IL-1β Converting Enzyme Is a Target for Nitric Oxide-Releasing Aspirin: New Insights in the Antiinflammatory Mechanism of Nitric Oxide-Releasing Nonsteroidal Antiinflammatory Drugs

Stefano Fiorucci, Luca Santucci, Giuseppe Cirino, Andrea Mencarelli, Luigi Familiari, Piero Del Soldato and Antonio Morelli
J Immunol November 1, 2000, 165 (9) 5245-5254; DOI: https://doi.org/10.4049/jimmunol.165.9.5245

Abstract

Caspase-1, the IL-1β converting enzyme (ICE), is required for intracellular processing/maturation of IL-1β and IL-18. NO releasing nonsteroidal antiinflammatory drugs (NSAIDs) are a new class of NSAID derivatives that spare the gastric mucosa. Here, we tested the hypothesis that NCX-4016, a NO-aspirin derivative, inhibits proinflammatory cytokine release from endotoxin (LPS)-challenged monocytes. Our results demonstrated that exposing LPS-stimulated human monocytes to NCX-4016 resulted in a 40–80% inhibition of IL-1β, IL-8, IL-12, IL-18, IFN-γ, and TNF-α release with an EC50 of 10–20 μM for IL-1β and IL-18. Incubating LPS-primed monocytes with NCX-4016 resulted in intracellular NO formation as assessed by measuring nitrite/nitrate, intracellular cGMP concentration, and intracellular NO formation. Exposing LPS-stimulated monocytes to aspirin or celecoxib caused a 90% inhibition of prostaglandin E2 generation but had no effect on cytokine release. NCX-4016, similar to the NO donor S-nitroso-N-acetyl-d-l-penicillamine, inhibited caspase-1 activity with an EC50 of ≈20 μM. The inhibition of caspase-1 by NCX-4016 was reversible by the addition of DTT, which is consistent with S-nitrosylation as the mechanism of caspase-1 inhibition. NCX-4016, but not aspirin, prevented ICE activation as measured by assessing the release of ICE p20 subunit. IL-18 immunoneutralization resulted in a 60–80% reduction of IL-1β, IL-8, IFN-γ, and TNF-α release from LPS-stimulated monocytes. Taken together, these data indicate that incubating human monocytes with NCX-4016 causes intracellular NO formation and suppresses IL-1β and IL-18 processing by inhibiting caspase-1 activity. Caspase-1 inhibition is a new, cycloxygenase-independent antiinflammatory mechanism of NO-aspirin.

Interleukin-1β is a proinflammatory cytokine, mainly released by macrophages and activated T cells that require a proteolytic enzyme for cleavage and release of its mature, active molecule (1). For IL-1β, a specific intracellular cysteine protease called the IL-1 converting enzyme (ICE)2 that cuts the IL-1β precursor into an active mature form (2) has been identified and cloned. In recent years, ICE has been recognized as a member of the growing family of intracellular cysteine proteases that share sequence homology with Ced-3, a nematode gene involved in the execution phase of apoptosis (3, 4, 5). The mammalian counterpart of the Ced-3 gene products includes at least 14 different endoproteases that have been renamed caspases to denote cysteine proteases acting after an aspartic acid residue. Caspase-1 denotes the original ICE (these terms are often used interchangeably) and has the greatest specificity for cleaving pro-IL-1β. Comparison of molecular structures suggests that the caspase family falls into three major groups: caspases that function primarily in cytokine maturation (e.g., caspase-1, -4, and -5); initiator caspases, involved in signaling early steps of extracellular regulated apoptosis (e.g., caspase-8, -9, and -10); and effector proteases involved in the execution phase of apoptosis (e.g., caspase-3, -6, and -7). Supporting the specialization of the three branches of caspase family, specific ICE inhibitors administered to mice exert poor antiapoptotic effects although they reduce inflammation as effectively as does blocking IL-1β activity with specific antagonists (6).

