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Published online before print October 11, 2006

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(Journal of Leukocyte Biology. 2007;81:272-283.)
© 2007 by Society for Leukocyte Biology

Requirement of tumor necrosis factor and nuclear factor-B in the induction by IFN- of inducible nitric oxide synthase in macrophages

Centro de Biología Molecular, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Cantoblanco, Madrid, Spain

¹Correspondence: Centro de Biología Molecular, CSIC-UAM, Universidad Autónoma de Madrid, Cantoblanco, E-28049 Madrid, Spain. E-mail: mfresno{at}cbm.uam.es

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
IFN- induces NO production, inducible NO synthase (iNOS) protein, and promoter expression in mouse macrophage cells. Mutation of IFN regulatory factor 1 responsive element, -activated site, as well as NF-B elements in the murine iNOS promoter strongly reduced IFN--induced iNOS transcriptional activity. The role of NF-B activation in iNOS induction by IFN- was corroborated by overexpression of the NF-B inhibitory protein IB, which inhibited iNOS promoter activity induced by IFN-. In addition, IFN- treatment induced p65 binding to the iNOS promoter by chromatin immunoprecipitation assay and NF-B binding to DNA by EMSA, although with a delayed kinetics, suggesting an indirect autocrine role for another cytokine produced in response to IFN-. It is interesting that we found that IFN- induced TNF- secretion, and the induction of iNOS expression by IFN- was abolished in primary peritoneal macrophages from TNF--deficient (TNF-^–/–) mice or in RAW 264.7 cells treated with anti-TNF- neutralizing antibodies. Moreover, exogenous addition of recombinant mouse TNF- restored iNOS expression induced by IFN- in TNF-^–/– mice. It is intriguing that NF-B binding to DNA in response to IFN- treatment was absent in TNF-^–/– mice. Taken together, our data suggest that the TNF- produced in response to IFN- is required for iNOS induction by activating NF-B transcription factor.

Key Words: inflammation • monocytes • cytokine response • iNOS

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Production of NO from activated macrophages is critical for the control of infections by a variety of viruses, bacteria, and protozoa [¹ ]. NO is produced in macrophages by the enzyme inducible NO synthase (iNOS). In contrast to constitutive NOS isoforms that become active upon intracellular Ca²⁺ elevations, iNOS regulation mainly takes place at the level of transcription [¹ , ² ]. Several stimuli can induce iNOS transcription. Among those, cytokines and bacterial products play a prominent role, as IFN- is a major inducer of iNOS transcription. TNF- is also a good inducer of NO production by macrophages, although it mostly activates iNOS induction in synergism with IFN- [^{1 2 3 4 5 6} ]. TNF- is produced by activated macrophages, and it protects against infection, promotes tissue remodeling, and activates inflammatory response [⁷ , ⁸ ].

Binding of IFN- to its receptor recruits two JAKs (JAK-1 and JAK-2), which phosphorylate STAT-1. This transcription factor then migrates to the nucleus and binds to the so-called -activated sites (GAS) on various promoters, inducing transcriptional activity [⁹ , ¹⁰ ]. Nevertheless, the transcriptional regulation induced by IFN- is more complex and in some cases, is mediated independently of STAT-1 activation [^{9 10 11} ]. IFN- activity is also mediated by IFN regulatory factors (IRFs), which constitute a family of transcription factors with a characteristic helix-turn-helix motif [¹² ].

The murine iNOS promoter region has been shown to contain binding sites for various transcription factors. Optimal expression depends on two regulatory regions: the proximal region, located between positions –48 and –209 bp, which contains the proximal NF-B and C/EBP sites, and the distal region, located between –913 and –1029 bp, which contains the distal NF-B (dNF-B), GAS, and IRF responsive element (IRF-E) sites [¹³ , ¹⁴ ]. It has been described that the distal region is acting by enhancing the transcriptional response elicited by the proximal region, in response to certain stimuli such as LPS and/or IFN- [^{15 16 17} ]. The IRF-E element located between –913 and –923 has been described as necessary for the binding of IRF-1 and subsequent enhancement of iNOS transcription [¹⁸ ]. IRF-1 and STAT-1 are necessary for iNOS transcription induced by IFN-. Thus, macrophages from mice genetically deficient in STAT-1 or IRF-1 are unable to induce iNOS expression in response to IFN- [¹⁴ , ^{18 19 20} ]. Moreover, the dNF-B site, located at positions –961 to –971, is required to produce a complete iNOS induction by LPS [¹⁶ ]. The proximal region is required for LPS inducibility and binds to several members of NF-B and C/EBP families of transcription factors [²¹ , ²² ]. TNF- is another good inducer of NF-B, and this transcription factor strongly synergizes with other factors induced by IFN- for a complete activation of the iNOS promoter [² ]. Besides, NF-B seems to interact with IRF-1, and this interaction may explain the synergistic action of TNF- or LPS and IFN- in iNOS induction [²³ ]. Although a report [¹⁷ ] has involved NF-B sites in IFN- induction of iNOS, the role of this factor is far from clear, as IFN- is not generally considered a good inducer of NF-B.

