Endotoxin-mediated nitric oxide synthesis inhibits IL-1beta gene transcription in ANA-1 murine macrophages

Rebecca A. Schroeder, Charles Cai, and Paul C. Kuo

Department of Surgery, Georgetown University Medical Center, Washington, District of Columbia 20007


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

On the basis of previous work demonstrating nitric oxide (NO)-mediated inhibition of nuclear factor-kappa B (NF-kappa B) DNA binding, we hypothesized that NO downregulates NF-kappa B-dependent interleukin-1beta (IL-1beta ) production in an ANA-1 macrophage model of lipopolysaccharide (LPS) stimulation. In the presence of LPS (100 ng/ml), levels of IL-1beta protein and mRNA were significantly upregulated with NO synthase inhibition. Using nuclear run-on analysis and transient transfection studies, IL-1beta gene transcription and IL-1beta promoter activity were also found to be increased with inhibition of NO production. Parallel transfection studies using an NF-kappa B long terminal repeat-reporter plasmid exhibited similar findings, suggesting an NO-mediated effect on NF-kappa B activity. Gel shift studies showed that LPS-associated NF-kappa B DNA binding was increased, both in the setting of NO synthase inhibition and in a reducing environment. Repletion of NO by addition of an S-nitrosothiol restored IL-1beta protein synthesis, mRNA levels, gene transcription, promoter activity, and NF-kappa B DNA binding to levels noted in the presence of LPS alone. Our studies indicate that NO may regulate LPS-associated inflammation by downregulating IL-1beta gene transcription through S-nitrosation of NF-kappa B.

S-nitrosation; inducible nitric oxide synthase; cytokine; nuclear factor-kappa B; lipopolysaccharide; interleukin-1beta


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

IN PATHOPHYSIOLOGICAL STATES that are characterized by the elaboration of proinflammatory cytokines, inducible nitric oxide synthase (iNOS) is expressed and nitric oxide (NO) is produced (20). This multifunctional free radical alters numerous biochemical functions, such as mitochondrial electron transport, activation of guanylyl cyclase, and expression of adhesion molecules. However, although it is apparent that NO production frequently accompanies inflammatory states, it is unclear whether NO is indifferent, promotes, or inhibits the inflammatory process. In this regard, we have demonstrated that NO S-nitrosylates a key thiol group in the DNA binding domain of nuclear factor-kappa B (NF-kappa B), a key transcription factor for the elaboration of multiple proinflammatory cytokines, such as interleukin-1beta (IL-1beta ) and tumor necrosis factor-alpha (TNF-alpha ) (1, 2, 6, 7). S-nitrosation of NF-kappa B at the redox-sensitive C62 residue of p50 is associated with inhibition of NF-kappa B DNA binding. Given the central role of NF-kappa B in proinflammatory gene transcription, S-nitrosation-associated inhibition of NF-kappa B-dependent promoter activity suggests a potential role for NO as a feedback inhibitor of the inflammatory process.

Excessive macrophage elaboration of IL-1beta plays a fundamental role in the pathogenesis of the sepsis syndrome (8). Transcriptional regulation is a major determinant of IL-1beta protein synthesis, and maximal transcription of the IL-1beta gene is NF-kappa B dependent (1, 2, 5, 8, 10). NF-kappa B therefore plays a central role in regulating IL-1beta production and subsequent IL-1beta -dependent inflammatory processes. Using a murine macrophage model of endotoxin [lipopolysaccharide (LPS)]-mediated NO production, we sought to test the hypothesis that endogenous synthesis of NO inhibits the synthesis of IL-1beta , an NF-kappa B-dependent proinflammatory protein, by S-nitrosation of NF-kappa B. Our results suggest a novel mechanism in which NO production feedback inhibits IL-1beta gene transcription in the context of LPS stimulation.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. ANA-1 macrophages were a gift from Dr. George Cox (National Cancer Institute, Frederick, MD). The murine IL-1beta promoter-chloramphenicol acetyltransferase (CAT) reporter gene plasmid construct and NF-kappa B-TNF-CAT reporter plasmid were gifts from Dr. Clifford J. Bellone (St. Louis University School of Medicine, St. Louis, MO). Human recombinant NF-kappa B p50 and the Gel Shift assay system, containing the NF-kappa B consensus oligonucleotide 5'-CGCTTGATGAGTCAGCCGGAA-3', were obtained from Promega, (Madison, WI). The rabbit anti-human NF-kappa B p50 monoclonal antibody (MAb) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The DIG gel shift kit was purchased from Boehringer Mannheim (Indianapolis, IN). LPS (Escherichia coli serotype 0111:B4), L-arginine, sodium nitrite, and nitrate reductase were obtained from Sigma (St. Louis, MO). DMEM, glutamine, penicillin-streptomycin, trypsin-EDTA, and endotoxin-free FCS were purchased from GIBCO BRL (Grand Island, NY). All other chemicals were of reagent grade.