IL-18, formerly termed IFN-γ-inducing factor (IGIF) is a proinflammatory cytokine structurally related to IL-1β (7). The existence of this cytokine was first demonstrated in Propionibacterium acnes preconditioned mice challenged with bacterial endotoxin (LPS) (8, 9). In this model of liver injury, IL-18 immunoneutralization prevents IFN-γ release and confers protection against acute toxicity induced by LPS (8). In addition to acting as a costimulus for IFN-γ production, IL-18 stimulates IL-1β, IL-2, IL-8, and TNF-α secretion from human PBMC, potentiates anti-CD3-induced T cell proliferation, and increases Fas ligand expression on NK cells (9). Similar to the IL-1β precursor, the IL-18 precursor (pro-IL-18) lacks a signal peptide (10, 11) and requires ICE/caspase-1 for cleavage and secretion (10, 11). The NH2-terminal amino acid sequence of the secreted form of murine IL-18 (10, 11) is consistent with cleavage after an aspartic acid residue (residue 35), a typical cleavage site for ICE. Relevant for inflammation is the fact that other caspases and particularly those cleaving intracellular proteins involved in apoptosis either did not cleave pro-IL-18 or required a 100-fold greater concentration of enzyme compared with ICE (10, 11). The fact that ICE-deficient mice release a reduced amount of IL-18 and IFN-γ in response to LPS is further evidence that IL-18 is generated through an ICE-dependent pathway and suggests that caspase-1 inhibition would be therapeutic for inflammatory diseases (12, 13).

NO-releasing nonsteroidal antiinflammatory drugs (NO-NSAIDs), are a recently described class of NSAID derivatives generated by adding a nitroxybutyl moiety through an ether linkage to the parental NSAID (14, 15, 16, 17, 18). These compounds exhibit a markedly reduced gastrointestinal toxicity, while retaining the antiinflammatory and antipyretic activity of parent NSAID. Although NO-NSAIDs spare the gastric mucosa, they inhibit prostaglandin generation and exert powerful antiapoptotic and antiinflammatory effects. Indeed, preliminary animal studies indicate that NO-NSAIDs are more effective than conventional NSAIDs in reducing inflammation and pain in arthritic rats (17, 19, 20). Previous studies from our laboratory have demonstrated that, similar to conventional NO donors, NO-releasing NSAIDs inhibit apoptotic pathways by causing the S-nitrosylation/inactivation of effector caspases, such as caspase 3 (20). Because inhibition of caspase-1 would be therapeutic for inflammation (6), we have designed the present study to investigate whether NCX-4016, an NO-aspirin derivative, inhibits caspase-1 activity and limits cytokine release from LPS-challenged monocytes.

Materials and Methods

Materials

Aspirin, S-nitroso-N-acetyl-d-l-penicillamine (SNAP), polyriboinosinic polyribocytidylic acid (Poly I:C), LPS from Escherichia coli (K235), FITC-conjugated anti-rabbit, and anti-hamster mAbs were purchased from Sigma (St. Louis, MO). Endotoxin-free, recombinant human (hr)IL-18, IL-1RA, and polyclonal goat anti-human IL-18 Ab were from Endogen (Woburn, MA). 2-(Acetyloxy)benzoic acid 3-(hydroxymethyl)phenyl ester, NCX-4017; 2-(acetyloxy)benzoic acid 3-(nitrooxymethyl)phenyl ester, NCX 4016 (NO-aspirin) were from Nicox (Sophia Antipolis, France). Celecoxib, 4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]be-nzene sulfonamide was synthesized by Dr. Francesca Benedini at Nicox Laboratories (Nicox Italia SpA, Milan, Italy) according to the method of Penning et al. (21). l-N6-(1-iminoethyl)-lysine (l-NIL) was from Biomol (Plymouth Meeting, PA). N-acetyl-Tyr-Val-Ala-Asp-aldehyde (Ac-YVAD-CHO), 7-amino-4-coumarin (AMC)- and 7-amino-4-trifluoro-methylcoumarin (AFC)-conjugated YVAD peptide were from Alexis Corporation (Lusanne, Switzerland). DAF-2 (4,5-diaminofluorescein) and DAF-diacetate (DAF-DA) were from Calbiochem (La Jolla, CA).

Monocyte isolation

Human PBMCs were obtained from normal individual donors to the Blood Bank Service of Perugia University Hospital. PBMCs were isolated by density gradient centrifugation (400 × g for 30 min at room temperature) through Ficoll-Hypaque (Pharmacia Biotech AB, Uppsala, Sweden). The major band, containing the mononuclear cells, was harvested with a yield typically of 1.5–2 × 108 cells per isolation. PBMC were washed by centrifugation three times using RPMI 1640 (Life Technologies Italia Srl, Milan, Italy) supplemented with 10% FCS, l-glutamine, penicillin (100 U/μl), streptomycin (100 U/μl), and gentamicin (10 μg/ml), and 2 × 107 cells were placed into each 75-cm2 tissue culture flask (Corning, Corning, NY) and incubated overnight at 37°C in an atmosphere containing 5% CO2 and 95% air. At the end of incubation, nonadherent cells were removed by washing and the cell layers (monocytes) incubated in RPMI complete medium until used for experimental treatments as indicated. Cell viability was checked before each experiment by trypan blue exclusion and was always >95%. Unless otherwise specified, this cell population was used throughout the study. In selected experiments, however, an enriched population of monocyte-derived macrophages (MDM) was prepared by a further purification by negative selection. PBMC-derived monocytes were incubated with a cocktail of mAbs against T and B cells followed by subsequent depletion using a secondary Ab conjugated to magnetic beads (Dynal, Lake Success, NY). Purified macrophages were plated in 24-well plates (Falcon Labware, Milan, Italy) at a concentration of 2.5 × 106 cells per well and maintained in the complete RPMI 1640 medium until used. The resulting adherent cells (macrophages) were >93% positive by nonspecific-esterase test (Sigma).

