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The Journal of Immunology, 1998, 160: 2889-2895.
Copyright © 1998 by The American Association of Immunologists

Triggering of Peritoneal Macrophages with IFN-{alpha}/ß Attenuates the Expression of Inducible Nitric Oxide Synthase Through a Decrease in NF-{kappa}B Activation1

Eduardo López-Collazo*, Sonsoles Hortelano*, Armando Rojas{dagger} and Lisardo Boscá2,*

* Instituto de Bioquímica (Centro Mixto Consejo Superior de Investigaciones Cientificas-Universidad Compluteuse de Madrid), Facultad de Farmacia, Universidad Complutense, Madrid, Spain; and {dagger} Centro de Química Farmacéutica, Habana, Cuba


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Triggering peritoneal macrophages with IFN-{gamma} and a low concentration of LPS induced the expression of the inducible form of nitric oxide synthase (iNOS). This process was significantly inhibited when IFN-{alpha}/ß was added during the initial 2 h after the start of IFN-{gamma}/LPS activation. Evaluation of the transcriptional activity using run-on assays indicated that IFN-{alpha}/ß inhibited the transcription of iNOS. Transfection experiments using a 1.7-kb promoter sequence corresponding to the 5' flanking region of the murine iNOS gene showed decreased promoter activity in the presence of type I IFNs. Analysis of the transcription factors that participate in iNOS expression revealed a marked decrease of NF-{kappa}B activation, a nuclear factor required for the transcription of this gene. The degradation of I{kappa}B{alpha} and I{kappa}Bß, which is required for the translocation of NF-{kappa}B to the nucleus, was inhibited in the presence of IFN-{alpha}/ß. However, the activity of other transcription factors such as IFN regulatory factor 1, which is involved in the expression of iNOS in response to IFN-{gamma}, was not affected by IFN-{alpha}/ß stimulation. These results suggest that in the presence of IFN-{alpha}/ß, the activity of the iNOS promoter is impaired, and this attenuated nitric oxide synthase expression could be important in pathophysiologic situations in which secretion of type I IFNs occurs.


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The ability of multiple proinflammatory cytokines and bacterial cell wall products to trigger the expression of inducible nitric oxide synthase (iNOS)3 in macrophages and other cell types has been widely described, and an important role for the synthesis of nitric oxide (NO) in host defense against pathogens and tumor cells has been recognized (1, 2, 3, 4). One aspect emerging from these studies is the existence of a broad synergism between several effector molecules in the process of transactivation of the iNOS gene (3, 5, 6). Indeed, this cooperative response could be of importance in the modulation of the process of iNOS expression under in vivo conditions (3, 7). The synergism between low doses of LPS and IFN-{gamma} has been studied in detail using mice lacking molecules involved in intracellular IFN-{gamma} signaling as well as mice with a disrupted IFN regulatory factor (IRF)-1 gene (8, 9, 10). Results from these models showed an impaired response of the peritoneal macrophages when IFN-{gamma} function was blocked (9, 10, 11).

The activity of iNOS is mainly controlled at the transcription level, and the characterization of the promoter of the murine gene showed the presence of numerous sites for the binding of several transcription factors, among them two {kappa}B sites and sequences for the binding of factors modulated by IFN-{gamma} and IFN-{alpha}/ß (5, 6). The two {kappa}B sites identified in the 1.7-kb promoter are critical for the inducibility of the gene, and mutation of one of them greatly decreases the promoter activity, whereas mutation of both sites abrogates the transcriptional activity (5, 6, 12, 13). It has been shown, using deletional analysis of the iNOS promoter, that the synergism between LPS and IFN-{gamma} requires the cooperation between the upstream 5' region of the promoter and the downstream {kappa}B site (5, 6). However, less is known regarding the role of IFN-{alpha}/ß in the transcription of iNOS, although potential sites for the binding of transcription factors modulated by type I IFNs are present in the promoter region of this gene (5, 6). Indeed, type I IFNs inhibit the IFN-{gamma}-dependent expression of iNOS (14). IFN-{alpha}/ß are synthesized by macrophages after stimulation with IFN-{gamma} and LPS (15). Moreover, IFN-{alpha}/ß efficiently restores the expression of iNOS induced by lipid A in macrophages that are deficient in their response to this bacterial product (16).