Here, we have analyzed the molecular mechanism of IFN--induced iNOS expression in macrophages. We have clearly shown that NF-B activation is involved in iNOS expression by IFN-. In addition, we have shown that production of TNF- is critical for IFN--induced iNOS expression, as its neutralization or its absence in macrophages from TNF- knockout mice strongly reduces iNOS expression induced by IFN-. Moreover, NF-B activation induced by IFN- is abolished in macrophages from TNF--deficient (TNF-^–/–) mice, indicating a critical role of endogenous TNF- in this response. Thus, this cytokine is required for IFN--induced iNOS expression acting in an autocrine manner through NF-B activation.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Antibodies
Antibodies against p65, c-rel (polyclonal rabbit antibodies), p50, actin (polyclonal goat antibody; 2 µg used in supershift experiments and at 1:1000 dilution in Western blot assays), and normal goat and rabbit serum were purchased from Santa Cruz Biotechnology (CA). The anti-iNOS mAb was obtained from Transduction Laboratories (BD Biosciences, San Jose, CA) and was used at 1:10,000 dilution. The neutralizing anti-TNF- antibody was purchased from R&D Systems (Minneapolis, MN) and used at 1 µg/ml.

Plasmids
Plasmid iNOS murine-luciferase (piNOSm-luc) was constructed by cloning murine iNOS 5'-flanking region (from –1584 to +161 bp) on a pGL3 basic vector (Promega, Madison, WI). For mutagenesis of the GAS and IRF-E elements, Quikchange^TM site-directed mutagenesis kit (Stratagene, La Jolla, CA) was used. The oligonucleotides used for mutagenesis of IRF-E and GAS elements were: 5'-CCC CTA ACA CTG TCA ATA Tgg CAC gg T CAT AAT GGA AAA TTC CAT GCC-3' and 5'-CCC TCT CTC TGT TTG TTC CTT ggC CCC TAc CAC TGT CAA TAT TTC AC-3', respectively. Mutant nucleotides are in lower case and underlined. The vector named del-333 (see Fig. 4B ) is a deletion construct that contains the 333-bp upstream of the transcription start of the iNOS gene. This construction was generated by SacI digestion of the piNOSm-luc plasmid and subsequent religation of the vector. The sequence of all constructs was confirmed by automatic sequencing. p2iNOS (+,+)-chloramphenicol acetyltransferase (CAT), p2iNOS (–,+)-CAT, p2iNOS (+,–)-CAT, and p2iNOS (–,–)-CAT constructs, which are mutated in dNF-B and/or proximal NF-B sites, were kindly provided by Dr. Lisardo Boscá (CNIC, Madrid, Spain) and described previously [¹⁷ ]. The IB expression vector (pCMV-IB) was described previously [²⁴ ]. The pNF₃ConA-luc reporter contains three tandem repeats of the human immunodeficiency virus-1 long-terminal repeat NF-B enhancer, upstream of the conalbumin promoter and the luciferase reporter gene (kindly provided by Dr. José Alcamí, Hospital 12 de Octobre, Madrid, Spain). pRL-TK-luc (Promega), which expresses Renilla luciferase, and pCMVß vector (Clontech, Palo Alto, CA), which expresses ß-galactosidase (ß-gal), were used for determining transfection efficiency.

View larger version (27K): [in this window] [in a new window]   Figure 1. iNOS expression is induced by IFN-. (A) Nitrite accumulation in supernatants of IFN--treated RAW 264.7 cells at the indicated doses. (B) RAW 264.7 macrophages were treated with IFN- (2.5 ng/ml) for the indicated times, and NO release to supernatants was measured by Griess reaction. (C) Cells were stimulated with IFN- (2.5 ng/ml) in a time-course assay, and determination of iNOS and ß-actin mRNA levels by semiquantitative RT-PCR was performed at the lineal zone of amplification. (D) Western blot analysis of iNOS protein expression was performed in RAW 264.7 cells treated with IFN- (2.5 ng/ml) for the indicated times. A representative experiment for each type of analysis is shown.

View larger version (36K): [in this window] [in a new window]   Figure 2. IFN- does not induce iNOS expression in peritoneal macrophages from IFN--R–/– mice. Primary peritoneal macrophages were isolated from normal control mice (129Sv) or IFN--R–/– and treated for 24 h with IFN- (2.5 ng/ml) alone or in combination with LPS (1 µg/ml) or TNF- (100 U/ml). Determination of iNOS protein levels was performed by Western blot analysis. Actin expression is shown as internal control.

