were performed by adding unlabeled double-stranded oligonucleotides at 100-fold molar excess over the labeled probe. The oligonucleotides used as probe or competitor are the following: IRF-1 SBE (5'-GATCGATTTCCCCGAAATGA-3') (21), mu iNOS SBE/ISRE (5'-TGTTTGTTCCTTTTCCCCTAACACTG-3') (17), mu iNOS IRF/ISRE (5'-CACTGTCAATATTTCACTTTCATAAT-3') (17) and mu iNOS IRF/ISRE mut (5'-CACTGTCAATATTTGACTTTCATAAT-3') (17).

Western blot assay
Whole cell extracts were prepared as described (22). Either 30 or 50 µg of whole cell extract was separated on 7.5% SDS–PAGE. Blots were incubated with rabbit polyclonal anti-STAT-1 (Transduction Laboratories, Lexington, KY) or anti-iNOS (UBI, New York, NY) antibodies (Santa Cruz Biotechnology) and then with anti-rabbit horseradish peroxidase-coupled secondary antibody (Amersham, Little Chalfont, UK) using the enhanced chemiluminescence system. Rabbit antiserum specific for tyrosine- and serine-phosphorylated STAT-1 isoforms were purchased from UBI, and used according to the manufacturer's instructions. For IRF-1 and IRF-2 expression, 20 µg of nuclear extracts for each condition was separated in 10% SDS–PAGE, and Western blot analysis was performed as above and developed by using anti-IRF-1 and IRF-2 antibodies (Santa Cruz Biotechnology). Anti-TFIIH antibody (Santa Cruz Biotechnology) was used as marker to control nuclear extracts.

Electroporation and enzymatic assays
Ten million cells were washed twice with cold PBS and resuspended in 1 ml of cold RPMI medium containing 10% FCS and 20 mM HEPES, pH 7.4. Then 30 µg of each construct was added and the suspension was allowed to sit on ice for 10 min with occasional mixing, and 250 µl of this cell–DNA suspension was then introduced into the electroporation chamber (4 mm space) and the following parameters were used for transfection: field strength 0.625 kV/cm; capacitance 960 µF; time constant ~65 µs. After 10 min on ice, cells were split and then treated as indicated. All the DNAs used were prepared according to the ENDO Free Quiagen product (Quiagen, Valencia, CA). piNOS-CAT construct contains the 1749 bp HincII fragment (–1588 to +161) of the mouse iNOS gene (17) inserted into the pCAT basic vector (Promega); a site-specific mutation in the IRF binding site present within iNOS promoter is contained in p1 IRFm (17); p10-5 iNOS-CAT (containing the region of promoter from –980 to +161) and pNhe iNOS-CAT (from –765 to +161) (a generous gift of E. Martin, Q. W. Xie and C. Nathan) were derived from piNOS-CAT. Determination of CAT activity was performed as previously described (17). The percentage of the conversion rate [product c.p.m.x100/(substrate c.p.m. + product c.p.m.)] was calculated after electronic autoradiography in an Instant Imager Instrument (Canberra Packard, Meriden, CT). The results were expressed as fold of induction in CAT activity seen in transfected cells treated with IFN-{gamma} or IFN-{gamma} plus IL-4 compared with that in transfected cells receiving control medium. Results are the means of three separate experiments with the SD. The WT-SBE construct (a generous gift of R. Pine) contains an oligonucleotide that includes the SBE sequence between –144 and –124 bp of the IRF-1 gene promoter linked to the luciferase reporter gene (21). Reagents from Promega were used to assay extracts for luciferase activity in a LUMAT LB9501 luminometer (EG & G Berthold, Bad Wildbad, Germany). The pSV2 CAT or RSV-luc plasmids were used as a control for transfection efficiency and for the specificity of cytokine treatment (23). All data are reported as fold induction, which was calculated by dividing the relative light units of each stimulated culture with that of the corresponding unstimulated control culture and averaging three independent experiments. The average is reported along with the SEM.