Induction of NO synthesis in ANA-1 macrophages. ANA-1 macrophages were maintained in DMEM with 0.4 mM L-glutamine, pyridoxine hydrochloride, 5% heat-inactivated, endotoxin-free FCS, and 1 mM L-arginine. LPS (0-1,000 ng/ml) was then added in the presence and absence of FCS (5%) to induce NO synthesis. In selected instances, the competitive substrate inhibitor of NO synthase NG-nitro-L-arginine methyl ester (L-NAME; 100 µM), superoxide dismutase (SOD; 100 U/ml), or the NO donor S-nitroso-N-acetylcysteine (SNAC; 100 µM), or a combination of these compounds, was added. After incubation for 6 h at 37°C in 95% O2-5% CO2, the supernatant was aspirated, and the cells washed twice with Dulbecco's PBS. After treatment with trypsin-EDTA, the macrophages were harvested for biochemical assays.

Measurement of NO. NO release from cells in culture was quantified by measurement of the NO metabolite nitrite by the technique of Snell and Snell (27). To reduce nitrate to nitrite, conditioned medium (200 µl) was incubated in the presence of 1.0 unit of nitrate reductase, 50 µM NADPH, and 5 µM FAD for 15 min at 37°C in the dark. Sulfanilamide (1%, 50 µl) in 0.5 N HCl (50% vol/vol) was then added to 50 µl of the treated medium. After a 5-min incubation at room temperature, an equal volume of 0.02% N-(1-naphthyl)ethylenediamine was added; after incubation at room temperature for 10 min, the absorbance at 570 nm was compared with that of an NaNO2 standard.

Determination of IL-1beta and TNF-alpha concentration. Cell culture medium levels of IL-1beta and tumor necrosis factor-alpha (TNF-alpha ) were determined by the University of Maryland Cytokine Core Laboratory with an ELISA technique utilizing murine MAbs.

Immunoblot analysis of IL-1beta and iNOS proteins. Isolated murine macrophages were washed three times in PBS and incubated with boiling 2× nonreducing electrophoresis sample buffer for 2 min. Separation was performed by SDS-12% PAGE, and then the products were electrotransferred to a polyester-supported nitrocellulose membrane for 90 min at 150 mA. The membrane was blocked overnight at 4°C in Tris-buffered saline (TBS) containing 3% BSA. Blocked membranes were incubated with the anti-mouse IL-1beta MAb or anti-rat iNOS MAb (Transduction Laboratories, Lexington, KY), washed three times in TBS-0.1% Tween, and incubated with biotinylated sheep anti-mouse IgG (Amersham, Arlington Heights, IL) for 1 h. After being washed three additional times, membranes were incubated with a strepavidin-horseradish peroxidase conjugate. After an additional washing, bound antibodies were detected by the ECL detection system (Amersham, Arlington Heights, IL). Blots were scanned, and the area under the curve was normalized to the murine IL-1beta or iNOS standard.

RNA extraction and RT-PCR. Total cellular RNA was extracted by a commercial modification of the phenol-chloroform-isoamyl alcohol extraction method (RNAgents total RNA isolation system; Promega) (14). On completion of RNA extraction, all samples were treated with 10 U/µl RNase-free DNase (Stratagene Cloning Systems, La Jolla, CA). Cellular RNA (0.5 µg) was reverse transcribed, and PCR was performed as previously described (14, 15). The sequences of primer pairs used to amplify mouse IL-1beta and TNF-alpha were 5'-CAGGATGAGGACATGAGCACC-3' and 5'-CTCTGCAGACTCAAACTCCAC-3' and 5'-ATGAGCACAGAAAGCATGATC-3' and 5'-TACAGGCTTGTCACTCGAATT-3', respectively. The primer sequences for beta -actin mRNA, used to determine constitutive mRNA expression, were 5'-CATCGTGGGCCGCTCTAGGCAC-3' and 5'-CCGGCCAGCCAAGTCCAGACGC-3'. A log-linear dose-response curve was determined for each set of primers to determine the appropriate number of amplification cycles. Verification of the amplified PCR products was performed by automated DNA sequencing (Applied Biosystems, Foster City, CA) for rat iNOS and beta -actin mRNA. PCR products for TNF-alpha , IL-1beta , and interferon-gamma (IFN-gamma ) have been previously verified (Stratagene). The PCR products were visualized by ultraviolet (UV) illumination after electrophoresis through 1.0% agarose (UltraPure; Sigma) and staining in Tris-borate-EDTA buffer containing ethidium bromide. DNA was visualized on a UV illuminator; gel photographs were scanned, and the area under the curve was normalized for beta -actin mRNA content.