Stimulation of cytokine production by PBMC-derived monocytes and MDM

PBMC-derived macrophages (2.5 × 106/ml) were cultured in flat-bottom 24-well plates with or without 1 μg/ml LPS in the presence or absence of the following: Ac-YVAD.CHO (0.1–200 μM), SNAP (0.1–200 μM), aspirin (0.1–200 μM), or NCX-4016 (0.1–200 μM), and cytokine was released in cell supernatants measured. To test whether cytokine release induced by LPS was mediated by IL-1β and/or IL-18, cells were incubated with 1 μg/ml LPS in the presence of IL-RA (10 μg/ml) or the goat anti-human IL-18 polyclonal Ab (50 μg/ml) and cytokine released in cell supernatants measured. In some experiments, monocytes were preincubated with or without 1 μg/ml Con A for 24 h and then exposed to hrIL-18 (10 ng/ml), and, after a further 24 h-period of incubation, IL-1β, IL-18, and IFN-γ released in cell supernatants measured. To investigate the role of endogenous NO in modulating cytokine release, MDM were preincubated with LPS for 12 h and then treated with 50 μg/ml Poly I:C alone or in combination with 10 μM l-NIL of NCX-4016 for 24 h and nitrite/nitrate and IL-1β released in cell supernatants measured (22). To further assess whether cytokine modulation caused by NCX-4016 was due to NO, we substituted the nitrooxymethyl phenyl ester group of NCX-4016 with an hydroxymethyl phenyl ester group. The structure of the resulting compound, NCX-4017, as well as the structure of aspirin and NCX-4016, is shown in Fig. 1⇓. We first tested whether this compound had any effect on cell viability. To ascertain this point, PBMC-derived monocytes were incubated with no agent, LPS alone, or LPS in combination with increasing concentrations, 1–200 μM, of NCX-4016 or NCX-4017 for 24 h and cell viability assessed through the analysis of propidium iodide stained nuclei at flow cytometry (16, 17). Briefly, macrophages were suspended in 0.1 M citrate buffer (pH 7.2) containing 0.1% Triton X-100 and 20 μg/ml propidium iodide incubated at 37°C for 30 min and fluorescence intensity measured at 515/620 nm wavelength pair using a flow cytometer analyzer (Beckman-Coulter, Fullerton, CA).

FIGURE 1.
FIGURE 1.

Box 1. Structure of aspirin, NCX-4016 (2-(acetyloxy>)benzoic acid 3-(nitrooxymethyl)phenyl ester), and NCX-4017 (2-(acetyloxy)benzoic acid 3-(hydroxymethyl)phenyl ester). Box 2. A, LPS stimulates IL-1β and IL-18 release. PBMC-derived monocytes were stimulated with increasing concentrations of LPS for 24 h. The data are mean ± SE of six donors. ∗, p < 0.01 vs cells incubated with the medium alone. B, Macrophages are the main source of cytokine released by PBMC-derived monocytes. PBMC-derived monocytes or MDM, prepared from the same set of donors, were incubated with or without 1 μg/ml LPS for 24 h, and cell supernatants were collected for cytokine determination. The data are mean ± SE of six donors. ∗, p < 0.01 vs cells incubated with the medium alone. C and D, NCX-4016, an NO-aspirin derivative, inhibits IL-1β and IL-18 release from LPS-challenged monocytes. Monocytes were stimulated with LPS (1 μg/ml) in the presence or absence of increasing concentrations of NCX-4016 or aspirin for 24 h. The data are mean ± SE of six donors. ∗, p < 0.001 vs cells incubated with the medium alone. ∗∗, p < 0.01 vs cells incubated with the LPS alone. Box 3. E and F, Substituting the nitrooxymethyl-phenyl ester group of NCX-4016 with an hydroxymethyl-phenyl ester results in a highly cytotoxic compound. PBMC-derived monocytes were incubated with LPS (1 μg/ml) alone or in combination with increasing concentrations of NCX-4016 or NCX-4017 for 24 h and cell viability assessed. The data are mean ± SE of six donors. ∗, p < 0.01 vs cells incubated with LPS alone.

