Expression of inducible NO synthase (iNOS) by macrophages is a prerequisite for the production of high output NO, which mediates many bactericidal and tumoricidal actions of these immune cells. The expression of iNOS in mammalian cells is governed predominantly by the transcription factor, NF-κB, which regulates the expression of many host defense proteins. In the present study, we characterize a novel, biphasic effect of NO on NF-κB activity in murine macrophages. This mechanism depends on the local concentration of NO and enables it both to up- and down-regulate the expression of host defense proteins including iNOS, cyclooxygenase-2, and IL-6. This biphasic activity of NO appears to play a pivotal role in the time course of activation of these immune cells and, by inference, in facilitating the initiation of a defense response against pathogenic stimuli and in its termination to limit tissue damage. This mechanism may explain at least in part the reported ability of NO to act in both a pro- and anti-inflammatory manner.
Nitric oxide production by the inducible isoform of NO synthase (iNOS)3 plays a pivotal role in numerous and diverse pathophysiological processes, particularly as a principal mediator of the microbicidal and tumoricidal actions of macrophages (1, 2, 3). Inducible NOS is expressed in many cell types in response to a diverse range of inflammatory cytokines, including IL-1β, IL-2, IFN-γ, TNF-α, and bacterial metabolites such as LPS (4, 5, 6). The inherent activity exhibited by iNOS results in the production of high output NO, which is cytotoxic and cytostatic to a number of pathogens and tumor cells; this is mediated via inhibition of various enzymes within target cells, including complexes I and IV of the mitochondrial respiratory chain (7, 8, 9), ribonucleotide reductase (10), aconitase (3), and GAPDH (11), and through DNA modification (12, 13).
In murine macrophages, regulation of iNOS expression is governed predominantly by the transcription factor NF-κB, which is expressed ubiquitously and is essential for the inducible expression of genes associated with immune and inflammatory responses (e.g., cytokines, adhesion molecules, antioxidant enzymes) (for review, see Refs. 14 and 15). NF-κB belongs to the larger NF-κB/Rel family of transcription factors that exist as homo- or heterodimers in the cytosol of most mammalian cells, sequestered in a quiescent state via interaction with a class of inhibitory proteins, termed IκBs. NF-κB can be activated by a number of immunological and pathological stimuli, including cytokines, oxidative stress, UV light, and bacterial and viral products. Following the appropriate stimuli, activation of an IκB kinase occurs, which results in phosphorylation of the IκB followed by polyubiquitination. These modifications result in rapid proteolysis of the IκB by the 26S proteosome, thereby releasing the active NF-κB complex, which translocates to the nucleus and binds the DNA at a specific consensus sequence (5′-GGGRNNYYCC-3′) to promote transcription of target genes. NF-κB consensus sites have been found in the promoter sequence of many proinflammatory proteins, including human and murine iNOS (6); accordingly, agents that modulate the activity of NF-κB have a pronounced effect on iNOS expression in response to different inflammatory stimuli (16, 17, 18, 19).
It is now becoming clear that NO plays a pivotal role in the regulation of gene expression. One key facet of this regulatory activity may be the control of iNOS induction. Such a mechanism would constitute a self-regulating pathway by which NO production from this NOS isoform could be fine-tuned, which is essential since iNOS is regulated transcriptionally rather than biochemically. Previous reports have suggested that NO has an inhibitory effect on iNOS expression (20, 21, 22, 23) and that this effect may be mediated at least in part by decreased NF-κB activity (22, 24, 25, 26, 27, 28). The mechanisms underlying this phenomenon are not clear, but may involve stabilization of the inhibitor protein IκBα (29) or nitrosation of the p50 subunit of NF-κB (which decreases its DNA-binding affinity) (30, 31, 32). In contrast, under certain conditions NO has been reported to augment the expression of host defense proteins, including cyclooxygenase-2 (COX-2) (33, 34, 35), TNF-α (36), and glutathione-synthesizing enzymes (37), possibly by potentiating NF-κB activity (38). Moreover, iNOS expression can be both up- and down-regulated in murine (ANA-1) macrophages by NO (39). Such observations hint that the action of NO in regulating gene expression is likely to be multifaceted and may involve both positive and negative effects. Moreover, the majority of the above observations have been made using exogenous NO sources, and the role of endogenous NO on NF-κB activity and proinflammatory protein expression remains to be elucidated.