NF-kappa B DNA binding analysis. Nuclear extracts from ANA-1 macrophages were incubated with boiling 2× nonreducing electrophoresis sample buffer for 2 min. In certain instances, dithiothreitol (DTT; 20 mM) was added before boiling to reduce any S-nitrosothiol bonds. Separation was performed on 5-15% gradient SDS-PAGE gels, and then the products were electrotransferred to a polyester-supported nitrocellulose membrane for 90 min at 150 mA. The membrane was blocked overnight at 4°C in TBS (10 mM Tris · HCl, pH 7.5, 150 mM NaCI) containing 3% BSA. Blocked membranes were incubated with a p50 MAb with known cross-reactivity to murine NF-kappa B p50 (primary anti-human NF-kappa B p50 MAb; Santa Cruz Biotechnology), washed three times in TBS-0.1% Tween, and incubated with biotinylated sheep anti-rabbit IgG (Amersham) for 1 h. After being washed three additional times, membranes were incubated with strepavidin-horseradish peroxidase conjugate. After an additional washing, bound antibodies were detected by the ECL detection system (Amersham). Blots were scanned with a computerized laser densitometer (Hoeffer Scientific Instruments, San Francisco, CA), and the area under the curve was normalized to the human NF-kappa B p50 standard.

Synthesis of SNAC. SNAC was synthesized by combining equimolar NaNO2 and N-acetylcysteine in 0.5 N HCl for 30 min at room temperature as previously described. Before use, the S-nitrosoprotein solution was neutralized to pH 7.0 with 0.1 N NaOH. Previous work has confirmed the presence of S-nitrosothiol bonds in the above species by 15N-NMR spectroscopy (28).

Transient transfection with murine IL-1beta promoter and reporter gene constructs. Transient transfection of ANA-1 macrophages was performed by electroporation with the Bio-Rad Gene Pulser (9). After cells were washed twice with media, 20 µg of plasmid DNA containing the IL-1beta promoter construct were added with 20 µg of beta -galactosidase reference plasmid to 107 cells in 1 ml of medium and the medium was transferred to two 0.4-cm cuvettes. After a single 250-V, 960-µF pulse, the cells were combined into a 60-mm dish containing 5 ml of complete medium. At least 24 h later, the medium was changed, and LPS, LPS-L-NAME, or LPS-L-NAME-SNAC was added. Approximately 24 h later, the cells were washed with ice-cold PBS, resuspended in 0.25 mM Tris (pH 7.8), and subjected to three cycles of freezing and thawing. Lysates were centrifuged (11,700 g for 10 min at 4°C); the supernatant was heated at 65°C for 10 min to inactivate CAT inhibitors and then centrifuged as described above. The supernatant was assayed for CAT activity by a CAT ELISA technique (Boehringer Mannheim). Transfection efficiency was normalized by cotransfection of a beta -galactosidase reporter gene with a constitutively active early simian virus 40 promoter. All values are expressed as picograms of CAT per milliliter.

Nuclear run-on assays. Nuclear run-on analysis was performed as previously described (14). Briefly, 100 µl of hepatocyte nuclei were incubated for 5 min at 30°C with 150 µCi of [alpha -32P]rUTP (800 Ci/mmol) in 100 µl of 10 mM Tris · HCl (pH 8.0), 5 mM MgCl2, 300 mM KCl, and 5.0 mM (each) ATP, CTP, and GTP. Labeled RNA was isolated by the acid-guanidinium thiocyanate method. Before ethanol precipitation, labeled RNA was treated with 0.2 M NaOH for 10 min on ice. The solution was neutralized by the addition of HEPES (acid free) to a final concentration of 0.24 M. After ethanol precipitation, the RNA pellet was resuspended in 10 mM N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (pH 7.4), 0.2% SDS, and 10 mM EDTA. Target DNA was spotted onto nylon membranes with a slot blot apparatus. Target DNA for murine IL-1beta was amplified by PCR based on the published sequence (1,800 bp; -1021 to -2821; GenBank X04964). beta -Actin and lambda -bacteriophage DNA served as positive and negative controls, respectively. Hybridization was performed at 42°C for 48 h with 5 × 106 cpm of labeled RNA in hybridization buffer [50% formamide, 4× sodium chloride-sodium citrate (SSC), 0.1% SDS, 5× Denhardt's solution, 0.1 M sodium phosphate (pH 7.2), and 10 µg/ml salmon sperm DNA]. After hybridization, the membranes were washed twice at room temperature in 2× SSC and 0.1% SDS, and three times at 56°C in 0.1× SSC and 0.1% SDS. The membranes were then exposed to X-ray film and scanned on a laser densitometer.