View larger version (12K): [in this window] [in a new window]   Figure 3. IFN- induces iNOS transcriptional activity. Time-course analysis of iNOS promoter activity. RAW 264.7 cells were transiently transfected with piNOSm-luc construct and 16 h after transfection, were treated with medium (C) or IFN- for the indicated times. The experiment shown is representative of three independent assays performed.

View larger version (17K): [in this window] [in a new window]   Figure 4. Analysis of the response elements involved in IFN--induced iNOS transcription. (A) Schematic representation of murine iNOS promoter. Distal and proximal regions are marked, and the main response elements are shown. (B) RAW 264.7 cells were transfected with the indicated mutant constructs and 24 h after transfection, were stimulated with IFN-. Luciferase activity was determined 16 h after treatment (left panel). Fold increase for the experiment is shown in the right panel. The experiment shown is representative of four independent assays performed. (C) Analysis of the mutation of NF-B sites on IFN- response. RAW 264.7 cells were transfected with wild-type (wt) iNOS promoter [p2iNOS (+,+)], dNF-B mutant [p2iNOS (–,+)], pNF-B mutant [p2iNOS (+,–)], or NF-B double mutant [p2iNOS (–,–)] constructs. Twenty-four hours after transfection, cells were stimulated with LPS (1 µg/ml) or IFN- (2.5 ng/ml) for an additional 16 h, and CAT activity was determined by CAT ELISA. (C, right panel) Results are expressed as percentage of promoter activity induced by the indicated stimuli, calculated over their respective controls in the absence of stimulation. Treatment of wild-type constructs with the indicated stimuli was taken as 100% of activation. Results shown are from one of the three performed with similar outcomes.

Nitrite and cytokine determination
Supernatants of control or IFN--treated cells were measured for TNF- presence using the Quantikine® M murine kit for mouse TNF- (R&D Systems), following the manufacturer’s instructions. The release of IL-1ß and IL-12 p40 cytokines to the supernatants was determined by Endogen mouse ELISA (Endogen, Woburn, MA), following the manufacturer’s instructions. For measuring nitrite accumulation, the Griess reaction was used [⁵ ].

Nuclear extracts and EMSA
Nuclear proteins were obtained as described [²⁵ ]. EMSAs were performed basically as described [²⁶ ] by using a NF-B consensus probe [²⁷ ]. Nuclear extracts (5 µg) were incubated with 2 µg poly (dI-dc) in 8 mM MgCl₂ for 10 min at room temperature. Radiolabeled probe (50,000 cpm) was added, and samples were incubated for an additional 15 min at 4°C. For competition studies, a 25-fold molar excess of the same cold probe (NF-B consensus probe) or nonspecific probe (AP-1 consensus probe, from Promega) was added 10 min before addition of the radiolabeled probe. For supershift assays, the antibodies were added to the samples 10 min before addition of the radiolabeled probe. Binding reaction was stopped by adding loading buffer, complexes were resolved by electrophoresis in a 4% SDS-polyacrylamide gel in 0.4x Tris-boric acid-EDTA buffer at 200 V for 3 h, and results were determined by autoradiography.

Chromatin immunoprecipitation assay
Cells (6x10⁶) were maintained in minimum serum complete medium (RPMI at 0.5% FCS) for 18 h before stimulation. After treatment with IFN- for 4 h, cells were fixed with 1% formaldehyde for 5 min at 37°C. Then, they were harvested and lysed in ice-cold lysis buffer [10 mM HEPES, 1.5 mM MgCl₂, 10 mM KCl, 0.5 mM DTT, 0.1% Nonidet P-40 (NP-40), 5 µg/ml leupeptin, 5 µg/ml aprotinin, 5 µg/ml pepstatin, and 1 mM PMSF] for 10 min at 4°C. Nuclei pellet was resuspended in nuclear lysis buffer (50 mM Tris-HCl, pH 8.1, 10 mM EDTA, 1% SDS, and protease inhibitors) and incubated on ice for 10 min. Chromatin was sheared by sonication (12 s each, three times at approximately one-fifth of the maximum power) and microfuged at 14,000 rpm for 10 min at 8°C. Extracts were diluted 10 times in dilution buffer (50 mM Tris-HCl, pH 8, 5 mM EDTA, 200 mM NaCl, 0.5% NP-40) and were precleared with a 25% suspension of salmon sperm DNA-saturated protein A agarose (Santa Cruz Biotechnology) for 3 h at 4°C. Precleared lysate was incubated with 5 µg of a rabbit specific antibody against p65 or with 1 µl normal rabbit serum (NRS) as a negative control overnight at 4°C. Immune complexes were collected adding salmon sperm DNA-saturated protein A agarose for 30 min at 4°C, washed three times with wash buffer (20 mM Tris-HCl, pH 8, 2 mM EDTA, 0.1% SDS, 1% NP-40, 500 mM NaCl), and followed by three additional washes in 20 mM Tris-HCl, 2 mM EDTA buffer. Extraction buffer (20 mM Tris-HCl, 2 mM EDTA, 2% SDS) was added to samples for eluting chromatin complexes, and the cross-link was reversed at 65°C overnight. Proteins were digested with 100 µg/ml Proteinase K for 2 h at 45°C, and DNA was extracted by using QIAquick PCR purification kit (Qiagen, Valencia, CA), following the manufacturer’s instructions. PCR amplifications were carried out by using Expand high fidelity kit (Roche, Nutley, NJ). The primers from iNOS promoter used for PCR were: primer 5', 5'-GTG AGT CCC AGT TTT GAA GTG ACT ACG TGC-3' (span at –250 bp from transcriptional start); primer 3', 5'-GGG CTC GAG GAC TAG GCT AGT CCG TGG AGT GAA C-3' (span at +161 bp from transcriptional start).