In the present study, we demonstrate that NO has a patent biphasic effect on NF-κB activity in murine macrophages and hence possesses the ability to both up- and down-regulate the expression of a number of proinflammatory proteins, including iNOS, COX-2, and IL-6. The dual effects of NO on NF-κB has a pronounced effect on the activation profile of immune cells and therefore has important implications for both the initiation and the suppression of an immune/inflammatory response. The dual effects of NO on NF-κB may also explain in part the ability of NO to exert both pro- and anti-inflammatory actions (40).
Materials and Methods
Salmonella typhosa (serotype 0901) LPS was purchased from Difco (Detroit, MI). Diethylamine-NONOate (DEA-NO) was obtained from Cayman Chemicals (supplied by Alexis, Nottingham, U.K.). All other chemicals were purchased from Sigma (Poole, U.K.).
Murine macrophages (RAW 264.7 cells) were purchased from American Type Culture Collection (TIB 71; Manassas, VA). Cells were cultured in suspension in a glass stirrer bottle in RPMI 1640 (with 25 mM HEPES) supplemented with 10% heat-inactivated New Zealand FCS (low endotoxin), 2 mM glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin (Life Technologies, Paisley, U.K.; complete medium). The cell cultures were maintained as a stirred culture at 37°C in a humidified incubator containing 5% CO2 in air.
In l-arginine-free experiments, medium was prepared using RPMI 1640 (with 25 mM HEPES) free from l-arginine (Life Technologies) supplemented with 10% dialyzed, heat-inactivated New Zealand FCS (low endotoxin), and glutamine, penicillin, and streptomycin as described above (l-arginine-free medium).
RAW 264.7 macrophages were incubated for 24 h (as above) in 75-cm2 flasks at a concentration of 5 × 106 cells/flask in 10 ml of complete medium (unless otherwise stated). Before activation with LPS (1 μg/ml), the medium was removed, and the cells were washed with 5 ml of PBS and replenished with complete medium or l-arginine-free medium. In certain experiments, cells were preincubated for 15 min with NG-nitro-l-arginine methyl ester (l-NAME; 1 mM) before activation with LPS. Flasks were then incubated as described above. At appropriate time points, medium was removed from flasks, and an aliquot was stored at −20°C for IL-6 and nitrite measurement. The remaining medium was discarded, cells were washed with 5 ml of PBS, and following addition of 1.5 ml of Accutase (TCS, Botolph Claydon, U.K.) flasks were incubated for an additional 10 min. Cells were dislodged and washed three times by centrifugation (200 × g, 5 min, 4°C) with PBS, and pellets were stored at −80°C.
Western blot analysis
Cell pellets were homogenized in 50 mM Tris-HCl (pH 7.5), 0.1 mM DTT, 0.2 mM EDTA, and 10 μg/ml protease inhibitor cocktail (benzamidine, leupeptin, aprotinin, and antipain) by sonication. The resulting lysate was centrifuged (105,000 × g, 30 min, 4°C), and the supernatant was retained. Protein concentrations were determined by protein assay (Bio-Rad, Hemel Hempstead, U.K.). Equal volumes of protein were subjected to 7.5% SDS-PAGE under reducing conditions. The proteins were transferred to nitrocellulose membranes (Pharmacia, Amersham, U.K.) with a semidry blotter (Pharmacia) at 120 mA for 60–90 min. The membranes were then incubated with shaking in 5% milk in wash buffer (PBS/0.1% Tween 20) for 1 h at room temperature. The membrane was washed twice (15 min/wash) in wash buffer before incubation overnight at 4°C with gentle shaking, with primary Ab (anti-iNOS (Transduction Laboratories, Lexington, KY), anti-COX-2 (Cayman Chemicals, Ann Arbor, MI), and anti-actin (Chemicon, Temesula, CA) diluted 1/2000 (anti-iNOS), 1/3000 (anti-COX-2), or 1/500 (anti-actin)) in 1% milk in wash buffer. The membrane was washed six times (5 min/wash) and then incubated, with gentle shaking for 2 h at room temperature, with HRP-conjugated anti-rabbit IgG (Vector Laboratories, Burlingame, CA) diluted 1/5000 in 1% milk in wash buffer. The membrane was washed as previously, and proteins were visualized using enhanced chemiluminescence (Amersham, Aylesbury, U.K.). Bands were quantified by densitometry (NIH Image).