In some cases, an important antagonism between IFNs has been observed when cells are simultaneously stimulated with IFN-{gamma} and IFN-{alpha}/ß. For example, IFN-{gamma} induces MHC class II expression, whereas IFN-{alpha}/ß down-regulates this process (17). In the same vein, synthesis and secretion of IFN-{alpha}/ß in the course of a viral infection decreases NO production, a process potentiated by IFN-{gamma}, especially in synergism with proinflammatory cytokines or bacterial products (18). In view of these data, we decided to investigate whether IFN-{alpha}/ß might modulate the expression of iNOS in response to IFN-{gamma} and a suboptimal dose of LPS. As our results show, challenging peritoneal macrophages with IFN-{alpha}/ß selectively antagonized the cooperative action of IFN-{gamma} on iNOS expression, mainly through a rapid decrease in NF-{kappa}B activity. However, this effect was absent when IFN-{alpha}/ß was added 2 h after IFN-{gamma}/LPS triggering or when macrophage stimulation was accomplished exclusively by increasing concentrations of LPS. These results suggest the existence of interference by IFN-{alpha}/ß in the mechanism of transactivation of the iNOS gene mediated by IFN-{gamma} and might be important in cases in which a continuous supply of IFN-{alpha}/ß occurs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals

Reagents were purchased from Sigma (St. Louis, MO) or Boehringer Mannheim (Mannheim, Germany). [{alpha}-32P]deoxyCTP, [{alpha}-32P]uridine triphosphate (UTP) and [14C]chloramphenicol were obtained from Amersham (Bucks, U.K.). IFN-{alpha}/ß and IFN-{gamma} were also from Boehringer. Serum and media were supplied by BioWhittaker (Walkersville, MD).

Treatment of animals and obtention of elicited peritoneal macrophages

10-wk-old BALB/c mice were maintained free of pathogens and bred in our colony. Mice were i.p. injected with 1 ml of thioglycolate broth 4 days before use (19). Peritoneal macrophages were prepared as follows: light ether-anesthetized mice (4–6 animals) were killed by cervical dislocation and injected i.p. with 5 ml of sterile RPMI 1640. The peritoneal fluid was carefully aspirated to avoid hemorrhage and kept at 4°C to prevent the adhesion of the macrophages to the plastic. After centrifugation at 200 g for 10 min at 4°C, the cell pellet was washed twice with 45 ml of ice-cold PBS. Cells were seeded at 1 x 106/cm2 in RPMI 1640 supplemented with 10% heat-inactivated FCS. After incubation for 1 h at 37°C in 5% CO2, nonadherent cells were removed by extensive washing with PBS. Experiments were conducted in phenol red-free RPMI 1640 supplemented with 0.5 mM arginine and 10% heat-inactivated FCS.

Determination of NO synthesis

NO release was determined spectrophotometrically by the accumulation of nitrite and nitrate in the medium (phenol red free). Nitrate was reduced to nitrite as previously described (19). Nitrite was determined with Griess reagent (19) by adding 1 mM sulfanilic acid and 100 mM HCl (final concentration). After an initial reading of the absorbance at 548 nm, naphthylenediamine (1 mM in the assay) was added and the absorbance was compared with a standard of NaNO2. Results were expressed as the amount of nitrite and nitrate released per milligram of cell protein.

RNA extraction and analysis

Total RNA (2–4 x 106 cells) was extracted following the guanidinium thiocyanate method (20). Equal amounts of RNA were denatured and size-separated by electrophoresis in a 0.9% agarose gel containing 2% formaldehyde and 3-(N-morpholino)-propanesulfonic acid buffer (21). The RNA was transferred to Nytran membranes (NY13-N; Schleicher & Schuell, Keene, NH) with 10x SSC under low vacuum conditions, and the membranes were prehybridized for 6 h at 42°C in 50% formamide, 0.25 mM NaCl, 0.1 mM sodium phosphate, 7% SDS, and 0.01% of salmon sperm. An 817-bp fragment (nucleotides 1–817) from the cDNA of macrophage iNOS was labeled with the Rediprime kit (Amersham, U.K.) and was used to detect the level of transcription (22). A 1.4-kb BamHI/HindIII fragment of the cDNA of I{kappa}B{alpha} was used as a probe to detect mRNA levels (22). A 420-bp fragment of I{kappa}Bß was synthesized by RT-PCR using samples of RNA obtained from testis due to the relative abundance in this tissue (23). The oligonucleotide primer sequences were 5'-GGACACAGCCCTGCACTTGG-3' (oligonucleotides 247–266) and 5'-GTAGCCTCCAGTCTTCATCA-3' (oligonucleotides 668–649) from the published murine cDNA (accession number U19799). The amplification product was cloned into a pGEM-T vector (Promega, Madison, WI) and sequenced to confirm the identity against that specified in the published data. The probes for I{kappa}B{alpha} and I{kappa}Bß specifically recognized the corresponding mRNA as deduced by the size of the mRNA and its relative abundance in various tissues (23). After hybridization with the probes, the membranes were washed once with 0.1x SSC and 0.1% SDS at room temperature for 10 min and then washed twice at 50°C for 30 min followed by quantification of the bands in a Fuji BAS1000 (Kangawa, Japan) autoradiograph and exposure to a micrograph film (Hyperfilm; Amersham, Little Chalfont, U.K.). Different exposure times of the films were used to ensure that bands were not saturated. Quantification of the film was performed by laser densitometry (Molecular Dynamics, Kemsing, U.K.) using hybridization with a ribosomal 18S probe as an internal control.