Statistical analysis. All values are presented as means ± SD of three or four experiments. Data were analyzed by one-way ANOVA, Student's t-test, or the Wilcoxon rank sum test, as appropriate. P values < 0.05 were considered significant.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The NO dose-response relationship with concentrations of LPS ranging from 1 to 1,000 ng/ml is depicted as a semilogarithmic plot in Fig. 1A. Increasing concentrations of LPS were associated with significantly increased NO production (P = 0.0001 by ANOVA). When L-NAME (100 µM) was added with LPS, NO production at all concentrations of LPS was ablated to a level that was no different from that noted in the absence of LPS, 22.3 ± 2.1 nmol/mg protein (P < 0.01 for LPS vs. LPS-L-NAME). The addition of SOD did not alter NO synthesis in a significant fashion. The concentration of IL-1beta in the cell culture medium was then determined (Fig. 1B). In the absence of LPS, the IL-1beta concentration was 1.5 ± 3.2 pg/mg protein. Logarithmically increasing concentrations of LPS (1-1,000 ng/ml) were associated with significant increases in IL-1beta production (P = 0.0001 by ANOVA). In the presence of L-NAME (100 µM), LPS-induced IL-1beta synthesis was increased two- to threefold (P < 0.01). NO was then made replete by the addition of the S-nitrosothiol SNAC (100 µM). In LPS-L-NAME-treated cells, the addition of SNAC restored IL-1beta production to levels that were not different from that noted with LPS alone. Similarly, the addition of SNAC to LPS-treated cells did not alter the extent of IL-1beta synthesis. IL-1beta production in the LPS-L-NAME-SOD-treated cells was not altered. These data indicate that inhibition of NO production increases IL-1beta levels in cell culture media after LPS stimulation. The effect of LPS-mediated NO synthesis on TNF-alpha was then analyzed (Fig. 1C). In the absence of LPS, TNF-alpha levels in the culture medium were 6.7 ± 10.1 pg/mg protein. The addition of LPS (1 ng/ml) resulted in a dramatic increase in TNF-alpha production. Subsequent concentrations of LPS (10, 100, and 1,000 ng/ml) were associated with incremental but significant increases in TNF-alpha levels (P = 0.01 by ANOVA). The addition of L-NAME (100 µM), SOD (100 U/ml), and/or SNAC (100 µM) with LPS did not alter the TNF-alpha production profile, indicating that NO synthesis does not alter the extent of TNF-alpha synthesis in ANA-1 murine macrophages. Subsequent studies were performed at an LPS concentration of 100 ng/ml.




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Fig. 1.   Effect of lipopolysaccharide (LPS) stimulation on nitric oxide (NO), interleukin-1beta (IL-1beta ), and tumor necrosis factor-alpha (TNF-alpha ) production in cell culture media of ANA-1 macrophages. Values are means ± SD of 4 experiments. A: semilogarithmic plot of NO-LPS dose-response relationships. Nitrite and nitrate production in LPS-stimulated ANA-1 macrophages was determined as described in MATERIALS AND METHODS. For LPS alone, P = 0.0001 by ANOVA; for LPS vs. LPS-NG-nitro-L-arginine methyl ester (L-NAME), P < 0.01 at all concentrations of LPS by Student's t-test. B: semilogarithmic plot of IL-1beta -LPS dose-response relationships. Cell culture medium IL-1beta levels in LPS-stimulated ANA-1 macrophages were determined as described in MATERIALS AND METHODS. P = 0.04 by ANOVA for LPS alone, LPS-L-NAME-S-nitroso-N-acetylcysteine (SNAC), LPS-L-NAME-superoxide dismutase (SOD), and LPS-SNAC; P = 0.001 by ANOVA for LPS-L-NAME; for LPS-L-NAME vs. LPS, LPS-SNAC, or LPS-L-NAME-SNAC, P < 0.01 at 10, 100, and 1,000 ng/ml LPS by Student's t-test. C: semilogarithmic plot of TNF-alpha -LPS dose-response relationships. Cell culture medium TNF-alpha levels in LPS-stimulated ANA-1 macrophages were determined as described in MATERIALS AND METHODS.