RNA isolation and RT-PCR
RNA was extracted from cells using a simple nucleic acid preparation Total RNA Isolation kit (Invitrogen, Groningen, The Netherlands). Random hexamers (0.15 ng/reaction; Life Technologies) were added to equal amounts of RNA made up to a 12-μl volume in diethylpyrocarbonate-treated water. The mixture was heated at 70°C for 10 min, then cooled immediately on ice. DTT (10 mM), first-strand buffer (supplied with enzyme), and PCR nucleotide mix (0.5 mM; Roche Molecular Biochemicals, Lewes, U.K.) were added to a final volume of 20 μl, and the mix was heated at 25°C for 10 min. RT was then conducted at 42°C for 55 min, with 200 U of Superscript II RNase H reverse transcriptase (Life Technologies) added after 2 min of incubation. The mixture was then heated at 70°C for 15 min and stored at −20°C.
PCR was performed on a Primus 96 Thermocycler (MWG Biotech, Milton Keynes, U.K.) in a reaction containing 0.2 mM PCR nucleotide mix (Roche Molecular Biochemicals), forward and reverse primers (1 μM each), 1× PCR buffer with MgCl2 (supplied with enzyme), 1 μl of cDNA, and 1 U of Taq DNA polymerase (Roche Molecular Biochemicals) made up to 25 μl with distilled water. The same master mix containing all reagents was used for each sample. Thermal cycling conditions were as follows: 95°C for 5 min, then 22 cycles of denaturation at 95°C for 1 min, annealing at 58°C for 2 min, and polymerization at 72°C for 2 min, followed by a final extension at 72°C for 10 min. PCR products were resolved by agarose gel electrophoresis (2% gel) and stained with ethidium bromide. Preliminary experiments were performed to ensure that the number of cycles used gave a product quantity that was on the linear portion of the PCR amplification curve (data not shown).
Primer sequences (Genosys, Cambridge, U.K.) were: iNOS sense, 5′-GCATTTGGGAATGGAGACTG-3′; iNOS antisense, 5′-GTTGCATTGGAAGTGAAGCGTTTC-3′; COX-2 sense, 5′-GAGGTACCGCAAACGCTT-3′; COX-2 antisense, 5′-TTATTGCAGATGAGAGACTG-3′; GAPDH sense, 5′-ATGGTGAAGGTCGGTGTGAACG-3′; and GAPDH antisense, 5′-GGCGGAGATGATGACCCGTTTGGC-3′.