Preparation of cytosolic and nuclear extracts

A modified procedure based on the method of Schreiber et al. was used (22, 24). The macrophage cell layers were washed twice with ice-cold PBS, and the plates were filled with 0.4 ml of Buffer A (20 mM Tris-HCl, pH 7.8; 5 mM MgCl2; 10 mM KCl; 0.5 mM EGTA; 0.5 mM DTT; 1 mM PMSF; and 10 µM leupeptin). The cells were scraped off the dishes using a rubber policeman and transferred to a 1.5-ml tube to which Nonidet P-40 was added to reach a 0.5% concentration. The tubes were gently vortexed for 15 s, and nuclei were sedimented by centrifugation at 8,000 g for 15 s. Aliquots of the supernatant were stored at -80°C (cytosolic extract), and the nuclei pellet was resuspended in 100 µl of Buffer A supplemented with 0.4 M KCl. Nuclear proteins were extracted by centrifugation at 13,000 g for 15 min, and aliquots of the supernatant were stored at -80°C. Proteins were measured using the Bio-Rad detergent-compatible protein reagent (Richmond, CA). All steps of cell fractionation were conducted at 4°C.

Electrophoretic mobility shift assays

Synthetic oligonucleotides were prepared using a Pharmacia oligonucleotide synthesizer: NF-{kappa}B, corresponding to the proximal {kappa}B motif of the iNOS promoter 5'-CCAACTGGGGACTCTCCCTTTGGGAACA-3'(5, 6, 22). Aliquots of 50 ng of these annealed oligonucleotides were end-labeled with Klenow enzyme in the presence of 50 µCi of both [{alpha}-32P]deoxyCTP and the other unlabeled deoxynucleoside triphosphate in a final volume of 50 µl. The oligonucleotides were precipitated with ethanol and extracted with phenol/chloroform. DNA probe (5 x 104 disintegrations per minute (dpm)) was used for each binding assay of nuclear extracts as follows: 5 µg of protein extract was incubated for 30 min at 4°C with the DNA and 1 µg of poly(dI-dC)/ml; 5% glycerol; 1 mM EDTA; 100 mM KCl; 5 mM MgCl2; 1 mM DTT; and 10 mM Tris-HCl, pH 7.8, in a final volume of 20 µl. The incubation mixture was applied to a 6% polyacrylamide gel that had previously been electrophoresed for 30 min at 100 V. Gels were run at 0.8 V/cm2 in 45 mM of Tris-borate followed by transference to 3MM Whatman paper, drying under vacuum at 80°C, and quantification of the band intensities in an autoradiograph. Analysis of competition with unlabeled oligonucleotides was performed using a 20-fold excess of dsDNA in the binding reaction and adding the nuclear extracts as the last step in the binding assay. Supershift assays were conducted after addition of the Ab (0.5 µg) to the binding reaction and incubation for 1 h at 4°C (22).

Western blot analysis

Cytosolic and nuclear extracts were obtained for the electrophoretic mobility shift assays (EMSAs) as described previously. After determining the protein content, samples containing equal amounts of protein were boiled in 250 mM Tris-HCl, pH 6.8; 2% SDS; 10% glycerol; and 2% ß-mercaptoethanol. Proteins (30 µg and 10 µg per lane of cytosolic and nuclear extracts, respectively) were size-separated in 10% SDS-PAGE. The gels were processed against the murine Ags as recommended by the manufacturer of the Abs (I{kappa}B{alpha}, I{kappa}Bß, p50, p65, c-Rel, and IRF-1; Santa Cruz Laboratories, Santa Cruz, CA), and after blotting onto a polyvinylidene difluoride membrane, proteins were revealed following the enhanced chemiluminescence technique (Amersham).

Transfection of macrophages and chloramphenicol acetyltransferase (CAT) assays

Recently isolated and adherent macrophages were transfected with DOSPER transfection reagent (Boehringer Mannheim) following the manufacturer’s instructions. After 6 h of culture, the medium was aspirated, and the dishes were washed twice with incubation medium. Macrophages were maintained overnight with incubation medium supplemented with 2% FCS and followed by stimulation with different factors. After incubation for 24 h, the medium was aspirated, and the cell layer was washed twice with ice-cold PBS. The plates were treated with 0.5 ml of 0.25 M Tris-HCl (pH 7.8) at 4°C, and the cells were scraped off the dishes. The extract was submitted to three cycles of freezing and thawing followed by centrifugation at 12,000 g for 10 min. The supernatant was heated at 65°C for 10 min and the CAT activity was measured by the synthesis of acetylated [14C]chloramphenicol according to the TLC method (22). As an internal standard to assess the purity of the plasmid preparations, cells were challenged with a fivefold amount of plasmid in the absence of transfection reagent and failed to induce NO synthesis.