RT-PCR
Total RNA was obtained by using TriZOL reagent (Invitrogen Life Technologies), following the manufacturer’s instructions. For RT-PCR, 1 µg total RNA was reverse-transcribed into cDNA and used for PCR amplification with specific oligonucleotides by using the two steps RT-PCR kit Gene Amp RNA PCR core kit (Perkin Elmer, Wellesley, MA). The sequence of each oligonucleotide used was as follows: iNOS sense, 5'-GAG AGA TCC GAT TTA GAG TCT-3' (span at 3' end of exon 12 of murine iNOS gene); iNOS antisense, 5'-GCA GAT TCT GCT GGG ATT TCA-3' (span at 3' end of exon 18 of murine iNOS gene); ß-actin sense, 5'-CTC TTT GAT GTC ACG CAC GAT TTC-3'; ß-actin antisense, 5'-GTG GGC CGC TCT AGG CAG CAA-3' (previously reported in ref. [²⁸ ]). Briefly, the PCR was amplified by 25 repeat denaturation cycles at 94°C for 45 s, annealing at 58°C for 45 s, and extension at 72°C for 45 s.

Western blot
Cells were harvested by centrifugation (1200 rpm/5 min) and washed twice with PBS. Lysis buffer (30 µl; 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 1 mM PMSF, 10 mM NaF, 10 mM Na₃VO₄, and 2 µg/ml each inhibitors leupeptin, aprotinin, and pepstatin A) was added to the cellular pellet and incubated for 30 min in ice. Whole cell extracts were obtained by centrifugation for 10 min at 14,000 rpm at 4°C, and protein concentration was determined by the bicinchoninic acid method (Pierce, Rockford, IL). Total protein (30 µg) was separated in a 10% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane (BioRad, Hercules, CA). The membranes were blocked in TBS-0.1% Tween 20 with 5% of skim milk, washed twice with TBS-0.1% Tween 20, and incubated with specific antibodies for 1 h at room temperature. Then, membranes were washed three times and incubated with a secondary antibody for 1 h at room temperature. After extensive washing, peroxidase activity was detected with SuperSignal® West Dura extended duration substrate, according to the manufacturer’s instructions (Pierce).

Transient transfection
RAW 264.7 cells were transiently transfected by using lipofectAMINE^TM Plus reagent (Invitrogen Life Technologies) following the manufacturer’s instructions. For iNOS promoter constructs piNOSm-luc, piNOS GAS mut-luc, and piNOS IRF-E mut-luc, 50 ng DNA per 10⁶ cells was used. For p2iNOS-CAT constructs, 10⁶ cells were transfected with 250 ng DNA. In cotransfection experiments, IB expression vector was used at 100 ng/10⁶ cells, and total DNA concentration was kept constant with the corresponding empty vector (pcDNA3). Sixteen to 24 h after transfection, cells were treated with LPS (1 µg/ml) and/or IFN- (2.5 ng/ml) for an additional 6–16 h. In experiments for measuring kinetic induction of iNOS promoter activity, cells were treated 16 h after transfection with IFN- for the indicated times. pRL-TK-luc (Promega) was used for determining transfection efficiency by measuring Renilla luciferase activity in the samples. Luciferase activity is thus represented as relative luciferase units (RLUs) firefly/RLUs renilla. In transfection experiments with p2iNOS-CAT, pCMVß vector (Clontech) was used for determining transfection efficiency by measuring ß-gal activity in the samples. In those experiments, CAT activity was determined by CAT ELISA assay (Roche) following the manufacturer’s instructions. CAT activity is thus normalized and represented as CAT activity/ß-gal activity.