Immunoblot analysis was performed on cellular protein extracts (Fig. 2). TNF-alpha protein expression in cellular extracts reflected that seen in the cell culture medium (data not shown). In the absence of LPS, no immunoreactive protein was detected. In contrast, in LPS-, LPS-L-NAME-, and LPS-L-NAME-SNAC-treated macrophages, immunoreactive TNF-alpha protein was readily detected. However, the presence or absence of NO, whether endogenous or exogenous in origin, did not alter the amount of TNF-alpha detected. In contrast, IL-1beta protein expression was noted to vary significantly with NO production. In the absence of LPS (and NO), no IL-1beta immunoreactive protein was detected. In LPS-treated cells, IL-1beta was detected. In the presence of both LPS and L-NAME (100 µM), the amount of IL-1beta synthesized was increased by approximately three- to fourfold over that found in the presence of LPS alone (P < 0.01). When NO was made replete in LPS-L-NAME-treated cells by the addition of SNAC (100 µM), IL-1beta production decreased to a level that was equivalent to that of LPS-treated cells. When SNAC (100 µM) was added to LPS-treated cells, the immunoreactive IL-1beta protein level did not change, reflecting that noted in the cell culture medium (data not shown). These data corroborate the cell culture medium findings regarding the influence of NO on IL-1beta protein production. An immunoblot analysis of iNOS protein expression was performed under these conditions. iNOS protein was detected in the LPS cells. Inhibition of iNOS by the addition of L-NAME resulted in a 35% increase in iNOS protein expression (P < 0.01 vs. LPS or LPS-L-NAME-SNAC). Again, the addition of SNAC to LPS-treated cells returned iNOS protein expression to a level equivalent to that for LPS treatment alone.



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Fig. 2.   IL-1beta and inducible nitric oxide synthase (iNOS) protein expression in ANA-1 macrophages. A: immunoblot analysis of IL-1beta and iNOS protein in whole cell lysates, as described in MATERIALS AND METHODS. Blot is representative of four experiments. B: histogram representation of densitometric analysis of IL-1beta and iNOS immunoreactive protein relative to standard. P < 0.01 for control vs. LPS, LPS-L-NAME, and LPS-L-NAME-SNAC for both iNOS and IL-1beta and P < 0.01 for LPS-L-NAME vs. LPS and LPS-L-NAME-SNAC for both iNOS and IL-1beta by Student's t-test. Values are means ± SD of 3 or 4 experiments.

Steady-state levels of IL-1beta mRNA were then analyzed semiquantitatively by RT-PCR (Fig. 3). In control cells, no iNOS or IL-1beta mRNA was detected. In the presence of LPS, iNOS and IL-1beta mRNA was expressed. Inhibition of NO synthesis by the addition of L-NAME (100 µM) was associated with a four- to fivefold increase in the normalized density of the IL-1beta mRNA band (P < 0.01 vs. LPS alone) and a two- to threefold increase in the iNOS density (P < 0.01 vs. LPS alone). When NO was made replete in LPS-L-NAME-treated cells by the addition of SNAC (100 µM), steady-state levels of IL-1beta mRNA production decreased to a level that was equivalent to that for LPS-treated cells. To determine the effect of NO on IL-1beta gene transcription, nuclear run-on studies were performed (Fig. 4). Inhibition of NO synthesis by the addition of L-NAME (100 µM) with LPS resulted in a 2.5-fold increase in IL-1beta gene transcription (P < 0.01). Again, repletion of NO by the addition of SNAC (100 µM) to LPS- L-NAME-treated cells was associated with IL-1beta gene transcription that was not different from that for cells exposed to LPS alone. The addition of SNAC to LPS-treated cells did not alter the extent of IL-1beta gene transcription as determined by the nuclear run-on assay (data not shown). These data indicate that NO synthesis inhibits IL-1beta gene transcription in LPS-treated macrophages.