Plasmids

The 1753-bp HincII fragment, corresponding to the 5'-flanking region of iNOS and fused to a promoterless CAT-reported gene ({kappa}B+,+ plasmid), was a generous gift from Drs. Q.W. Xie and C. Nathan (New York, NY). Mutated {kappa}B promoter plasmids were generated by PCR using oligonucleotide primers in which two GG bases of each {kappa}B motif were replaced by a CC pair and were kindly given by Dr. T.J. Evans (London, U.K.) ({kappa}B-,- plasmid) (see Refs. 13, 22). The vectors were sequenced to confirm their fidelity. A kSV2CAT plasmid, in which the CAT gene is driven by the SV40 early promoter and enhancer, was used as a control of efficiency in transfection assays (22).

In vitro elongation of nascent RNA (run-on assay)

Macrophages (107 cells) were stimulated for 1 h with combinations of IFNs and LPS, and nuclei were isolated as described (25). Elongation of RNA was accomplished in the presence of an excess of ATP, CTP, GTP, and 2 µM UTP (150 µCi per assay). After 30 min of reaction at 30°C, the RNA was extracted in phenol/chloroform and partially purified using spin columns (Boehringer Mannheim). Equal amounts of radioactivity (107 dpm) were incubated with 2 µg of plasmid containing iNOS (pUC vector) (see 22 or plasmid lacking the iNOS cDNA insert and then linked to a Nytran membrane (NY13). After incubation at 65°C for 36 h, the membranes were washed, and the radioactivity was determined by fluorescence in a Fuji BAS1000 autoradiograph.

Statistical analysis

The data shown are the means ± SEM of three or four experiments. Statistical significance was estimated with Student’s t test for unpaired observations. A p value of <0.05 was considered significant. In studies of Northern blot analysis, linear correlations between increasing amounts of input RNA and signal intensity were observed (with correlation coefficients >0.9).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IFN-{alpha}/ß antagonizes the effect of IFN-{gamma} on NO synthesis

Stimulation of cultured macrophages with IFN-{gamma} induced a moderate synthesis of NO, a process which potentiated when it was combined with a suboptimal dose of LPS. However, when cells were treated with IFN-{alpha}/ß, a significant decrease in the response to IFN-{gamma} alone or IFN-{gamma} plus LPS, but not to LPS alone, was observed (Fig. 1GoA). Moreover, pretreatment of the cells with IFN-{alpha}/ß for 20 min before stimulation with IFN-{gamma} plus LPS resulted in a synthesis of NO identical with that observed when added simultaneously (Fig. 1GoA). The time course of NO release by cells treated with IFN-{gamma} and a low dose of LPS in the absence or presence of IFN-{alpha}/ß is shown in Figure 1GoB. As this figure shows, the inhibitory action of IFN-{alpha}/ß on NO synthesis persisted during the period of sampling assayed (up to 24 h). Moreover, when macrophages were stimulated with increasing concentrations of LPS, the presence of IFN-{alpha}/ß did not significantly affect the NO production (Fig. 1GoB, inset).



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  		FIGURE 1. IFN-{alpha}/ß antagonizes the effect of IFN-{gamma} on NO synthesis. Cultured peritoneal macrophages were stimulated for 24 h with 50 U/ml of IFN-{gamma}, 100 ng/ml of LPS, or 100 U/ml of IFN-{alpha}/ß, either alone or in various combinations. When cells were pretreated with IFN-{alpha}/ß, it was added 20 min before stimulation (cross-hatched bar). Nitrate was reduced, and the amount of nitrite was measured using Griess reagent (panel A). The time course of NO release was measured in macrophages stimulated in the absence ({triangleup}) or presence of 100 U/ml of IFN-{alpha}/ß ({blacktriangledown}), 100 ng/ml of LPS plus 50 U/ml of IFN-{gamma} ({square}), or a combination of both supplemented with 100 U/ml of IFN-{alpha}/ß ({blacksquare}) (panel B). The inset shows the NO release of cells incubated for 24 h with various doses of LPS and in the absence ({circ}) or presence of 100 U/ml of IFN-{alpha}/ß (•). The results show the mean ± SEM of three experiments. * denotesp < 0.01 with respect to the corresponding condition in the absence of IFN-{alpha}/ß.