Statistical analysis
Values in figures are expressed as mean ± SD of three independent experiments in duplicate, unless otherwise specified. Student’s two-tailed t-test was used to compare means between groups. A value of P < 0.05 was considered to be statistically significant.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
IFN- induces iNOS expression in RAW 264.7 cells
To study the molecular mechanism by which IFN- regulates iNOS expression in macrophages, we analyzed nitrite accumulation as well as iNOS mRNA and protein levels induced by IFN- treatment in RAW 264.7 cells. As shown in Figure 1A , NO production by RAW 264.7 macrophages was first detected at 24 h of stimulation with 1 ng/ml IFN- and increased in a dose-dependent manner up to 5 ng/ml. For the subsequent experiments, RAW 264.7 cells were treated with 2.5 ng/ml IFN-, as that dose produced a medium increase in nitrite concentration (15.9 µM). Next, NO synthesis was determined at different time-points (Fig. 1B) . Nitrite accumulation in supernatants was first detected at 16 h of stimulation and increased in a time-dependent manner up to 48 h of IFN- treatment (Fig. 1B) . When iNOS mRNA expression was determined by semiquantitative RT-PCR (Fig. 1C) , we found that iNOS mRNA was first detected at 4 h and increased up to 48 h of IFN- treatment. That induction was followed by the expression of iNOS protein (Fig. 1D) . A weak expression could be observed at the first 6 h but was clearly detected after 8 h of treatment with IFN-. iNOS protein levels were maintained until 48 h of IFN- stimulation.

Previously, it has been described that IFN- treatment, in the absence of another stimulus such as LPS, did not induce NOS and iNOS mRNA expression. To determine if iNOS induction was a specific response to IFN- treatment, we analyzed iNOS protein expression in primary peritoneal macrophages from 129Sv control mice (129Sv) or IFN--R^–/– mice. As shown in Figure 2 , IFN- treatment induced iNOS expression in wild-type mice, and there was no expression detected in IFN--R^–/– macrophages. As a control, LPS stimulation induced iNOS expression in both types of macrophages (129Sv or IFN--R^–/– macrophages). A strong, synergic induction was observed in 129Sv control macrophages when IFN- stimulation was combined with LPS or TNF- treatment, whereas it was not detected in IFN--R^–/– macrophages. These results demonstrate clearly that iNOS induction is mediated by the action of IFN-.

Transcriptional regulation of the iNOS promoter by IFN-
To investigate if the above effects were taking place at a transcriptional level, RAW 264.7 cells were transiently transfected with the piNOSm-luc vector, expressing a luciferase reporter gene under the control of the murine iNOS promoter, and were stimulated with IFN- for the indicated times (Fig. 3 ). Although this assay requires some accumulation of luciferase protein to be detected, we found an almost negligible activity in the first 4 h, a small increase in iNOS promoter activity from 6 h to 10 h of stimulation, and a greater increment from 10 h to 24 h of IFN- treatment.

The distal region (–1029/–913) of the murine iNOS promoter contains the GAS and IRF-E sites (Fig. 4A ), which have been involved in conferring IFN- responsiveness [¹³ , ¹⁵ , ¹⁸ , ²⁹ ], although a role of the dNF-B and NF-B proximal elements in IFN- response has also been suggested [¹⁷ ]. To determine the relative contribution of the different elements of the iNOS promoter to the transcriptional activity induced by IFN-, RAW 264.7 cells were transfected with different constructs of the promoter mutated in GAS or IRF-E sites or dNF-B or NF-B proximal elements (Fig. 4B and 4C , respectively). As expected, stimulation by IFN- was reduced strongly by mutation of the GAS or the IRF-E sites (Fig. 4B) , similarly to the diminished response to IFN- observed when the complete distal region was deleted (del-333). As a control, transcriptional activity induced by LPS was partially inhibited when the distal region was deleted. It is interesting that RAW 264.7 macrophages transfected with constructs containing mutations in the dNF-B or the proximal NF-B elements showed a diminished response to IFN- stimulation (Fig. 4C) . Similar reductions were observed when LPS was used as a typical inductor of iNOS transcriptional activity mediated by NF-B. It is intriguing that although the mutation of the proximal NF-B site produced a reduction of approximately 50% in the IFN- induction, the dNF-B mutation completely abrogated the IFN--induced iNOS promoter activity. Taken together, the above results suggest that all of these sites are necessary for a complete IFN- response.

NF-B involvement in iNOS induction by IFN-
As IFN--induced iNOS promoter activity was strongly diminished when the NF-B sites were mutated, we further investigated the role of NF-B in the IFN- response. First, we determined the effect of IFN- treatment on the transcriptional activity driven by three tandem repeats of the HIV NF-B enhancer (NF₃ConA-luc). As shown in Figure 5A , IFN- treatment produced a significant increase in NF-B-driven transcription. Besides, NF-B transcription factors are bound to IB proteins, which retain them in an inactive form in the cytoplasm [³⁰ ]. Therefore, we overexpressed IB in RAW 264.7 cells to test its effect on the piNOSm-luc promoter. As shown in Figure 5B , 5I B overexpression strongly reduced IFN--induced iNOS promoter activity, suggesting that NF-B is involved in the IFN- response.