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Fig. 3.   Effect of NO on steady-state IL-1beta mRNA levels in ANA-1 macrophages. A: RT-PCR analysis of iNOS, IL-1beta , and beta -actin mRNA levels. Gel is representative of four experiments. B: histogram representation of relative IL-1beta and iNOS cDNA expression levels normalized to beta -actin, as described in MATERIALS AND METHODS. P < 0.01 for control vs. LPS, LPS-L-NAME, and LPS-L-NAME-SNAC for IL-1beta and iNOS and P < 0.01 for LPS-L-NAME vs. LPS and LPS-L-NAME-SNAC for IL-1beta and iNOS by Student's t-test. Values are means ± SD for 4 experiments.




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Fig. 4.   Effect of NO on IL-1beta gene transcription in ANA-1 macrophages. A: nuclear run-on blot of IL-1beta gene transcription. Target DNA for murine IL-1beta was amplified by PCR on the basis of published sequence (1,800 bp; -1021 to -2821; GenBank X04964). beta -Actin and lambda -bacteriophage DNA served as positive and negative controls, respectively. Gel is representative of 3 experiments. B: histogram representation of LPS-induced IL-1beta gene transcription normalized to beta -actin. P < 0.01 for control vs. LPS, LPS-L-NAME, and LPS-L-NAME-SNAC and P < 0.01 for LPS-L-NAME vs. LPS and LPS-L-NAME-SNAC by Student's t-test. Values are means ± SD of 3 experiments.

Transient transfection studies were performed with the murine IL-1beta promoter to determine whether the effect of NO was dependent on the IL-1beta promoter region or a noncontiguous enhancer/repressor region (Fig. 5). The IL-1beta murine promoter plasmid contains 4,093 bp of the 5'-flanking sequence containing the entire first exon, first intron, and untranslated portion of the second exon. This has been previously shown to have strong inducibility by LPS (over 30-fold) (9). As a positive control for LPS induction of NF-kappa B-dependent transcriptional activity, parallel experiments were performed by transfection of a CAT plasmid containing three multimerized NF-kappa B sites upstream from a TNF-alpha minimal promoter (9). After exposure to LPS (100 ng/ml), LPS-L-NAME (100 µM), LPS-SNAC (100 µM), or LPS-L-NAME-SNAC, levels of IL-1beta promoter and NF-kappa B inducibility were determined by CAT expression. Transfection of a mock plasmid containing only a CAT reporter revealed no detectable CAT protein in the presence of LPS stimulation. Full-length IL-1beta promoter transfection was associated with minimal CAT expression under unstimulated control conditions. Stimulation with LPS (100 ng/ml) resulted in significantly increased CAT expression, over sixfold greater than that of the control (P < 0.01 vs. control). Compared with that resulting from LPS treatment, CAT expression was increased an additional fivefold in LPS-L-NAME-treated cells (P < 0.01 vs. LPS alone). Repletion of NO by exogenous administration of the S-nitrosothiol SNAC decreased IL-1beta promoter activity to a level that was not different from that for LPS-treated cells. Last, IL-1beta promoter activity in LPS-SNAC-treated cells was not different from that in ANA-1 macrophages treated with LPS alone. Results from the parallel experiments with the NF-kappa B long terminal repeat (LTR) CAT reporter plasmid mirrored those for the IL-1beta promoter. Inhibition of LPS-mediated NO synthesis with L-NAME significantly increased NF-kappa B LTR activity. This activity was decreased to baseline levels with repletion of NO in LPS-L-NAME-SNAC-treated cells. These data suggest that inhibition of NO synthesis increases LPS-induced IL-1beta promoter activation and NF-kappa B LTR activation.


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Fig. 5.   Effect of LPS-mediated NO production on activity of transfected IL-1beta promoter and NF-kappa B long terminal repeat (LTR) plasmids. Shown is a histogram representation of normalized chloramphenicol acetyltransferase (CAT) expression. Mock plasmid contains CAT reporter gene alone. P < 0.01 for control vs. LPS, LPS-L-NAME, LPS-L-NAME-SNAC, and LPS-SNAC and P < 0.01 for LPS-L-NAME vs. LPS, LPS-L-NAME-SNAC, and LPS-SNAC for both IL-1beta and NF-kappa B LTRs by Student's t-test. Values are means ± SD for 3 experiments.