 
Since iNOS activity is mainly controlled at the transcriptional level, the effect of IFN-{alpha}/ß on iNOS mRNA was investigated. As Figure 2Go shows, a time-dependent expression of iNOS was observed in response to a combination of IFN-{gamma} and LPS. However, when stimulation with IFN-{gamma}/LPS included the presence of IFN-{alpha}/ß, the corresponding iNOS mRNA decreased 63 and 42% at 3 and 6 h, respectively. Moreover, in contrast to IFN-{gamma}, IFN-{alpha}/ß was completely inefficient in promoting the transcription of the iNOS gene during the 1-, 3-, and 6-h periods of time analyzed. To further investigate the mechanism responsible for the decrease of iNOS mRNA levels by IFN-{alpha}/ß, an experiment was performed in which cells were stimulated with IFN-{gamma} plus LPS or with a saturating concentration of LPS (1 µg/ml), and IFN-{alpha}/ß was added at various times. As Figure 3GoA shows, IFN-{alpha}/ß affected only the mRNA levels of cells stimulated with IFN-{gamma} plus LPS. Moreover, addition of IFN-{alpha}/ß 20 min before stimulation or during the initial 2-h period after IFN-{gamma}/LPS challenge was required for the inhibition to be observed. Interestingly, IFN-{alpha}/ß inefficiently modified the iNOS mRNA levels in response to a high concentration of LPS. In view of these results, and to ascertain that IFN-{alpha}/ß effectively attenuated the transcription of the gene, run-on experiments were conducted in this experimental model. As Figure 3GoB shows, iNOS RNA elongation in isolated nuclei from control or IFN-{alpha}/ß-treated cells was negligible. A marked elongation was observed in IFN-{gamma}/LPS-treated cells and, as expected from the results reported in Figure 3GoA, the presence of IFN-{alpha}/ß produced a 71% reduction in UTP incorporation into nascent iNOS RNA.



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  		FIGURE 2. IFN-{alpha}/ß decreases iNOS mRNA levels in macrophages activated with IFN-{gamma}/LPS. Peritoneal macrophage cultures were stimulated with distinct combinations of LPS (100 ng/ml), IFN-{gamma} (50 U/ml), and IFN-{alpha}/ß (100 U/ml). Total RNA was prepared at the indicated times and was analyzed by Northern blot. The amount of iNOS mRNA was calculated after normalization for the 18S ribosomal RNA content of each sample. Results show the mean ± SEM of four experiments. * denotes p < 0.01 with respect to the corresponding condition in the absence of IFN-{alpha}/ß.

 


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  		FIGURE 3. IFN-{alpha}/ß inhibits iNOS transcription. The levels of iNOS mRNA were measured at 6 h after stimulation with 1 µg/ml of LPS ({square},{blacksquare}) or 50 U/ml of IFN-{gamma} and 100 ng/ml of LPS ({circ},•). Open and solid symbols correspond to experiments in the absence or presence of 100 U/ml of IFN-{alpha}/ß, respectively, which was added to the culture at the indicated times. Negative time values correspond to IFN-{alpha}/ß treatment before LPS and IFN-{gamma} stimulation (panel A). Run-on experiments on nascent RNA were conducted using equal amounts of incorporated UTP (107 dpm) by nuclei isolated from cells stimulated for 1 h with the indicated stimuli (panel B). Controls of the vector lacking the full length iNOS cDNA were included in hybridization (pUC). Results show the mean ± SEM of three experiments (panel A), or a representative experiment of two (panel B). * and ** denote p < 0.05 and p < 0.01, respectively, with regard to the condition in the absence of IFN-{alpha}/ß.

 
IFN-{alpha}/ß attenuates the activation of the 1.7-kb iNOS promoter region

The preceding results show a significant inhibition of iNOS transcription by IFN-{alpha}/ß upon challenge of macrophages with IFN-{gamma}/LPS. To gain insight into the mechanism mediating this inhibition, the effect of IFN-{alpha}/ß on the promoter activity of iNOS was investigated. Cells were transfected with a plasmid containing a 1.7-kb fragment corresponding to the 5'-flanking region of iNOS, and linked to a CAT-reporter gene. As Figure 4Go shows, IFN-{gamma}/LPS promoted the expression of CAT, but in the presence of IFN-{alpha}/ß the reporter activity was inhibited by 89%. Interestingly, this inhibition of the promoter by IFN-{alpha}/ß was quantitatively more important than the effect on the synthesis of NO (64% decrease; Fig. 1GoB), which suggests that this promoter fragment is more sensitive to the action of IFN-{alpha}/ß than the natural domain. Moreover, when cells were transfected with a plasmid mutated in the two {kappa}B motifs of the sequence, the reporter activity was completely abolished, indicating that these {kappa}B sites were essential for the transcriptional activation in cells stimulated with IFN-{gamma} and in the absence or presence of a suboptimal dose of LPS.