Cells (7 × 106) were seeded in complete medium in 10-cm tissue culture dishes, then incubated and activated as described above. Nuclear extracts were prepared by washing the adhered cells with cold PBS and adding cold buffer A (10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, and 0.2% Nonidet P-40). The dish was scraped, and the reaction volume was centrifuged (1850 × g, 1 min, 4°C). Supernatants were discarded, and the nuclear pellets were resuspended in 60 μl of buffer C (20 mM HEPES (pH 7.9), 25% glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 0.5 mM DTT, and 0.2 mM EDTA) and left on ice for 30 min. A protease inhibitor cocktail tablet (Roche) was added to buffers A and C. The solution was centrifuged (1850 × g, 2 min, 4°C), and the supernatants were recovered as nuclear extracts. The protein concentration was measured by the Bio-Rad protein assay. Samples were aliquoted and stored at −20°C. A double-stranded oligonucleotide containing an NF-κB consensus sequence (5′-GGGGACTTTCC-3′; Promega) was end labeled using [γ-32P]ATP and T4 polynucleotide kinase (Promega). Probes were purified using Centri-sep spin columns (Princeton Separations, Sigma, Genosys, Cambridge, U.K.). For each experiment protein DNA-binding reactions were performed using equal amounts of nuclear extract protein (2–3 μg) and labeled oligonucleotide in the presence of incubation buffer (1 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 50 mM NaCl, 10 mM Tris-HCl (pH 7.5), 0.05 μg/μl poly(dI-dC), and 4% glycerol) for 30 min on ice. To ensure specificity of probe binding, certain experiments were conducted in the presence of a 100-fold molar excess of unlabeled (cold) NF-κB consensus oligonucleotide (data not shown.). Protein-DNA complexes were resolved in 5% polyacrylamide gels, electrophoresed for 1 h at room temperature in 0.5× TBE (45 mM Tris-borate and 1 mM EDTA, pH 8.0). Gels were exposed to x-ray film overnight at −80°C. Bands were quantified by densitometry (NIH Image).
IL-6 and nitrite measurement
IL-6 was measured using the Quantikine M mouse IL-6 quantitative colorimetric sandwich ELISA from R&D Systems (Abingdon, U.K.) according to the manufacturer’s instructions. Nitrite accumulation was determined by mixing equal volumes of cell culture medium and Griess reagent (0.5% sulfanilamide, 0.05% naphthylethylenediamine dihydrochloride, and 2.5% H3PO4) with absorbance (A540–A620) read on a Molecular Devices 96-well microplate reader (Menlo Park, CA); standard curves were constructed with known concentrations of NaNO2.
All statistical analyses were performed using GraphPad Prism (GraphPad Software, San Diego, CA). Densitometric analyses were performed using NIH Image. All data are plotted graphically as mean values, with vertical bars representing SEMs. Student’s t test was used to assess differences between experimental conditions. A p < 0.05 was taken as an appropriate level of significance.
Effect of inhibition of endogenous NO synthesis on iNOS and COX-2 protein expression
Activation of RAW 264.7 cells with LPS (1 μg/ml) produced a time-dependent expression of iNOS and COX-2 mRNA and protein. In both cases, protein expression peaked between 12 and 24 h and had decreased by 75% of maximum by 48 h (Figs. 1 and 2). mRNA expression for both proteins followed a very similar pattern but peaked between 9 and 12 h (data not shown). In cells cultured in l-arginine (substrate)-free medium, the time course of iNOS and COX-2 protein expression was significantly altered, such that peak expression was delayed until 48 h, and substantial levels of protein were still present at 96 h (Figs. 1 and 2). mRNA expression was delayed in a similar manner, peaking at 24–48 h (data not shown). This protracted rise and fall in protein expression was also observed in the presence of the NOS inhibitor l-NAME (Figs. 1 and 2). However, the effect of l-NAME was not as marked as that seen with cells cultured in l-arginine-free medium. Peak iNOS and COX-2 protein expression in response to LPS was reduced significantly in cells in which endogenous NO production was inhibited; in cells cultured in l-arginine-free medium, iNOS and COX-2 proteins levels reached 75.4 ± 7.8% (n ≥ 3; p < 0.05) and 80.4 ± 12.6% (n ≥ 3; p < 0.05) of control values, respectively.