To determine whether the effect of NO occurs at the level of NF-kappa B DNA binding, gel shift analysis was performed with ANA-1 macrophages (Fig. 6). In certain instances, DTT (20 mM) was added to the nuclear extracts to reduce any potential sulfur-NO (S-NO) bonds. In control cells, no NF-kappa B DNA binding was found. The addition of LPS (100 ng/ml) resulted in NF-kappa B DNA binding, which was confirmed in supershift studies with a MAb to NF-kappa B p50. NF-kappa B binding in LPS-L-NAME was significantly increased over that found in LPS-treated cells; the further addition of SNAC to LPS-L-NAME-treated cells restored NF-kappa B DNA binding to a level equivalent to that for LPS-treated cells. The addition of the reducing agent DTT (20 mM) to LPS-treated cells significantly augmented NF-kappa B binding to its consensus DNA binding element. These data indicate that inhibition of NO synthesis significantly increases NF-kappa B DNA binding. The addition of DTT in the setting of NO synthesis augments NF-kappa B binding, suggesting that inhibition of NF-kappa B DNA binding may be the result of S-nitrosation of NF-kappa B, as we and others have previously demonstrated.(6, 7, 16)


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Fig. 6.   Effect of LPS and NO on NF-kappa B DNA binding. Representative gel shift analysis of NF-kappa B binding in setting of LPS-stimulated NO production. Gel is representative of 3 experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we hypothesized that LPS-mediated NO production inhibits production of the NF-kappa B-dependent protein IL-1beta . Our results indicate that inhibition of NO production in the setting of LPS stimulation 1) increases IL-1beta protein expression in the cell culture supernatant and cell lysates, 2) increases steady-state levels of IL-1beta mRNA, 3) augments IL-1beta gene transcription, 4) increases IL-1beta promoter activity, and 5) promotes NF-kappa B DNA binding. The addition of the S-nitrosothiol SNAC as an exogenous NO donor restores IL-1beta protein expression, gene transcription, and NF-kappa B DNA binding to levels noted with LPS alone. Finally, gel shift analysis showed that NF-kappa B DNA binding in LPS-treated cells is significantly increased in the presence of the reducing agent DTT. This would suggest that S-nitrosation of NF-kappa B with resulting inhibition of DNA binding is a plausible mechanism underlying decreased expression of the NF-kappa B-dependent protein IL-1beta . Given the central position of NF-kappa B in proinflammatory gene transcription, S-nitrosation-associated inhibition of NF-kappa B-dependent promoter activity suggests a potential role for NO as a feedback inhibitor of specific components in the inflammatory process, such as IL-1beta .

We and others have previously demonstrated that ex vivo biochemical modification of NF-kappa B p50 inhibits DNA binding as determined by the gel shift assay. In particular, Matthews and co-workers (16) showed that NO donors inhibit the DNA binding activity of p50 and that a p50 mutant with a serine substitution (C62S) was significantly resistant to DNA binding inhibition; they also showed, by electron spray ionization mass spectrometry, that the C62 residue in p50 was S-nitrosylated by NO. We characterized the biochemical kinetic effects of p50 S-nitrosation and found that NO decreased the dissociation constant by fourfold, from 1.0 × 1010 M-1 to 0.29 × 1010 M-1 (6). Using the ANA-1 macrophage model, we have subsequently demonstrated that inhibition of the endotoxin-mediated synthesis of NO enhances NF-kappa B p50 DNA binding without altering NF-kappa B activation and nuclear translocation (7). Immunoprecipitation studies confirm the presence of S-NO bonds in p50 isolated from endotoxin-stimulated cells and, conversely, the absence of S-NO bonds in the presence of the iNOS inhibitor L-NAME. The p50 isolated in this fashion retains functional DNA binding properties, mirroring that found in our ex vivo kinetic studies. Transcription of the NF-kappa B-dependent genes for iNOS and macrophage colony-stimulating factor (M-CSF) was enhanced in the setting of NO inhibition. In total, these results strongly suggest a role for S-nitrosation as a specific modulator of NF-kappa B DNA binding, with its associated effects on NF-kappa B-dependent gene transcription. In this study, using ANA-1 macrophages we demonstrate that NO alters IL-1beta gene transcription and, ultimately, IL-1beta protein expression.