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  		FIGURE 4. IFN-{alpha}/ß inhibits the reporter activity of macrophages transfected with a 1.7-kb iNOS promoter linked to a CAT gene. Macrophages were transfected by lipofection with a plasmid containing a 1.7-kb fragment of the iNOS promoter linked to a CAT gene ({kappa}B+,+plasmid; solid bars). Alternatively, cells were transfected with a mutated promoter lacking NF-{kappa}B binding to both {kappa}B sites ({kappa}B-,- plasmid; open bars). Cells were treated for 24 h with the indicated stimuli, and CAT activity was measured. CAT activity was expressed as the percentage of the activity of cells transfected with a kSV2CAT plasmid. CAT activity in kSV2CAT-transfected cells was independent of cell stimulation (data not shown). Results show the mean ± SEM of three experiments. * and ** denote p < 0.05 andp < 0.001, respectively, vs nonstimulated cells transfected with the {kappa}B+,+ plasmid.

 
NF-{kappa}B activity is inhibited by IFN-{alpha}/ß in IFN-{gamma}/LPS-activated cells

To better characterize the mechanisms governing the inhibition exerted by IFN-{alpha}/ß, the activity of some relevant transcription factors involved in the expression of iNOS was analyzed. As previously mentioned, activation of NF-{kappa}B has been considered a necessary requirement for the expression of iNOS (12, 13, 22). As Figure 5Go shows (left panel), the NF-{kappa}B activity induced by IFN-{gamma} was notably inhibited at 1 h in the presence of IFN-{alpha}/ß. This included both the upper (p50/p65) and lower (p50/p50) complexes. When cells were activated with IFN-{gamma}/LPS, the intensity of both bands increased with respect to IFN-{gamma} treatment. However, the binding of NF-{kappa}B in the presence of IFN-{alpha}/ß was inhibited, although to a lesser extent when compared with IFN-{gamma}-stimulated cells. Moreover, supershift assays of nuclear extracts from IFN-{gamma}/LPS-activated cells using anti-p50, -p65, and -c-Rel Abs revealed that the two complexes observed corresponded to p50/p65 and p50/p50 dimers for the upper and lower bands, respectively. A quantitative analysis of the band intensities is shown in Figure 5Go (right panel). When the levels of IRF-1 were measured in parallel with NF-{kappa}B binding, this transcription factor was detected in these cells, and no significant changes were observed in all conditions assayed (Fig. 5Go, bottom).



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  		FIGURE 5. NF-{kappa}B activity decreases in IFN-{gamma} and LPS-stimulated cells treated with IFN-{alpha}/ß. Macrophages were treated with the indicated stimuli (100 U/ml IFN-{alpha}/ß, 50 U/ml IFN-{gamma}, and 100 ng/ml LPS), and nuclear extracts were prepared at 1 h. EMSAs were conducted using the labeled (5 x 104 dpm), proximal {kappa}B sequence of the iNOS promoter as a probe and 5 µg of nuclear protein extracts (left panel). Supershift assays were prepared from IFN-{gamma}/LPS nuclear extracts incubated with 0.5 µg of the indicated Ab, and the retained protein complexes were labeled with a point arrow (central panel). Quantitative analysis of the upper and lower {kappa}B complexes was conducted by densitometric measurement of the bands. IRF-1 levels were determined by Western blot analysis using the same samples assayed for EMSA. Results show the mean ± SEM of four experiments (right panel). * denotes p < 0.005 vs cells stimulated in the absence of IFN-{alpha}/ß at 1 h.

 
NF-{kappa}B is retained in the cytosol through interaction with inhibitory proteins termed I{kappa}B (26). Since NF-{kappa}B activation is dependent upon I{kappa}B phosphorylation and degradation, the measurement of I{kappa}B levels offers additional information regarding the mechanism of NF-{kappa}B activation (26, 27). When the time-course of the I{kappa}B{alpha} and I{kappa}Bß levels was determined in macrophages stimulated with IFN-{gamma} and LPS, a rapid and important degradation of both I{kappa}B proteins was observed. However, the levels of I{kappa}B{alpha} and I{kappa}Bß were notably higher when IFN-{alpha}/ß was present in the incubation (Fig. 6Go, left). When stimulation was accomplished in the presence of increasing amounts of IFN-{alpha}/ß, a concentration-dependent increase of I{kappa}B{alpha} and, to a lower extent, I{kappa}Bß, was detected at 1 h (Fig. 6Go, central). In addition to this observation, the mRNA levels of both I{kappa}B{alpha} and I{kappa}Bß were measured, and an IFN-{alpha}/ß-dependent decrease in the corresponding mRNA content at 1 h was observed (Fig. 6Go, right panel). These results support the decreased NF-{kappa}B activity observed in the presence of IFN-{alpha}/ß that show a relatively rapid recovery of I{kappa}B levels, which might contribute to reset NF-{kappa}B activity retaining the newly synthesized Rel proteins in the cytosol where they are inactive.