RNA extraction and analysis
Cells (108) were treated as indicated and subsequently washed twice with PBS. Total RNA was isolated by the guanidinium–cesium chloride method (22). RNase protection was performed on 5 µg of total RNA hybridized for 18 h to the RNA probes (3x105 c.p.m.) as previously described (22). To obtain the pGEM4Z IRF-1 construct, the plasmid pUC IRF-1 (a generous gift of T. Taniguchi) was digested with AccI and the 483 bp long fragment was cloned into the same sites of pGEM4Z (Promega, Madison, WI). To generate the 32P-labeled antisense IRF-1 RNA probe, the plasmid (pGEM4Z IRF-1) was linearized with EcoRI and transcribed by T7 polymerase. The pTRI-GAPDH mouse antisense control template (Ambion, Austin, TX) was used as an internal standard to establish the relative amount of RNA loaded.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
IL-4 inhibits the expression of iNOS at the transcriptional level in IFN-{gamma}-treated RAW264.7 cell line
It has been previously demonstrated that IL-4 can suppress iNOS expression in primary mouse macrophages treated with IFN-{gamma} (24). To investigate the molecular mechanisms underlying this negative regulation we studied the effects of IL-4 on the IFN-{gamma}-induced expression of iNOS in the murine macrophage cell line RAW264.7. This mature macrophage cell line was selected because of its ability to be electroporated and manipulated easily. The consequences of IL-4 treatment on iNOS expression in this cell system were studied by Western blot analysis with extracts prepared from cells stimulated with IFN-{gamma} or with IL-4 for 16 h, or from cells pre-treated for 1 h with IL-4 and then stimulated with IFN-{gamma} for further 16 h (Fig. 1AGo). Under these conditions, IFN-{gamma}-induced iNOS expression was strongly suppressed by IL-4, while IL-4 itself had no effect on iNOS expression. Similarly, analysis of iNOS mRNA expression showed stimulation by IFN-{gamma} and down-regulation of IFN-{gamma}-mediated iNOS mRNA induction by IL-4 (data not shown).



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Fig. 1. Analysis of IL-4 and IFN-{gamma} treatment on iNOS expression. (A) iNOS protein content was evaluated by Western blot analysis. RAW264.7 cells were left untreated (–) or were stimulated for 16 h with IFN-{gamma} or IL-4, or pre-treated for 1 h with IL-4 and then treated for an additional 16 h with IFN-{gamma}. (B) iNOS constructs, containing different deletions of the promoter fused to the CAT reporter gene, were transiently transfected into RAW264.7 cell line. The expression of CAT gene was measured after stimulation of transfected cells with medium alone, with IFN-{gamma} for 16 h or in cells pre-treated for 1 h with IL-4 cells before stimulation with IFN-{gamma}. Fold of induction refers to the level of CAT activity detected in unstimulated cells transfected with the different iNOS promoter constructs to which a value of 1.0 was assigned. The data shown are the means ± SD of CAT activity normalized to a RSV-luc reporter vector.

 
To see whether these effects were due to changes in iNOS transcription, we transiently transfected the RAW264.7 cell with different constructs containing a CAT reporter gene under the control of 5'-flanking regions of the murine iNOS promoter. The expression of the CAT gene was measured in cells stimulated for 16 h with control medium, IFN-{gamma} or in cells pre-treated with IL-4 for 1 h before adding IFN-{gamma}. As shown in Fig. 1Go(B), in RAW264.7 cells transfected with piNOS-CAT, containing a 1588 bp promoter fragment, IFN-{gamma} treatment causes a 5.2-fold induction of CAT activity as compared to untreated cells. This stimulatory effect of IFN-{gamma} was clearly reduced after pre-treatment of cells with IL-4. Deletion of the flanking region from –1588 to –980 bp (p10-5 iNOS-CAT) did not cause significant changes concerning the inducibility of the promoter construct by IFN-{gamma} (4.7-fold of induction) and its inhibition by IL-4. Further deletion of ~200 nucleotides from –980 to –765 (pNhe iNOS-CAT) completely abolished induction of CAT activity after IFN-{gamma} stimulation, suggesting that the fragment from –980 to –765 contains putative regulatory elements responsive to IFN-{gamma}.