To ascertain the extent of inhibition of endogenous NO synthesis by l-NAME and l-arginine-free medium, nitrite accumulation in the culture medium was determined. Under control conditions, activation of RAW 264.7 cells resulted in the production of ∼35 μM nitrite over a 96-h period. Cells cultured in l-arginine-free medium did not produce any significant concentrations of nitrite over the same time period (Fig. 3). However, in the presence of l-NAME (1 mM), nitrite accumulation was significantly inhibited, but still reached a peak of about 15 μM (∼45% of control; Fig. 3). l-NAME itself did not cause any nitrite accumulation in the absence of LPS (data not shown).
Effect of inhibition of endogenous NO synthesis on IL-6
To verify the effect of inhibition of endogenous NO synthesis on the cellular response to LPS, the production of a third inflammatory mediator, IL-6, was assessed. Accumulation of IL-6 in the supernatant following activation of murine macrophages with LPS peaked at 72–96 h, after which time no further production was observed. In the presence of l-NAME (1 mM) or in cells cultured in l-arginine-free medium, production of IL-6 was significantly retarded at time points up to 12 h, but exceeded levels seen in controls at later time points (Fig. 4). As was observed with the expression of iNOS and COX-2 protein, the effect of l-NAME on IL-6 production was reduced compared with that in l-arginine-free medium. However, a similar pattern of activity of endogenous NO on IL-6 production as that seen with iNOS and COX-2 mRNA and protein was revealed.
Effect of inhibition of endogenous NO synthesis at different time points during macrophage activation
To study the differential activity of NO in the early (9 h) and late (24 h) phases of macrophage activation, endogenous NO synthesis was inhibited at different times following LPS stimulation. In the first series of experiments, l-NAME (1 mM) was present throughout; in this case, expression of iNOS protein 9 h after activation with LPS was significantly reduced compared with the control value (Fig. 5,A). In contrast, if inhibition of endogenous NO synthesis was established with l-NAME (1 mM) at 9 h subsequent to LPS activation, expression of iNOS protein at 24 h was markedly higher in treated cells (Fig. 5 A).
Effect of inhibition of endogenous NO synthesis NF-κB activity
To examine whether the effect of inhibition of endogenous NO synthesis on iNOS and COX-2 mRNA/protein expression and IL-6 production might be the result of a modification of the transcription factor NF-κB, EMSAs were conducted. Under control conditions, activation of RAW 264.7 cells with LPS resulted in a time-dependent increase in NF-κB activity, which peaked between 1–2 h and approached baseline values after 24 h (data not shown). Studies were conducted to examine the effect of inhibition of endogenous NO synthesis at different time points following cellular activation with LPS. Addition of l-NAME (1 mM) to cells 15 min before LPS resulted in a significant decrease in NF-κB activity as measured at 90 min (Fig. 5,B). In contrast, addition of l-NAME (1 mM) 9 h subsequent to LPS elicited a marked increase in NF-κB activity as assessed at 24 h (Fig. 5 B).
Effect of exogenous NO on NF-κB activity
To investigate more extensively the effect of NO on NF-κB activity in murine macrophages, the NO donor DEA-NO was used as an exogenous source of NO. DEA-NO releases NO spontaneously in aqueous solution with a half-life of ∼3 min at 37°C (41). Addition of DEA-NO at increasing concentrations (30 nM to 300 μM) to the RAW 264.7 cells immediately before addition of LPS caused a distinct biphasic effect on NF-κB activity as assessed by EMSA 90 min subsequent to activation (Fig. 6). At lower concentrations (30 nM to 3 μM) DEA-NO caused a significant enhancement of NF-κB activity, whereas at higher concentrations (30 μM to 300 μM) DEA-NO produced an inhibition of NF-κB activity (Fig. 6). The peak NO levels attained from this range of DEA-NO concentrations are given in Table I; such figures correspond closely with those reported previously (42).
|30 nM||300 nM||3 μM||30 μM||300 μM|
|Peak [NO] (nM)||6.5 ± 1.6||21.8 ± 4.7||85.6 ± 14.8||528.9 ± 246.0||2067.8 ± 1053.9|
|30 nM||300 nM||3 μM||30 μM||300 μM|
|Peak [NO] (nM)||6.5 ± 1.6||21.8 ± 4.7||85.6 ± 14.8||528.9 ± 246.0||2067.8 ± 1053.9|
Peak [NO] levels produced by increasing concentrations of DEA-NO (30 nM–300 μM) as measured by a Clarke-type electrode (World Precision Instruments, Sarasota, FL) in 1 ml complete medium containing 106 RAW 264.7 cells. Data are expressed as mean ± SE from at least four separate experiments.