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  		FIGURE 6. Time- and dose-dependent inhibition by IFN-{alpha}/ß of I{kappa}B{alpha} and I{kappa}Bß degradation in macrophages stimulated with IFN-{gamma}/LPS. Cells were treated with 50 U/ml of IFN-{gamma}, 100 ng/ml of LPS, and, except where otherwise indicated, 100 U/ml of IFN-{alpha}/ß. The amount of I{kappa}B{alpha} and I{kappa}Bß was determined by Western blot analysis using equal amounts of protein and specific Abs that recognized two proteins of 26- and 42-kDa, respectively (left panel). Dose-dependent inhibition of I{kappa}B degradation (central panel) and up-regulation of mRNA levels (right panel) were measured at 1 h after activation. The amount of I{kappa}B{alpha} (open bars) and I{kappa}Bß (solid bars) protein and RNA at 1 h are given in each panel. Results show the mean ± SEM of three experiments. * and ** denote p < 0.05 andp < 0.005, respectively, vs the maximal level in each case (no condition, central panel; IFN-{gamma}/LPS, right panel).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this work, we have analyzed the effect of IFN-{alpha}/ß on the expression of iNOS in macrophages activated with suboptimal concentrations of LPS and IFN-{gamma}, a model in which cooperation between these two factors is well known (2, 3, 4, 5, 6). However, the data regarding the effect of type I IFNs on iNOS transcription are rather controversial: IFN-{alpha}/ß is induced and secreted by macrophages after triggering with IFN-{gamma}, LPS, or combinations of both, and the kinetics of the process are well known (15, 27, 28). Challenge of the murine macrophage cell line J774 with LPS induces the synthesis of IFN-{alpha}/ß after 3 h of treatment (27), a time at which we have observed that the addition of IFN-{alpha}/ß has minimal effects on the expression of iNOS. Moreover, if the culture medium is depleted of IFN-{alpha}/ß with a neutralizing Ab, the synthesis of NO markedly decreases, indicating that type I IFNs might participate in an autocrine loop intended to maintain iNOS levels in LPS-activated J774 cells. However, other investigators have reported an inhibitory effect of exogenously added IFN-{alpha}/ß on IFN-{gamma}-dependent iNOS expression (18, 29, 30). According to our data, these heterogeneous results can be explained in terms of the time at which IFNs are delivered: acting simultaneously, both IFN-{alpha}/ß and IFN-{gamma} exhibit antagonism in their actions, probably because of an altered sequence of activation of IFN-{gamma}-dependent signaling molecules, a process that is overcome if the addition or release of IFN-{alpha}/ß is delayed.

The activity of iNOS is mainly regulated at the expression level and, therefore, the study of the transcriptional control of its induction has been a field of intense research over the past few years (3, 5, 6). Numerous sequences specific for the binding of transcription factors involved in host defense have been identified in the 5' flanking region of the gene (5, 6). In this context, a pivotal role for the two {kappa}B sites, extending from position -85 to -76 and from -971 to -962 in the murine iNOS promoter, has been confirmed using deletional analysis or selective base mutation of the binding sequences (5, 6, 13, 22). In view of this requirement of NF-{kappa}B activation for the expression of iNOS, we investigated whether the simultaneous treatment with type I and type II IFNs might result in decreased NF-{kappa}B activity, which could contribute to the observed attenuation of iNOS transcription in these conditions. Indeed, this was the case, and the amount of p50/p65 (the transcriptionally active form) present in the nucleus 1 h after IFN-{gamma} and LPS stimulation was significantly reduced. In agreement with the decrease of NF-{kappa}B activity, IFN-{alpha}/ß prevented the degradation of I{kappa}B{alpha} and I{kappa}Bß in a time- and concentration-dependent fashion. However, when macrophages were stimulated with a saturating concentration of LPS in the presence of IFN-{alpha}/ß (data not shown), a complete degradation of I{kappa}B{alpha} and I{kappa}Bß was observed, which suggests that type I IFNs partially inhibit the IFN-{gamma} signaling pathway, including IFN-{gamma}-dependent I{kappa}B targeting, but do not inhibit the I{kappa}B{alpha} or I{kappa}Bß proteolytic process (26, 31). The mechanism by which IFN-{alpha}/ß decreases NF-{kappa}B activation remains to be established, although it might include the alteration of specific signaling pathways that participate in the I{kappa}B phosphorylation, such as the activation of the dsRNA-dependent protein kinase which, at least in vitro, efficiently phosphorylates I{kappa}B{alpha} (32, 33, 34).