View larger version (11K): [in this window] [in a new window]   Figure 5. NF-B involvement on iNOS transcriptional activity induced by IFN-. (A) RAW 264.7 cells were transiently transfected with NF3ConA-luc construction, which contains three NF-B sites in tandem. Twenty-four hours after transfection, cells were treated with IFN- for an additional 16 h. The mean ± SD of three independent experiments is shown. *, P< 0.05. (B) NF-B inhibitory protein IB or control empty vector () was transiently transfected in RAW 264.7 cells together with piNOSm-luc construction. Cells were treated with medium or IFN- for 16 h, and luciferase activity was determined. The experiment shown is representative of three independent assays performed.

View larger version (42K): [in this window] [in a new window]   Figure 6. Analysis of NF-B activation by IFN-. (A and B) RAW 264.7 cells were treated with IFN- for the indicated times, and nuclear extracts were obtained for performing EMSA assays with a NF-B consensus probe. Competition studies (A) or supershift analysis (B) were performed. Solid arrowheads indicate specific NF-B complexes, determined by a competition assay with excess of unlabeled probe (Comp.). Nuclear extracts from cells treated with LPS for 1 h were used as a control for NF-B binding. Supershifted complexes are indicated with open arrowheads. NGS, normal goat serum; ns comp., nonspecific competitor (AP-1 consensus probe). (C) Chromatin immunoprecipitation (ChIP) assay. p65 transcription factor was immunoprecipitated (IP) from RAW 264.7 cells stimulated with IFN- for 4 h, as described in Materials and Methods.

Endogenously produced TNF- is required for iNOS induction by IFN-
As NF-B is a rapid response transcription factor by all known inducers [³¹ ], its slow induction by IFN- suggested that the effect was indirect. A possibility could be the existence of an autocrine mechanism involving the action of an IFN--induced cytokine on iNOS expression, which could activate NF-B. To test this hypothesis, we first determined the IFN--induced release of some cytokines to macrophage supernatants. Among the various cytokines tested (Fig. 7A ), only TNF- was increased significantly in a time-dependent manner upon IFN- treatment in RAW 264.7 cells. Other macrophage cytokines such as IL-1ß or IL-12 (Fig. 7A) could not be detected upon IFN- induction in those conditions.

View larger version (16K): [in this window] [in a new window]   Figure 7. TNF- production is required for iNOS induction by IFN- in RAW 264.7 macrophages. RAW 264.7 cells were treated with medium or IFN- (2.5 ng/ml). In neutralization assays, anti-TNF- (TNF Ab) neutralizing antibody (1 µg/ml) was added simultaneously with IFN-. ns, nonspecific (normal goat serum). (A) Cytokine production by RAW 264.7 at different time-points of IFN- stimulation. TNF-, IL-1ß, and IL-12 p40 production was determined by ELISA. (B) Nitrite accumulation in supernatants of stimulated RAW 264.7 macrophages was determined 24 h after IFN- treatment. (C) Western blot analysis of iNOS expression induced by IFN- at 24 h after treatment in the presence or absence of anti-TNF- neutralizing antibody. A densitometric analysis is shown in the bottom of the figure.

To corroborate the role of endogenous TNF- in IFN--induced iNOS expression, we obtained primary peritoneal macrophages from mice genetically deficient in TNF-. As shown in Figure 8A , iNOS protein induction by IFN- and subsequent NO production were detected after 16 h of IFN- treatment in wild-type mice, whereas they were absent in mouse peritoneal macrophages from TNF-^–/– mice at any time-point tested. Moreover, when we analyzed the iNOS protein induction by different doses of IFN- (Fig. 8B) , we found that there was no iNOS expression at any dose of IFN- tested in macrophages from TNF-^–/– mice, in contrast with the result obtained in wild-type macrophages. These results suggest that TNF- secretion induced by IFN- is necessary for iNOS expression. To corroborate this further, we analyzed whether exogenous addition of TNF- could restore iNOS induction in IFN--treated macrophages from TNF-^–/– mice. In wild-type macrophages, TNF- addition had no significant effect on iNOS protein levels or NO production, alone or in combination with IFN- (Fig. 8C) . By contrast, in TNF-^–/– macrophages, where there was no iNOS expression in response to IFN- treatment, addition of exogenous TNF- in combination with IFN- restored iNOS protein levels and NO release. Together, those results clearly indicate that the autocrine effect of TNF- secretion is necessary for iNOS expression induced by IFN-.

View larger version (35K): [in this window] [in a new window]   Figure 8. Lack of iNOS induction by IFN- in TNF-–/– mice. (A) Primary peritoneal macrophages were isolated from normal, wild-type or TNF-–/– mice and were treated with IFN- for indicated times. Western blot analysis of iNOS protein expression (left panel) and nitrite accumulation determination (right panel) is shown. (B) Peritoneal macrophages were isolated from wild-type mice or TNF-–/– mice and were treated with different doses of IFN- for 24 h. Analysis of iNOS protein expression was performed by Western blot assay. (C) Effect of exogenous TNF- on iNOS protein expression (left panel) and nitrite accumulation (right panel) in wild-type and TNF-–/– mice. Primary peritoneal macrophages obtained from wild-type mice or TNF-–/– mice were stimulated with medium (C), IFN- (2.5 ng/ml), and/or TNF-–/– at indicated doses for 24 h. Representative experiments from five independent assays performed are shown. Actin expression was analyzed as an internal control.