Analysis of transcription factors involved in the regulation of the iNOS promoter in IL-4- and IFN-{gamma}-treated cells
The –980 to –765 bp iNOS promoter region (Fig. 2AGo) includes: (i) one SBE, (ii) two ISRE able to bind ISGF3, (iii) one IRF-1 binding site that has been recently reported to be essential for iNOS activation in murine macrophages and (iv) one NF-{kappa}B site (1517,19). To identify the regulatory elements responsible for the inhibition by IL-4 of IFN-{gamma}-induced iNOS expression EMSA experiments were performed using specific probes containing the SBE and the ISRE consensus sequences present in the iNOS promoter (IRF/ISRE and SBE/ISRE respectively) (Fig. 2AGo). Nuclear extracts from cells treated with medium alone, with IFN-{gamma} for 4 h or from cells pre-treated for 1 h with IL-4 before the addition of IFN-{gamma} were incubated with the IRF/ISRE probe. As shown in Fig. 2Go(B), treatment of RAW264.7 cells with IFN-{gamma} (Fig. 2BGo, lane 2) induced the binding activity of IRF-1 to its consensus motif as confirmed by competition experiments with an excess of cold wild-type (Fig. 2BGo, lane 3) or mutated (Fig. 2BGo, lane 4) ISRE/SBE oligonucleotides or by the addition of anti-IRF-1 antibodies (Fig. 2BGo, lane 7). IL-4 alone did not induce any IRF-1 binding activity (Fig. 2BGo, lane 6). However, when cells were pre-treated with IL-4, IRF-1 binding activity was drastically reduced (Fig. 2BGo, lane 5) as compared to treatment with IFN-{gamma} alone (Fig. 2BGo, lane 2).



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Fig. 2. Analysis of transcription factors binding to the iNOS promoter in IL-4- and IFN-{gamma}-treated cells. (A) Schematic structure of the iNOS promoter. The inserts show the positions of the binding sites for NF-{kappa}B, STAT-1 and IRF-1. Analysis of IRF–ISRE protein (B) and SBE–ISRE protein complexes (C). Nuclear cell extracts were prepared from RAW264.7 cells treated with medium alone, IFN-{gamma} (for 4 h in B and for 15 min in C) or from cells pre-treated for 1 h with IL-4 before the addition of IFN-{gamma} (for 4 h in B and for 15 min in C). The extracts were incubated with the radiolabeled IRF–ISRE and SBE–ISRE oligonucleotides, and analyzed by EMSA. Where indicated, anti-IRF-1 ({alpha}-IRF-1) and anti-STAT-1 ({alpha}-STAT-1) antibodies were included in the reactions. Competition assays were performed using 100-fold molar excess of unlabeled oligonucleotides whose sequences are indicated in Methods.

 
In EMSA experiments using the SBE/ISRE oligonucleotide (Fig. 2CGo), STAT-1 binding was induced after 15 min of IFN-{gamma} treatment (Fig. 2CGo, lane 2) while no complexes were activated by IL-4 (Fig. 2CGo, lane 3). Moreover, 1 h of pre-treatment with IL-4 did not affect STAT-1 binding activity in cells treated with IFN-{gamma} (Fig. 2CGo, lane 4). The addition of anti-STAT-1 antibodies (Fig. 2CGo, lane 5) confirms the presence of STAT-1 alone in the complex activated in cells treated with the two cytokines, indicating that STAT-6 does not bind to the SBE sequence present within the iNOS promoter. All together these results suggest that the inhibitory effect of IL-4 on IFN-{gamma}-stimulated iNOS gene transcription can be primarily ascribed to a reduced binding activity of IRF-1 since STAT-1 binding to SBE/ISRE sequences remained unaffected by IL-4 treatment. Keeping with these results, transfection experiments performed with a CAT construct containing the portion of iNOS promoter mutated in the IRF-1 binding site (p1 IRFm) confirmed the requirement of IRF-1 binding activity to achieve the full activation of iNOS gene transcription by IFN. No effect of IL-4 could therefore be detected using this IRF-1 mutated iNOS construct (data not shown).

Mechanisms underlying the mutual regulation of the iNOS promoter by IL-4 and IFN-{gamma}
Since IRF-1 is essential for iNOS transcription (16,17) and IRF-2 can antagonize the trans-activating potential of IRF-1 (25), we further investigated the role of IRF transcription factors in iNOS gene regulation by IL-4 and IFN-{gamma} (Fig. 3Go). Nuclear cell extracts were prepared from cells treated with IL-4 and different doses of IFN-{gamma} for 4 h. By Western blot analysis, we found that IRF-1 was undetectable in control (Fig. 3Go, upper panel, lane 1) and in IL-4-treated (Fig. 3Go, upper panel, lane 2) cells, whereas it was strongly induced by IFN-{gamma} treatment (Fig. 3Go, upper panel, lanes 3 and 4). Pre-treatment with IL-4 reduced IRF-1 protein induction by a low dose (10 U/ml) of IFN-{gamma} (Fig. 3Go, upper panel, lane 5), whereas it was ineffective when a higher dose of IFN-{gamma} (100 U/ml) was used (Fig. 3Go, upper panel, lane 6). Conversely, IRF-2 was expressed constitutively and was only slightly induced after 4 h by a high dose of IFN-{gamma} (100 U/ml) in a manner not affected by IL-4 (Fig. 3Go, middle panel). These observations indicate that the inhibitory effect exerted by IL-4 on iNOS gene transcription is not mediated by IRF-2 but appears to involve primarily a decreased expression of IRF-1 under our experimental conditions (i.e. treatment with 10 U/ml IFN-{gamma} for 4 h). The blots were also probed with antibodies raised against TFIIH to control the quality of nuclear extracts (Fig. 3Go, lower panel).