In addition to the effects on NF-{kappa}B, IFNs may regulate the activity of the iNOS promoter through the recruitment of specific transcription factors that bind to at least 17 consensus motifs: IRF-binding element, IFN-{gamma} activation site (GAS), IFN-stimulated response element (ISRE), and IFN-{gamma} response element sites (9, 15, 16). We have investigated the possible role of IRF-1 as a likely candidate to modulate IFN-{gamma}-dependent responses (9, 35, 36), although IFN-{alpha}/ß was unable to modify the high content of IRF-1 present in these cells. These results suggest that IRF-1 function is probably masked by the interaction with other regulatory proteins such as IRF-2.

The complex structure of the iNOS promoter, in terms of the abundance of putative regulatory sites, make it difficult to develop a detailed functional analysis and is better covered in transfection assays following the activity of the intact promoter linked to a reporter gene. In this way, the significant inhibition of CAT expression elicited by IFN-{alpha}/ß in IFN-{gamma}/LPS-treated cells indicates a negative contribution of type I IFNs to the promoter activity of the region analyzed. Moreover, using similar conditions of iNOS expression in response to IFN-{gamma} and LPS, an important contribution of transcription factors regulated by IFN-{gamma} is evident (9, 13, 36). For example, the two ISRE sequences juxtaposed to the distal {kappa}B site act as enhancers, especially the distal motif, and in this way IFNs can modulate iNOS transcription (6, 9, 13, 36, 37). Additionally, a certain cell specificity in the synergism of IFN-{gamma} and LPS on iNOS transcription has been reported: in macrophages, the activity of the responsive element to LPS and cytokines is located in the -890 to 1002 region of the promoter, whereas in vascular smooth muscle cells, a segregation between the elements involved in the cooperative response to IFN-{gamma} (-1029 to -913) and LPS (-85 to -75) has been observed (13). Similar cooperative interactions between ISRE and {kappa}B motifs activated in response to IFN-{gamma} and LPS have been described in the promoter region of other genes involved in the mediation of inflammatory responses (38). More recently, an important decrease in the iNOS promoter activity was observed when macrophages were transfected with constructs containing mutated sequences of the GAS motifs (39). Interestingly, the specific interaction of STAT1{alpha} with the GAS site has been recognized as a necessary requirement for the optimal induction of iNOS in response to IFN-{gamma}/LPS (39).

The signal transduction pathways activated by type I and type II IFNs are known in detail and involve the phosphorylation of latent transcription factors of the STAT family (STAT1 and STAT2) (40, 41). The ability of the STAT proteins to form distinct complexes, such as homodimers and heterodimers that exhibit specific interaction with the DNA binding motifs, may contribute to the antagonism observed between IFNs. This situation has been reported for several genes regulated by ISRE sequences that are induced either by type I or type II IFNs (42, 43). In this way, this mechanism of regulation of IFN-dependent gene expression might coexist with other types of controlling NF-{kappa}B activation by IFNs.

In summary, our results add new insight to the mechanism by which IFN-{alpha}/ß modulates iNOS expression, i.e., by exerting an inhibitory action over the signaling induced by IFN-{gamma}. Since type I IFNs are induced during the course of viral infections, it is possible that they can modulate NO synthesis in a manner which improves macrophage antiviral functions. In this regard, a complex regulatory response in terms of iNOS expression has been observed in macrophages during the course of viral infection (30, 44), and the observation that some viruses encode soluble forms of IFN-{alpha}R (45) points to a specific function of type I IFNs in host defense against viral infections.


   Acknowledgments
 
The authors thank Dr. S. Lamas for critical reading of the manuscript, O.G. Bodelón for technical support, E. Lundin for help in the preparation of the manuscript, and Drs. Q.W. Xie and C. Nathan for the generous gift of the iNOS cDNA probe.


   Footnotes
 
1 This work was supported by Grants PM95-007 and BIO95-2071-E from the Comisión Interministerial de Ciencia y Tecnología, Spain. Back

2 Address correspondence and reprint requests to Dr. Lisardo Boscá, Instituto de Bioquímica, Facultad de Farmacia, 28040 Madrid, Spain. Back

3 Abbreviations used in this paper: iNOS, inducible nitric oxide synthase; NO, nitric oxide; IRF, IFN regulatory factor; GAS, IFN-{gamma} activation site; ISRE, IFN-stimulated response element; NF-{kappa}B, nuclear factor {kappa}B; EMSA, electrophoretic mobility shift assay; UTP, uridine triphosphate; dpm, disintegrations per minute; CAT, chloramphenicol acetyltranferase. Back

Received for publication June 6, 1997. Accepted for publication November 17, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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