Cytokine ELISA and PGE2 measurement

IL-1β, IL-8, IL-12, IL-18, TNF-α, and IFN-γ concentrations in cell supernatants were measured using a commercial ELISA kit (Endogen, and R&D Systems, Minneapolis, MN) using the standard procedure recommended by manufacturers. Cytokine concentrations were calculated from the standard curves using the GraphPad Prism software (GraphPad Software, San Diego, CA) and results expressed as pg/ml. Each kit was specific and showed negligible cross-reactivity with several other cytokines (data furnished by the manufacturer). PGE2 levels were measured in cell supernatants using a commercially available enzyme immunoassay system (Amersham Pharmacia Biotech, Buckinghamshire, U.K). PGE2 concentrations were calculated from the standard curve and results expressed as pg/ml. The kit was specific and showed negligible cross-reactivity with several other eicosanoids (data furnished by the manufacturer).

ICE/caspase-1-like (YVADase) activity

ICE-like protease activity, YVADase activity, was assessed by measuring the proteolytic cleavage of fluorogenic substrates YVAD.AMC according to previous published methods (17, 23). In brief, after incubation with the appropriate agent, macrophages were precipitated by centrifugation, and cytosolic extracts were prepared by repeated freezing and thawing in 300 μl extraction buffer (12.5 mM Tris, pH 7.0, 1 mM DTT, 0.125 mM EDTA, 5% glycerol, 1 mM PMSF, 1 μg/ml leupeptin, 1 μg/ml pepstatin, and 1 μg/ml aprotinin). Cell lysates were then diluted with the buffer (50 mM Tris, pH 7.0, 1 mM DTT, 0.5 mM EDTA, 20% glycerol) and incubated at 37°C in the presence of 14 μM the selective ICE-inhibitor acetyl-Asp-Glu-Val-Asp-aminomethylcoumarin (Ac-YVAD.CHO) for 15 min at 37°C to ensure equilibrium. The reaction was then initiated by addition of 14 μmol/L YVAD.AMC and followed for 20 min. Fluorescent AMC formation was measured at excitation 360 nm, emission 460 nm using a Hitachi 2000 fluorometer (Hitachi, Milan, Italy). A standard curve was constructed using AMC as standard and human recombinant ICE as previously described (17, 23). Protein content was analyzed using the Bio-Rad assay (Bio-Rad Laboratories, Hercules, CA).

Western blot analysis of ICE p20 subunit cleavage

PBMC-derived monocytes (1×107) were lysed in 100 μl of lysis buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 2 mM EDTA, 1 mM PMSF, 40 μg/ml aprotinin, 20 μg/ml leupeptin) at 4°C. Then, 10 μl of sample were run on either 10 or 8 16% linear gradient polyacrylamide gels (Bio-Rad). Gels were transferred to nitrocellulose membrane (Hybond-C extra; Amersham). Membranes were blocked in 5% milk powder in PBS, and probed with either anti-FLAG Ab (M2; Sigma) or a rabbit polyclonal anti-caspase-1 Ab (PharMingen) both at dilutions of 1:1000. The secondary Ab was goat anti-mouse IgG HRP-conjugated Ab (PharMingen) used at a dilution of 1:1000 (23).

Detection of intracellular NO formation in monocyte-incubated NCX-4016

Intracellular NO formation in NCX-4016-treated monocytes was conducted according to the method of Kojima et al. and Nakatsubo et al. using DAF-DA (24, 25). Briefly, 1 × 106/ml were loaded by suspending them in PBS in the presence of 10 μM DAF-DA and incubating at 37°C for 30 min. Cells were washed twice in iced buffer solution and samples added to a quartz cuvette stirring continuously and the temperature thermostatically maintained at 37°C using a Hitachi 2000 (Hitachi) fluorescence spectrophotometer. Samples were preincubated with 1 mmol/L l-NIL for 30 min to suppress endogenous NO generation and then stimulated with 1 μg/ml LPS alone or in combination with increasing concentrations of SNAP or NCX-4016, excited 395-nm wavelengths with a 10-nm slit and the intensity of fluorescence emitted at 515 nm recorded. NO generation was expressed in arbitrary unit of absorbance. NO generation was also assessed indirectly by measuring intracellular cGMP concentrations ([cGMP]i) (18). PBMC-derived monocytes (1.0 × 107) were incubated for a variable amount of time with aspirin, SNAP, or NCX-4016 (see Fig. 8⇓C), and, at prefixed time points, incubation was stopped by adding acetic acids. cGMP concentration on cells lysates was then measured by specific enzyme immunoassay kit (Cayman Chemical, Ann Arbor, MI), as previously described (26). The detection limit was 0.9 pmol/ml. Values of each experimental sample were expressed as a ratio to the control value.