Endogenously produced TNF- is responsible for NF-B activation induced by IFN-

View larger version (23K): [in this window] [in a new window]   Figure 9. TNF- is required for NF-B activation and affects iNOS transcription induced by IFN-. (A) RAW 264.7 cells were transiently transfected with piNOSm-luc construct and 24 h after transfection, were treated with IFN- and with or without anti-TNF- (TNF Ab) neutralizing antibody. Luciferase activity was determined 16 h after treatment. Results are expressed as percentage of iNOS promoter activity in which IFN- stimulation was taken as 100% of activation. A representative experiment of three independent assays performed is shown. (B) Nuclear extracts were isolated from primary peritoneal macrophages from normal (wt) or TNF-–/– mice, treated with LPS for 30 min or IFN- for the indicated times. An EMSA was performed with NF-B consensus probe.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
iNOS-derived NO production by activated macrophages is a key event in the control of many infectious diseases [¹ , ³² ]. IFN-, a pleiotropic cytokine responsible for macrophage activation and differentiation, is one of the major inducers of iNOS, as IFN- ^–/– macrophages are unable to synthesize iNOS [³² ]. Here, we have investigated the molecular mechanism by which IFN- induces iNOS transcription, with an emphasis on the role of NF-B in this induction. Two elements in the murine iNOS promoter, IRF-E and GAS elements, where IRF-1 and STAT-1 bind, respectively, have been described previously as responsible for the IFN--induced iNOS expression [^{13 14 15} , ¹⁸ ]. In agreement with those studies, we have observed that mutation of the GAS element or IRF-E element strongly inhibited iNOS transcriptional activity induced by IFN-. We have also found an important role of the proximal and dNF-B elements in iNOS transcriptional activation by IFN-, as mutation of those sites drastically inhibited IFN--induced promoter activity. Mutation of the proximal NF-B site diminished the IFN- response approximately 50%. This result indicates that this site also partially participates in IFN--induced iNOS expression, likely as a cooperating factor to link proximal and distal regions for performing an enhanceosome complex (Fig. 10 ). However, mutation of the dNF-B site, like mutation of GAS or IRF-E elements, strongly inhibits IFN--induced iNOS transcriptional activity. An identical result was obtained when the complete distal region was deleted (Fig. 4B) . Together, all these data suggest that an intact distal region for eliciting an optimal and complete iNOS transcriptional activation by IFN- is necessary, probably as the transcription factors bound to GAS, IRF-E, and dNF-B sites have to interact among them for accomplishing an active transcriptional complex (Fig. 10) . Paludan et al. [³³ ] previously showed similar results. These authors found that all the motifs in the distal region were necessary for full responsiveness to IFN- and the synergistic response elicited by HSV-2-induced TNF- production. Moreover, they showed that the proximal NF-B site played an important role for inducing optimal promoter activity.

View larger version (27K): [in this window] [in a new window]   Figure 10. Working model for iNOS transcriptional induction by IFN- in macrophages. IFN- stimulation induces an early response that allows the binding of IRF-1 and STAT-1 to IRF-E and GAS sites of the mouse iNOS promoter. At the same time, IFN- stimulation induces TNF- secretion, which in an autocrine manner, activates NF-B binding to its corresponding elements in the promoter. The interaction between IRF-1 and NF-B bound to the proximal NF-B site produces a bend in DNA that allows a more efficient binding of transcriptional coactivators as CREB-binding protein (CBP)/p300 and the basal transcription.

In apparent contrast with our results, several authors have reported that IFN- treatment alone did not induce iNOS expression. The main reasons for discrepancies between those results and ours are first, the sensitivity of the methods used for detecting iNOS expression and second, the time of treatment used. Thus, in the work of Lorsbach et al. [³⁹ ], the authors analyzed iNOS expression by Northern blot, and they were not able to detect iNOS expression induced by IFN- treatment. In our study, we used a more sensible technique for determining iNOS mRNA levels such as RT-PCR, which permits detection of a lower number of mRNA molecules expressed than the Northern blot technique. In the work of Lowestein et al. [¹⁵ ], they did not observe iNOS transcriptional induction by IFN- after 8 h of treatment, determined by transfection assays with the murine iNOS promoter. At this time of stimulation, the promoter activity induced by IFN- is lower than that observed at 16 h of treatment, as we determined by time-course analysis of iNOS promoter activity (Fig. 3) . For this reason, all the promoter activity studies carried out in this work were performed at 16 h of IFN- treatment.