Effect of exogenous NO on iNOS and COX-2 mRNA and protein expression
To investigate whether the biphasic effect of NO on NF-κB activity was reflected by an analogous change in the expression of proteins regulated by this transcription factor, the effect of DEA-NO on iNOS and COX-2 mRNA and protein expression was studied 24 h after activation. Mirroring the effects observed with NF-κB, both iNOS and COX-2 mRNA and protein expression were affected in a biphasic manner by DEA-NO (30 nM to 300 μM) given concomitantly with LPS. As before, lower concentrations of DEA-NO (30 nM to 3 μM) caused a potentiation of mRNA and protein expression, whereas higher concentrations (30 μM to 300 μM) elicited an inhibitory effect (Fig. 7). DEA-NO (30 nM to 300 μM) alone was unable to induce iNOS protein expression (data not shown).
Cell viability, as measured by trypan blue exclusion, was not altered significantly by any of the experimental conditions (data not shown).
Recent reports have revealed an important regulatory role for NO in the control of gene expression, primarily an inhibitory effect on proteins associated with host defense. In the present study the importance of NO as a regulator of host defense gene expression has been extended significantly with the observation that NO has an explicit biphasic effect on the activity of NF-κB. Via this mechanism, which depends on its local concentration, NO is able both to turn on and turn off the expression of a diverse range of proteins regulated by this transcription factor and to modulate the activation profile of immune cells. In murine macrophages stimulated with LPS, expression of iNOS and COX-2 follows a distinctive pattern, characterized by a rapid onset that peaks at ∼12 h followed by a swift cessation of protein expression (43, 44). This study shows that when macrophages are activated in l-arginine-free medium, which is known to lead to the complete suppression of endogenous NO synthesis (45), the time course of activation changes considerably, with the peak response being significantly delayed and expression of inducible proteins maintained for prolonged periods. Interestingly, incubation of cells in normal medium, but in the presence of l-NAME to inhibit endogenous generation of NO, leads to a similar, but less pronounced, change in the profile of activation; this can be explained by the fact that complete suppression of nitrite accumulation (and hence NO production) was not achieved under these conditions. Nevertheless, this correlation between nitrite accumulation and modulation of iNOS/COX-2 expression provides further evidence that the activation profile of the macrophages is closely linked to NO production. Moreover, peak protein expression in cells cultured in l-arginine-free medium or exposed to l-NAME was reduced compared with the control value. This suggests that if the initial NO-mediated facilitation of the response to LPS is not present, then the magnitude of the activation is diminished.
To investigate this phenomenon further, experiments were conducted in which cells were activated in the presence of l-NAME, or this intervention was established 9 h after activation. The results from these studies substantiated the initial observations, since the presence of l-NAME at the early stages of activation decreased iNOS expression, whereas the same conditions established at 9 h potentiated iNOS expression. Thus, at earlier time points NO augments activation, producing a sharp rise and peak in protein expression, while at later time points, NO has an inhibitory effect on the cells, such that a rapid termination of activation is brought about.
The present study is therefore in agreement with reports that ascribe to NO a regulatory role on gene expression and may explain in part why NO has been reported both to inhibit the expression of proinflammatory proteins (e.g., iNOS (23, 39), COX-2 (28), cytokines (24, 46, 47, 48, 49), adhesion molecules (25, 50), MHC class II molecules (51), and thioredoxin (52) and enhance their expression (e.g., iNOS (39), COX-2 (33, 34, 35), TNF-α (36), and glutathione-synthesizing enzymes (37)). This study also demonstrates clearly that regulation of gene expression by NO is inherently versatile, encompassing both up- and down-regulation.