Effect of SNAP and NO-aspirin on ICE/caspase-1-like proteases

To investigate whether inhibition of ICE/caspase-1 like proteases exerted by NCX-4016 was due to protein S-nitrosation, lysates obtained from monocytes incubated with LPS alone or in combination with 100 μM NCX-4016, aspirin, or SNAP were incubated with DTT (20 mM) to remove the thiol-bound NO. The DTT and excess of NO were then removed by passing the sample through a Sephadex G-25 column preequilibrated with the lysis buffer and enzyme activity assessed (17, 27). In another set of experiments, lysates obtained from monocytes incubated with LPS alone or in combination with NCX-4016 were exposed to DTT, 20 mM, and/or HgCl2, 5 mM, for 40 min on ice, and caspase-1-like activity was measured (17, 20, 27). To confirm the formation of nitrosylated thiol, lysates obtained from monocytes incubated with LPS alone or in combination with NCX-4016 or SNAP were exposed to 5 mM HgCl2 for 10 min on ice to displace bound NO group and NO released in cell supernatants measured using DAF-2 as previously described (24, 25).

Nitrite/nitrate assay

Nitrite/nitrate concentrations in cells supernatants were measured by a fluorometric detection kit (Cayman Chemical). The lower detection limit, as reported by the manufacturer, was ≈4 pmol/well.

Data analysis

All values in the figures and text are expressed as mean ± SE of n observations. Data sets were compared with a ANOVA and Student’s t test when appropriate (28).

Results

NO-aspirin inhibits LPS-stimulated cytokine release from monocytes/macrophages

Incubation of PBMC-derived monocytes with LPS for 24 h resulted in a concentration-dependent stimulation of IL-1β and IL-18 release in cell supernatants with a peak occurring at 1 μg/ml (Fig. 1⇑A). Monocytes were the major source of cytokines production in this cell preparation as demonstrated by the fact that PBMC-derived monocytes and MDM released approximately the same amount of IL-1β and IL-18 in response to 1 μg/ml LPS (Fig. 1⇑B). As shown in Fig. 1⇑, C and D, exposure of PBMC-derived monocytes or MDM (data not shown) to NCX-4016, an NO-aspirin derivative, resulted in a concentration-dependent inhibition of IL-1β and IL-18 release induced by 1 μg/ml LPS. Inhibition of IL-1β and IL-18 release was significant at 10 μM (p < 0.05 for both cytokines), reached the half-maximum at 21 ± 6.3 and 25 ± 5.1 μM, respectively, and plateaued at 100 μM (Fig. 1⇑, C and D). In contrast to NCX-4016, equimolar concentrations of aspirin failed to inhibit IL-1β and IL-18 release. Indeed, at concentrations of 100 and 200 μM it potentiated IL-1β (but not IL-18) release induced by LPS (Fig. 1⇑, C and D; p < 0.05). Because plasma aspirin concentrations measured after oral administration of low-moderate doses (i.e., 300–900 mg) of aspirin range from 80 to 240 μM, all the following experiments were conducted using the concentration of 100 or 200 μM (29). Although incubating the LPS-treated macrophages with 1–200 μM NCX-4016 had no effect on monocytes viability, NCX-4017 caused a time- and concentration-dependent decrease in cell viability, and, at the concentration of 100–200 μM, it caused 80% cell death (Fig. 1⇑, Box 3, E and F). Thus, the substitution of the NO moiety of NCX-4016 with an hydroxyl group results in a highly cytotoxic compound.