Here, we have found an obligatory role of TNF- in this induction, as macrophages from TNF-^–/– mice have no NO production or iNOS induction in response to any dose of IFN- tested. Those results were also corroborated in RAW 264.7 macrophages by the use of neutralizing anti-TNF- antibodies. A previous report has shown that neutralizing anti-TNF- antibodies decreased IFN--induced NO production by murine macrophages activated with IFN- [⁴⁰ ], which is in perfect agreement with our results. The autocrine role of TNF- seems to be taking place at least at the transcriptional level, as TNF- neutralization is inhibiting iNOS transcriptional activity induced by IFN-. Furthermore, we have demonstrated the obligatory role of endogenously produced TNF- in IFN--induced NF-B activation, as peritoneal macrophages from TNF-^–/– mice did not show NF-B activation by IFN-. Some studies have described a role for endogenously produced TNF- in iNOS induction. In these studies, TNF- production was elicited by the infection with several pathogens such as HSV-2 [⁴¹ ] or Leishmania major [⁴² ], and this TNF- was acting in an autocrine manner and collaborating with IFN- in iNOS induction. Furthermore, it has been shown that TNF- plays an important role in host resistance to L. major [³⁵ , ⁴² , ⁴³ ]. It has been demonstrated that development of leishmanicidal activity by IFN--activated macrophages requires the autocrine effect of TNF- by inducing the production of NO, responsible for the intracellular destruction of the parasites [⁴² ]. In addition, TNF- and iNOS induction is involved in the control of the lesion size developed in a model of experimental cutaneous leishmaniasis [⁴³ ]. Otherwise, a similar autocrine role for TNF- has been demonstrated for IFN- induction of other genes such as high mobility group box 1 protein [⁴⁴ ], CD40 [⁴⁵ ], or cyclooxygenase 2 [²⁵ ] in murine macrophages. This cooperative effect of cytokines through autocrine secretion is commonplace in cytokine activity. Taken together, all these results suggest that the IFN- response is mediated by the concerted action of the factors activated or induced directly by IFN- signaling and indirectly through the activity of endogenously produced TNF-, which in turn leads to NF-B activation and full expression of several genes including iNOS.

Many studies have revealed the synergistic role of TNF- and IFN- in the induction of many promoters such as iNOS, IRF-1, and Class I MHC [²³ , ^{46 47 48} ]. The basis of this synergism lies in the interaction between STAT-1 or IRF-1 induced by IFN- with NF-B activated by TNF-. Our results are in agreement with this and will contribute to explain the general phenomenon of the synergism of IFN- and TNF- in iNOS induction. The synergic induction of iNOS by IFN- and exogenously added TNF- is mediated by a physic interaction between IRF-1 and p65/p50 dimers, bound to the IRF-E in the distal region and to the proximal NF-B site in the iNOS promoter, respectively. This interaction is accompanied by bending of DNA, which is postulated to be necessary for accomplishing a productive enhanceosome complex [²³ ]. It is tempting to speculate that this interaction might occur in our system and could be necessary for a complete iNOS expression. In addition, it is important to note that the collaboration between the factors, which bind to the distal region of the iNOS promoter (including NF-B), is absolutely required for iNOS induction by IFN-.

In summary, our results indicate that the TNF- produced in response to IFN- is required for iNOS induction by activating the NF-B transcription factor and support a working model of IFN- induction of iNOS expression, which is shown in Figure 10 . IFN- signaling activates the STAT-1 transcription factor, which binds to the GAS elements present in iNOS and IRF-1 promoters, induces the synthesis of IRF-1, which binds to the IRF-E site in the distal region of the iNOS promoter, and induces the early iNOS expression at low levels. At the same time, IFN- induces the synthesis and secretion of TNF-, which in an autocrine manner, binds to its receptors in cellular surface, triggering a signal that culminates in NF-B activation and binding to the NF-B proximal and distal sites in the iNOS promoter. The simultaneous binding of all these transcription factors may then allow the recruitment of transcriptional coactivators such as CBP/p300 and the formation of an active, transcriptional complex for enhancing transcriptional synergy.

	ACKNOWLEDGEMENTS

 
This work was supported in part by grants from Programa Nacional de Salud of Spain (SAF 2004-05109 SAF 2005-02220), Fondo de Investigaciones Sanitarias (RIS G04/173, RECAVA C03/01, and RICET C03/04), Comunidad Autónoma de Madrid (08.3/0023.1/2001 and SAL/2001/2004), Laboratorios del Dr. Esteve, Integrated Project EICOSANOX (LSH-CT-2004-005033), and MAIN network of excellence from the 6th EU Framework Program European Commission, 6th Framework Program, and Fundación Ramón Areces. We thank those who kindly provided plasmids and essential reagents that made this work possible. We thank Dr. M. A. Iñiguez for helpful discussions. We also thank María Chorro, María Cazorla, and Gloria Escribano for excellent technical assistance.

Received September 23, 2005; revised January 17, 2006; accepted September 5, 2006.

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TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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