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Fig. 3. Effects of IL-4 and IFN-{gamma} on IRF gene expression. IRF-1 (upper panel) and IRF-2 (middle panel) protein content was evaluated by Western blot analysis. Nuclear cell extracts were prepared from cells treated for 4 h with IFN-{gamma} or IL-4, or from cells pre-treated for 1 h with IL-4 and for an additional 4 h with IFN-{gamma}. As a control of nuclear extracts, the membranes were stripped and incubated with anti-TFIIH antibody (lower panel).

 
To explore whether the reduced IRF-1 protein content observed in cells treated with IL-4 and IFN-{gamma} could be attributed to a reduced gene transcription, we analyzed the expression of IRF-1 mRNA (Fig. 4AGo). IRF-1 mRNA was induced by IFN-{gamma} (Fig. 4AGo, lane 4), whereas IL-4 treatment was not effective at all (Fig. 4AGo, lane 3). A 1 h pre-treatment of cells with IL-4 before stimulation with IFN-{gamma} reduced IRF-1 mRNA expression (Fig. 4AGo, lane 2) as compared to cells treated with IFN-{gamma} alone (Fig. 4AGo, lane 4). To test if this expression was regulated at the transcriptional level as described for ANA-1 cells (10), we measured in vivo the activity of the SBE element known to mediate the transcriptional induction of IRF-1 by IFN-{gamma} (Fig. 4BGo). RAW264.7 cells were transiently transfected with luciferase construct (WT-SBE) containing the SBE motif present in the IRF-1 promoter (21). Luciferase activity was induced 9-fold after stimulation of transfected RAW264.7 cells with IFN-{gamma} treatment for 16 h, whereas pre-treatment for 1 h with IL-4 reduced the IFN-{gamma} induced activity by ~50%. This result confirms the ability of IL-4 to inhibit transcription of the IRF-1 gene even if other mechanisms could by activated by IL-4, and in turn could be responsible for the pronounced reduction of IRF-1 mRNA and protein content observed in IL-4-treated cells.



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Fig. 4. Regulation of IRF-1 gene transcription by IL-4 and IFN-{gamma}. (A) RAW264.7 cells were treated for 4 h with IFN-{gamma} or IL-4 or pre-treated for 1 h with IL-4 and for an additional 4 h with IFN-{gamma} as indicated. Total RNA was extracted and analyzed by RNase protection with riboprobes for IRF-1 and GaPDH as indicated in Methods. (B) In vivo activity of the SBE sequence present on the IRF-1 promoter. A construct containing the WT-SBE sequence of the IRF-1 promoter fused to the luciferase reporter gene (20 µg) was transiently transfected into RAW264.7 cells as described in Methods. Expression of the luciferase gene was measured after stimulation of transfected cells for 16 h with medium, IL-4, IFN-{gamma} or in cells pre-treated for 1 h with IL-4 before adding IFN-{gamma} for an additional 16 h. Fold of induction refers to the level of luciferase activity detected in untreated cells transfected with WT-SBE construct. Transfection efficiencies were normalized for the amount of plasmid uptake using the pSV2 CAT (5 µg) construct as a control. Numerical values were calculated as described in Methods. The results are expressed as mean ± SD of luciferase activity obtained from three experiments.

 
The current model of IFN-{gamma}-mediated transcriptional induction involves activation of STAT-1 by tyrosine and serine phosphorylation following ligand–receptor interaction (26). We thus investigated if IL-4 treatment could alter the phosphorylation status of STAT-1, thus inhibiting IFN-{gamma}-stimulated transcription (Fig. 5AGo). Cells were starved for 4 h in FCS-deprived medium before cytokine treatments. Thereafter, Western blot analysis was performed in total cell extracts from cells untreated, treated with IL-4 for 1 h or with IFN-{gamma} for 15 min, or from cells pre-treated with IL-4 for 1 h before adding IFN-{gamma} for an additional 15 min. The blots were probed with antiserum recognizing specifically STAT-1 phosphorylated at Ser727 or at Tyr701 residues. Neither Ser727 nor Tyr701 phosphorylation of STAT-1 induced by IFN-{gamma} was affected in RAW264.7 cells pre-treated with IL-4, indicating that other mechanisms are involved in the inhibition of the IFN-{gamma} signaling.