To investigate whether inhibition of cytokine release was specific for IL-1β and IL-18 or extended to other cytokine/chemokines, we have then examined the effect of NCX-4016 and aspirin on proinflammatory cytokine/chemokine (IL-1β, IL-8, IL-12, IL-18, IFN-γ, and TNF-α) released from by monocytes challenged with LPS. Exposure of PBMC-derived monocytes to 1 μg/ml LPS for 24 h significantly increased IL-1β, IL-8, IL-12, IL-18, IFN-γ, and TNF-α concentrations in cell supernatants (Fig. 2⇓, A–F). In this experimental setting, NCX-4016, at the concentration of 100 μM, almost completely prevented IL-1β, IL-12, IL-18, and IFN-γ release (p < 0.001), and caused an ≈40% reduction of TNF-α and IL-8 generation (Fig. 2⇓, A–F). In contrast, coincubating monocytes obtained from the same set of donors with 100 μM aspirin had no effect on IL-8, IL-12, IL-18, and IFN-γ release, although it significantly potentiated the effect of LPS on IL-1β and TNF-α release (Fig. 2⇓, A–F).

FIGURE 2.
FIGURE 2.

A–F, NCX-4016 prevents cytokine release from LPS-challenged monocytes. PBMC-derived monocytes were stimulated with 1 μg/ml LPS alone or in combination with 100 μM NCX-4016 or aspirin for 24 h. Each incubation was conducted in duplicate and each assay in duplicate. The data are mean ± SE of at least six donors. ∗, p < 0.001 vs cells incubated with the medium alone; ∗∗ and ∗∗∗, p < 0.01 vs cells incubated with LPS alone.

Because most of the antiinflammatory properties of aspirin are mediated through the inhibition of cyclooxygenase 1 and 2 (COX-1 and COX-2), we have then assessed whether NCX-4016 inhibits PG generation from LPS-stimulated monocytes. Indeed, as shown in Fig. 3⇓A, at the concentration of 100 μM, aspirin and NCX-4016 caused an ≈90% inhibition of PGE2 generation induced by LPS (p < 0.001). Because PGE2 generation from LPS-stimulated monocytes is a measure of COX-2 activity, but aspirin and NO-aspirin are preferential COX-1 inhibitors, we have assessed whether COX-2 inhibition was involved in the effect exerted by the two compounds. To ascertain this point, we incubated the cells with celecoxib, a selective COX-2 inhibitor (21). However, although incubating PBMC-derived monocytes with 10 μM celecoxib caused an ≈90% inhibition of PGE2 generation, it had no effect on cytokine release (Fig. 3⇓, A and B). Taken together these data indicate that NCX-4016 inhibits cytokines release through a COX-independent mechanism.

FIGURE 3.
FIGURE 3.

A, Inhibition of cytokine release by NCX-4016 is COX-independent. PBMC-derived monocytes were incubated with 1 μg/ml LPS alone or in combination with 100 μM aspirin and NCX-4016 or 10 μM celecoxib for 24 h. The cell supernatants were then collected for prostaglandin assays. Each incubation and each assay was conducted in duplicate. The data are mean ± SE of six donors. ∗, p < 0.001 vs cells incubated with the medium alone. ∗∗, p < 0.001 vs cells incubated with LPS alone. B, Celecoxib had no effect on cytokine release induced by bacterial endotoxin. Monocytes were stimulated with 1 μg/ml LPS in the presence or absence of 10 μM celecoxib for 24 h. The data are mean ± SE of six donors. ∗, p < 0.001 vs cells incubated with the medium alone.

Cytokine release from LPS-stimulated monocytes is regulated by an ICE-dependent pathway

Because maturation of pro-lL-1β and pro-IL-18 into mature cytokines requires the cleaving activity of ICE-like peptidases, we have assessed whether ICE is required to generate mature IL-1β and IL-18 from LPS-challenged monocytes. As illustrated in Fig. 4⇓A, exposure of PBMC-derived monocytes to 1 μg/ml LPS for 24 h resulted in a 7- to 8-fold increase of YVADase activity (ICE-like cleaving activity) on monocyte lysates. In these experimental conditions, incubating LPS-challenged monocytes with increasing concentrations, 0.1–100 μM of Ac-YVAD. CHO, an highly selective caspase-1 inhibitor, resulted in a dose-dependent inhibition of mature IL-1β, IL-18, and IFN-γ release (Fig. 4⇓B) with an IC50 of 0.2–0.7 μM. Moreover, at the dose of 100 μM, Ac-YVAD. CHO caused a 40% reduction of TNF-α release, from 2432.3 ± 156.6 to 1578.4 ± 234.0 pg/ml (p > 0.05), although it had no effect on IL-8 and IL-12 release (data not shown).

FIGURE 4.
FIGURE 4.