Since NF-κB is known to be involved in iNOS expression in murine and human macrophages, EMSAs were used to demonstrate that the effects of NO observed in the present study were exerted via an action on NF-κB. Inhibition of endogenous NO production elicited a marked inhibitory effect on NF-κB activity measured 90 min after exposure to LPS. However, following inhibition of endogenous NO synthesis 9 h subsequent to activation, NF-κB activity at 24 h was markedly enhanced. This pattern of activity mirrors that observed with iNOS protein expression, implying that the effects of NO are mediated via modulation of NF-κB activity. To explore this possibility further, the effect of exogenous NO on NF-κB activity was investigated. The biphasic nature of NO-mediated modulation of NF-κB activity was clearly evident under these conditions. The NO donor DEA-NO produced a concentration-dependent influence on NF-κB, which comprised enhancement at concentrations between 30 nM and 3 μM followed by inhibition at concentrations of 30–300 μM. The peak levels of NO produced by these concentrations of DEA-NO range from 1 nM to 2 μM, which correspond approximately to levels of NO produced by constitutive NOS under physiological conditions and iNOS under pathological conditions, respectively. Thus, both constitutive and inducible NOS isoforms might be involved in the up- and down-regulation of NF-κB activity and gene expression in vivo. Certainly, the effects of DEA-NO could not be attributed to toxicity, since cell viability was not significantly changed by any of the concentrations used. To ensure that these actions of NO on NF-κB activity were reflected by similar differences in mRNA and protein expression, we looked at both these parameters for iNOS and COX-2. Indeed, in both cases the expression of mRNA and protein in the presence of increasing concentrations of DEA-NO mirrored the biphasic pattern observed with NF-κB, suggesting that the NO-mediated alteration in NF-κB activity did manifest itself as a significant change in proinflammatory protein expression in these cells. These observations are supported by a previous report that DEA-NO can both up- and down-regulate iNOS expression in ANA-1 murine macrophages (39). Moreover, the production of IL-6 (an inflammatory cytokine that is regulated via the action of NF-κB(53)) was influenced in a similar manner by NO, such that following inhibition of endogenous NO synthesis, IL-6 production was significantly retarded following activation with LPS over the first 9–12 h and then was potentiated to a level above that seen in control cells at 24–48 h. This provides further evidence that a diverse range of proteins that are regulated via the activity of NF-κB are modulated in a biphasic manner by NO; whether the expression of other proinflammatory proteins that are governed by NF-κB (e.g., TNF-α) is also subject to the same biphasic regulation by NO remains to be determined.
The mechanisms invoked by NO to bring about a biphasic regulation of NF-κB activity are currently under investigation; several possibilities exist (Fig. 8). NO induces and stabilizes IκBα (29), which prevents nuclear localization of NF-κB. NO has also be shown to nitrosate a specific cysteine residue (Cys62) on the p50 subunit of NF-κB to reduce its DNA-binding capacity (30, 31, 32). Furthermore, reactive oxygen species are believed to be important in the activation of NF-κB (54, 55, 56), and it might be hypothesized that the rapid interaction of NO with these oxygen derivatives results in quenching and diminished activation of NF-κB. Each of these process would down-regulate NF-κB transcriptional activity, and one or more may underlie the inhibitory effect on NO reported in the present study. The activation of NF-κB by NO is more difficult to explain. The most intriguing possibility is via activation of p21ras. This intracellular signaling protein has a redox active cysteine residue that is obligatory for Ras activation in response to oxidative stress (57). Nitrosation of this cysteine causes the protein to become constitutively active. Since Ras has been implicated in NF-κB activation (58, 59), in particular in response to oxidative stress and reactive oxygen species, it is possible that nitrosation of p21ras is responsible at least in part for augmentation of NF-κB activity by NO. However, the source of the initial NO that expedites the host defense response is not clear, but may be from constitutive or inducible NOS. Many immune cells, including macrophages, are endothelial NOS positive (60, 61), and the small quantities of NO released by this NOS isoform may be crucial in the early stages of an immune response. Alternatively, the first iNOS protein expressed may only generate small local concentrations of NO equivalent to constitutive NOS activity.