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Fig. 5. Activation of the STAT transcription factors binding to the SBE motif present within the IRF-1 gene promoter. (A) Whole-cell extracts were prepared from RAW264.7 treated as indicated for 15 min or pre-treated for 1 h with IL-4 and treated for an additional 15 min with IFN-{gamma}. Extracts (30 µg) were subjected to Western blot analysis as described in Methods. The tyrosine- and serine-phosphorylated STAT-1 isoforms were detected respectively with anti-pY701-STAT-1 and anti-pS727-STAT-1 antiserum. (B) Nuclear cell extracts were prepared at different time points from cells stimulated with IL-4 and, where indicated, treated for an additional 15 min with IFN-{gamma}. Proteins (10 µg) were subjected to EMSA analysis with IRF-1 SBE sequence as described in Methods.

 
A kinetic study was then performed to analyze IL-4-induced STAT-6 activation and whether STAT-6 activation may affect STAT-1 binding to the same SBE motif (Fig. 5BGo). Nuclear extracts were prepared from cells stimulated with IL-4 at different time points and then, where indicated, treated with IFN-{gamma} for 15 min. A rapid induction of STAT-6 binding activity was observed between 30 and 60 min after stimulation with IL-4, whereas a decrease was detected after longer treatments. Interestingly, STAT-6 activation did not affect STAT-1 binding activity to the consensus sequence at all examined time points as compared to cells treated with IFN-{gamma} alone. Although IL-4 does not impair protein phosphorylation or binding activity of STAT-1, it is highly suggestive that it reduces STAT-1-driven gene transcription (Fig. 4Go) through the activation of STAT-6. These results are in accordance with data published by Ohmori and Hamilton (10) that describe the ability of STAT-6 to counterbalance the stimulatory potential of STAT-1.


    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Regulation of inflammatory responses involves intercellular communication through a network of secreted cytokines. To avoid detrimental inflammatory and cytotoxic reactions of activated macrophages, the production of pro-inflammatory cytokines (tumor necrosis factor-{alpha}, IL-1{alpha}, IL-1ß and IL-12) and chemokines as well as of reactive oxygen and nitrogen intermediates, and the expression of cell surface molecules (Fc{gamma}R and ICAM-1) are tightly regulated (5,24,2729). This regulation is at least partially dependent on the balance between proinflammatory (IFN-{gamma} and IL-2) and anti-inflammatory (IL-4 and IL-10) cytokines. Although IL-4-mediated anti-inflammatory function has been found to include both post-transcriptional and translational events, the transcriptional regulation represents the major target (10,2732). Among the genes that are differentially regulated by IFN-{gamma} and IL-4, IRF-1 represents a key transcription factor that regulates several immune processes, such as T cell selection and maturation, development of NK cells and Th1 cells, and maturation of macrophages (22,3335). In particular, a model that involves competition between STAT-6 and STAT-1 for occupancy of common SBE binding site has been proposed to explain the mechanism utilized by IL-4 to inhibit IFN-{gamma}-induced gene expression of IRF-1 (10, 11). Conversely, IFN-{gamma} antagonizes the IL-4-induced gene transcription promoting the expression of the inhibitory protein SOCS-1, which can repress the IL-4-induced STAT-6 binding activity (36). Moreover, the transcriptional regulation of genes expressed in activated macrophages is also achieved by a complex structure of promoters that contains different regulatory motifs responsive to several signals (13,14,37,38) (such as cytokines and bacterial products). In this respect, the presence of several IFN-{gamma}-responsive elements in the iNOS promoter, including ISRE and SBE sites, clearly indicates the importance of IFN-{gamma} for the maximal stimulation in macrophages under different pathological conditions (12,3941). Keeping with these results, the well-characterized transcription factors involved in IFN-{gamma} signaling, STAT-1 and IRF-1, have been previously demonstrated to play a role in iNOS gene regulation (16,17,19).