A, ICE-inhibition prevents cytokine release from LPS-challenged monocytes. PBMC-derived monocytes were stimulated with LPS, 1 μg/ml, in the presence or absence of 0.1–100 μM Ac-YVAD.CHO, a selective ICE inhibitor for 24 h and ICE-like (YVADase) activity measured. Each incubation and each assay was conducted in duplicate. The data are mean ± SE of six donors. ∗, p < 0.001 vs cells incubated with medium alone. ∗∗, p < 0.01 vs cells incubated with the LPS alone. B, The ICE inhibitor, Ac-YVAD.CHO, inhibits cytokine release from LPS-challenged monocytes. Monocytes were stimulated with 1 μg/ml LPS in the presence or absence of 100 μM Ac-YVAD.CHO for 24 h. The data are mean ± SE of six donors. ∗, p < 0.01 vs cells incubated with the medium alone. ∗∗, p < 0.01 vs cells incubated with the LPS alone.

NCX-4016 inhibits ICE activity

As illustrated in Fig. 5⇓A, incubating monocytes with NCX-4016, but not with aspirin or NCX-4017 (data not shown), resulted in a concentration-dependent inhibition of ICE-like proteases with an IC50 of 18.3 ± 5.6 μM (Fig. 5⇓B). Maximal inhibition of 78.2 ± 6.4% was observed at the concentration of 100 μM of NCX-4016. Because, upon activation, proteolytical cleavage of caspase-1 releases two subunits of p20 and p17 KD, which heterodimerize to form the active protease (23), we have then investigated whether NCX-4016 reduced the appearance of the p20 subunit in the cytosol of LPS-challenged monocytes, and as shown in Fig. 5⇓C, we found that, in contrast to aspirin, the NO-derivative significantly reduced the amount of p20 subunit released upon LPS stimulation.

FIGURE 5.
FIGURE 5.

A, NCX-4016 inhibits ICE activation induced by LPS. YVADase activity was measured in lysates obtained from PBMC-derived monocytes stimulated with LPS, 1 μg/ml, in the presence or absence of 100 μM NCX-4016 or aspirin for 24 h. The data are mean ± SE of six donors. ∗, p < 0.01 vs cells incubated with medium alone. ∗∗, p < 0.01 vs cells incubated with the LPS alone. B, YVADase inhibition by NCX-4016 is concentration-dependent. The data are mean ± SE of six donors. ∗, p < 0.01 vs cells incubated with LPS alone. The EC50 value was calculated by regression analysis. C, NCX-4016 prevents pro-ICE cleavage. Western blot analysis conducted on monocyte cell lysates (see Materials and Methods). Lane A, Cells incubated with medium alone; lane B, cells incubated with LPS 1 μg/ml for 24 h; lane C, cells incubated with LPS together with 100 μM NCX-4016; and lane D, cells incubated with LPS together with 100 μM aspirin. Data shown are representative of five other determinations.

Because ICE is not directly involved on IFN-γ, IL-8, IL-12, and TNF-α processing, but IL-1β and IL-18 modulate IFN-γ, IL-8, and TNF-α release, experiments were performed to assess whether inhibition of IL-1β and/or IL-18 production accounts for the inhibitory effect exerted by NCX-4016 on these cytokines. As demonstrated in Fig. 6⇓A, incubating monocytes with 50 μg/ml of rabbit anti-human IL-18 polyclonal antiserum, but not with 10 μg/ml IL-1RA, caused a 60–80% reduction of IL-1β, IFN-γ, IL-8, and TNF-α (but not IL-12) release induced by LPS, suggesting that inhibition of IL-18 production was responsible for the inhibitory effect exerted by NCX-4016 on these cytokine/chemokines. Supporting this view, incubating PBMC-derived monocytes with 10 ng/ml hrIL-18 directly stimulates IL-1β, IL-8, and TNF-α (but not IL-12) release from human monocytes (Fig. 6⇓B), and potentiated IFN-γ release from concanavallin A pretreated monocytes, from 2234.2 ± 156.5 to 3651.2 ± 311.6 pg/ml (p < 0.001). The effect of IL-18 on IFN-γ was not the result of endotoxin contamination as assessed by coincubation of IL-18 with polymyxin B (2.5 μg/ml) (data not shown). Taken together, these data suggest that NCX-4016 inhibits cytokine release through ICE-dependent (IL-1β, IL-8, IL-18, IFN-γ, and TNF-α) and ICE-independent (IL-12) pathways.

FIGURE 6.
FIGURE 6.