The biphasic effect of NO on NF-κB activity and immune cell activation may underlie several observations in the literature that have remained unexplained. Perhaps most important, the present findings may explain the ability of NO to exert a dual effect on the activity of Th1 cells during inflammatory episodes (62, 63). This effect is mediated at least in part via modulation of the production and release of IL-12, a cytokine regulated by NF-κB (63, 64, 65, 66). Consequently, the differential effect of NO could be explained by the biphasic effect of NF-κB activity described in the current study; this would confer on NO a powerful mechanism by which to regulate the activation profile of Th1 cells. Biphasic regulation of NF-κB by NO may also underlie control of osteoclast function, which appears to be regulated by a combination of constitutive and inducible NOS activity in vitro and in vivo (67, 68). Reports suggest that constitutive NOS (i.e., low NO) augments osteoclast function, and inducible NOS (i.e., high NO) is inhibitory. Since NF-κB has an obligatory role in osteoclast development and function (69, 70, 71), it seems likely that this bidirectional effect of NO is the result of the presently reported activity on NF-κB. It should also be noted that NO has a marked influence on IL-6 activity in an analogous biphasic manner, and since IL-6 plays a crucial role in bone resorption and osteoclast activity (72), this provides an additional route via which NO may govern osteoclast function.
The novel, autocrine pathway characterized in this study involving a biphasic effect of NO on NF-κB activity is easy to reconcile with the needs of host defense to produce a rapid response to a pathogenic stimulus that is shut down promptly so as to minimize (host) tissue damage. Initially, NO-mediated potentiation of NF-κB activity will promote the expression of adhesion molecules and proinflammatory cytokines to facilitate cellular recruitment to the site of infection/damage and activate these cells to eradicate the pathogenic insult. In due course many of these cells will express iNOS, and as a consequence the concentration of NO in the vicinity will rise and exert an inhibitory effect on NF-κB activity. At this point adhesion molecule expression would be down-regulated, and proinflammatory cytokine production arrested to efficiently halt the immunological process. Undoubtedly, the process of inflammation and host defense comprises a plethora of signaling and effector molecules, and the observed effects of NO on NF-κB activity are unlikely to be the sole determinants of the expression of proinflammatory proteins such as iNOS, COX-2, and IL-6. For instance, posttranscriptional modifications (i.e., destabilization of iNOS mRNA by TGF-β (73)) and other transcription factors/signaling pathways mobilized following macrophage activation (i.e., AP-1 (74, 75)) will undoubtedly influence protein expression; however, since in both humans (76) and rodents (6, 77) NF-κB activation is imperative for cellular activation and iNOS (and other proinflammatory protein) expression, the present study indicates that the biphasic effect of NO on NF-κB may represent an essential framework for regulation of the entire process. Moreover, this phenomenon may explain why many studies have reported both positive and negative actions of NO in host defense; the time of intervention with exogenous NO donors/NOS inhibitors and the local concentration of NO are critical in determining whether this molecule may augment or suppress an inflammatory response. This biphasic effect of NO will have a pronounced effect on cellular recruitment/infiltration, immune cell function, tissue repair, and antioxidant protection and therefore promotes NO to the forefront of the regulation of host defense.
We thank E. A. Higgs for editorial assistance.
A.J.H. is the recipient of a Wellcome Trust Career Development Fellowship. L.C. is the recipient of a Medical Research Council Ph.D. Research Studentship.
Abbreviations used in this paper: iNOS, inducible NO synthase; COX-2, cyclooxygenase-2; DEA-NO, diethylamine-NONOate; l-NAME, NG-nitro-l-arginine methyl ester.