In this paper, we evaluated the molecular mechanisms involved in the ability of IL-4 to suppress IFN-{gamma}-stimulated transcription of the iNOS gene focusing on the activation of IRF-1 and STAT binding activities (Fig. 6Go). Following IFN-{gamma} treatment iNOS expression is induced through the activation of STAT-1 and IRF-1. IL-4 treatment counteracted the IFN-{gamma}-inducible transcription of the iNOS gene, affecting neither the IFN-{gamma} activated Jak–STAT pathway nor inducing STAT-6 binding activity to the SBE within the iNOS promoter (Fig. 2CGo). Conversely, a clear inhibition of IFN-{gamma}-stimulated IRF-1 binding activity was evaluated by EMSA using an oligonucleotide corresponding to the IRF/ISRE regulatory sequence within the iNOS promoter (Fig. 2BGo). As found in previous studies (10), both STAT-1 and STAT-6 activated after combined treatment of RAW264.7 cells with IFN-{gamma} and IL-4 respectively are able to bind to the SBE sequences of the IRF-1 promoter (Fig. 5BGo) resulting in inhibition of gene transcription. Since STAT-1 is an inducer of IRF-1 transcription while STAT-6 appears to have the opposite effect (10,11), it is reasonable that IL-4 down-regulates IRF-1 expression via induction of STAT-6 activity without affecting STAT-1 phosphorylation and/or binding activity (Fig. 5Go). In accord with the data published by Ohmori and Hamilton (10), our results suggest that activated STAT-6, binding to the SBE motif, reduces the trans-acting force of STAT-1 on the IRF-1 gene promoter. The possibility that in vivo competition might exist between these two transcription factors for the binding to IRF-1 SBE motif, cannot be clarified by EMSA experiments performed in vitro. In fact, EMSA experiments are carried out with an excess of labeled probe which is opposite to the conditions present in the cells. Moreover, we cannot exclude that other mechanisms may play a role in the inhibition of iNOS expression by IL-4 as recently published by Paludan and co-workers (25). The authors provided evidence that IL-4 can inhibit IFN-{gamma}-stimulated iNOS transcription by elevating the level of IRF-2 which competes with IRF-1 binding. This apparently conflicting result might depend on the different doses of IFN-{gamma} used. In fact, the 4 h treatment with IL-4 and low doses of IFN-{gamma} (10 U/ml) clearly allowed us to examine a reduced expression of IRF-1 without increasing the IRF-2 level both as binding activity (Fig. 2Go) and as protein content (Fig. 3Go). Conversely, higher doses of IFN-{gamma} (100 U/ml) induce both IRF-1 and IRF-2 expression in a manner not affected by IL-4 treatment (Fig. 3Go). Thus, different molecular mechanisms can be involved in the IL-4-mediated inhibition of IFN-{gamma} functions, depending on the microenvironmental conditions which lead to macrophage activation.



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Fig. 6. A schematic model of the mechanism underlying the opposite effect of IL-4 and IFN-{gamma} on iNOS expression. (A) Following exposure to IFN-{gamma}, the Jak-1 and Jak-2 kinases become phosphorylated in tyrosine and, then, phosphorylate STAT-1. The activated STAT-1 homodimers bind to specific SBE sequences present in different gene promoters, such as iNOS and IRF-1. Hence, IRF-1 acts as an intermediate transcription factor to regulate iNOS gene transcription. (B) Thus, in IL-4-treated cells STAT-6 activation inhibits IRF-1 expression and, in turn, iNOS expression.

 
These results together with the data obtained with IRF-1–/– mice (16,42) suggest once again the importance of IRF-1 in the regulation of iNOS and that anti-inflammatory cytokines, such as IL-4, may target this transcription factor to down-regulate inflammatory processes in macrophages.


    Acknowledgments
 
We are grateful to Richard Pine for critical comments and helpful discussion on the manuscript. We are grateful to Sabrina Tocchio and Romina Tomasetto for editorial assistance, and Eugenio Morassi for preparing illustrations. This work was supported by grants from Special Project AIDS and `1% Project' of the Istituto Superiore di Sanità, and the Austrian National Bank NB 6981.


    Abbreviations
 
EMSA electrophoretic mobility shift assay
iNOS inducible NO synthase
IRF IFN regulatory factor
ISRE IFN-stimulated responsive factor
Jak Janus kinase
SBE STAT-binding element
STAT signal transducers and activators of transcription

    Notes
 
Transmitting editor: T. Taniguchi

Received 29 October 1999, accepted 7 March 2000.


    References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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