These observations correlate well with others relating NO synthesis and the functional results of NF-kappa B inhibition. NF-kappa B activation is a prerequisite for the transcription of a variety of proinflammatory mediators and markers, including IL-1beta , iNOS, M-CSF, TNF-alpha , and vascular cell adhesion molecule (VCAM) (21, 25, 26). In particular, a number of previous studies have documented the regulatory interplay between NO and IL-1beta synthesis in a variety of experimental models (4, 12, 13, 17, 18, 24, 29). Inhibition of NO synthesis is associated with increased IL-1beta protein expression. Until recently, an underlying mechanism has not been elucidated. In models of macrophage synthesis of NO, Kim and colleagues (12) have demonstrated that NO suppresses IL-1beta protein processing by inhibition of caspase-1, a key enzyme required for IL-1beta maturation and release. The inhibition of caspase-1 activity is reversed in the presence of DTT. This, in combination with previous work by the same group, indicates S-nitrosation of caspase proteases as a potential mechanism (11). These results notwithstanding, Kim and colleagues also demonstrate that inhibition of NO synthesis is associated with 1) increased IL-1beta and TNF-alpha mRNA expression and 2) increased IL-1beta protein expression but unchanged TNF-alpha protein expression. Our findings certainly corroborate the results of Kim and coworkers and indicate that S-nitrosation may regulate the activity of key regulatory proteins involved at multiple levels of protein synthesis, including transcription and posttranslational processing.

Previous work has demonstrated that S-nitrosation of NF-kappa B p50 is associated with decreased gene transcription and synthesis of the NF-kappa B-dependent proteins iNOS and M-CSF (7). However, the effects of S-nitrosation are not generalized to all NF-kappa B-dependent proteins, as evidenced by our observation that LPS-stimulated TNF-alpha elaboration is not altered by inhibition of NO synthesis. Posttranscriptional regulatory mechanisms may obviate the effects of transcriptional inhibition.

Although TNF-alpha mRNA levels are increased in the presence of NO inhibition, as shown by Kim et al. (12), additional posttranscriptional mechanisms in the macrophage may obviate these effects. In contrast, other investigators have demonstrated that inhibition of NO synthesis is associated with increased expression of both IL-1beta and TNF-alpha proteins (17, 18, 29). Although these differences in the effect of NO on TNF-alpha biology may be the result of differences in the experimental models, inhibition of IL-1beta synthesis by NO is a recurring theme in the work of many groups. The results from the present study suggest that NF-kappa B DNA binding properties, IL-1beta gene transcription, and IL-1beta protein expression are significantly decreased in the presence of NO. The underlying mechanism is consistent with our previous observations concerning S-nitrosation of NF-kappa B and its effects on DNA binding and gene transcription (7).

As a result of its participation in redox chemistry, NO is a pluripotent regulator of multiple cellular functions (19). The formation of S-nitrosothiols exemplifies these pathways of NO oxidation, which lead to surrogate NO-like bioactivity and result in allosteric receptor modification, inhibition of sulfhydryl enzyme activities, and downregulation of transcriptional activators. With respect to NF-kappa B activity, studies utilizing exogenous NO donors have shown that NF-kappa B DNA binding is inhibited (3, 21, 22). In a system of human vascular endothelial cells, Peng and colleagues (21, 22) showed that exogenous NO stabilized NF-kappa B by inhibiting the dissociation of the Ikappa B inhibitor while simultaneously increasing Ikappa B mRNA levels. These investigators also found that NO minimally altered activation of nuclear binding proteins AP-1, GATA-2, and GATA-3 (21, 22). Recently, Calmels and colleagues (3) also demonstrated NO-mediated inhibition of p53 DNA binding resulting from NO-induced conformational and functional modifications, suggesting S-nitrosation, given the absence of an Ikappa B correlate for p53. Of added interest is the relative specificity of S-nitrosation for NF-kappa B- and, perhaps, p53-associated activity. Nuclear proteins, such as IRF, GATA, AP-1, oct-2, and STAT, do not exhibit NO-induced alteration in activity (6, 21, 22). Future identification of a pool of NO-modulated nuclear protein transcription factors would allow elucidation of the pathways of NO-mediated transcriptional regulation.

These results indicate that NO may play a direct regulatory role in the inflammatory response. Our study suggests a novel mechanism in which NO production feedback inhibits IL-1beta gene transcription in the context of LPS stimulation. Although initial studies in the field suggested that iNOS expression and NO production were associated with deleterious biological effects, more recent studies have elucidated a feedback inhibitory role for iNOS and NO in the elaboration of IL-1beta , IFN-gamma inducing factor, IL-6, and VCAM-1 (12, 23, 24). Under certain inflammatory states, we propose that NO may function to inhibit by feedback the expression of specific NF-kappa B-dependent proteins in an autoregulatory inducible fashion.


    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: P. C. Kuo, Dept. of Surgery, Georgetown Univ. Medical Center, 4 PHC; 3800 Reservoir Rd. NW, Washington, DC 20007 (E-mail: kuop{at}gunet.georgetown.edu).

Received 11 January 1999; accepted in final form 9 May 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Cell Physiol 277(3):C523-C530
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