A, IL-18 immunoneutralization reduces cytokine release from LPS-challenged monocytes. PBMC-derived monocytes were stimulated with LPS, 1 μg/ml, in the presence or absence of 50 μg/ml anti-IL-18 polyclonal Ab, 10 μg/ml IL-RA, or 10 μg/ml control IgG for 24 h. The data are the mean ± SE of four donors. ∗, p < 0.01 compared with medium. ∗∗, p < 0.01 compared with LPS alone. B, IL-18 directly stimulates cytokine release from PBMC-derived monocytes. PBMC-derived monocytes were stimulated with 10 ng/ml IL-18 for 48 h. After the incubation, the cell supernatants were removed and assayed for IL-1β, IL-8, and TNF-α concentrations. The data represent the mean ± SE of four donors. ∗, p < 0.01 compared with medium.

We have then examined whether the effect of NCX-4016 on IL-1β release could be reproduced by endogenous NO. Because human monocytes were unable to produce detectable amount of NO in response to LPS, LPS-treated MDM were coincubated with or without 10 μg/ml Poly I:C for 24 h to stimulate endogenous NO generation. In this experimental setting, although LPS and Poly I:C alone failed to cause the appearance of nitrite/nitrate in cell supernatants, the combination of the two resulted in a significant increase of nitrite/nitrate generation (p < 0.005). Coincubating the cells with l-NIL almost completely abolished this effect (Fig. 7⇓A). Although LPS and Poly I:C individually stimulated IL-1β release, Poly I:C did not potentiate IL-1β release induced by LPS (Fig. 7⇓B). Coincubating the cells with 10 μM l-NIL had no effect on IL-1β release induced by LPS, although it significantly increased the cytokine release induced by Poly I:C alone or in combination with LPS (p < 0.05). Moreover, l-NIL increased ICE-like activity in MDM incubated with Poly I:C alone or in combination with LPS, but failed to modulate ICE-like proteases in cells treated with LPS alone (Fig. 7⇓C). Incubating the cells with NCX-4016, 100 μM, markedly inhibited IL-1β release induced by Poly I:C. Thus, driving human MDM to produce endogenous NO results in partial ICE inhibition.

FIGURE 7.
FIGURE 7.

Endogenous NO modulates cytokine release and ICE activity. A–C, MDM were prestimulated with LPS (1 μM) for 12 h and then with 50 μg/ml Poly I:C in the presence or absence of 10 μM l-NIL or 100 μM NCX-4016 for a further 24-h period. The cell supernatants were then collected for nitrite/nitrate and IL-1β assay, whereas MDM lysates were harvested for ICE (YVADase activity) measurement. The data are the mean ± SE of six donors. ∗, p < 0.01 compared with medium. ∗∗, p < 0.01 compared with LPS alone. ***, p < 0.01 compared with LPS + Poly I:C.

NO-aspirin is metabolized by monocytes to generate intracellular NO

Although NCX-4016 does not release spontaneously NO when incubated at 37°C with the medium alone (data not shown), incubating monocytes with NCX-4016 resulted in a concentration- and time-dependent increase in nitrite/nitrate released in cell supernatants (Fig. 8⇓, A and B). However, in contrast to the effect of SNAP, which caused a rapid increase of nitrite/nitrate generation, nitrite/nitrate production from NCX-4016-treated monocytes required ≈3–6 h to reach the steady state (Fig. 8⇓B). Confirming the fact that nitrite/nitrate generation was due to the cellular metabolism of the NO-aspirin derivative, incubating the cells with NCX-4016 or SNAP, but not aspirin, resulted in a time-dependent increase in intracellular fluorescence generated by monocytes loaded with the cell-permeable NO-reactive fluorochrome DAF-DA (Fig. 8⇓C). Moreover, incubating PBMC-derived monocytes with 200 μM of SNAP and NCX-4016, but not aspirin, resulted in a time-dependent increase in [cGMP]i, although to a different extent. Indeed, incubating the cells with SNAP caused a rapid and short-lasting accumulation of cGMP, whereas peak of [cGMP]i (≈10-fold increase) induced by NCX-4016 appeared after 3–6 h, but lasted for 12–24 h (Fig. 8⇓D). Incubating the cells with 100 μmol/L ODQ, to inhibit cyclic guanylyl cyclase, completely prevented the stimulatory effect exerted by SNAP and NCX-4016 on [cGMP]i (Fig. 8⇓, E and F). Thus, NCX-4016 penetrates monocyte cell membranes and is metabolized to release free NO and/or NO-derived compounds.

FIGURE 8.
FIGURE 8.

Incubating human monocytes with NCX-4016 and SNAP, but not aspirin, results in a concentration-dependent generation of intracellular NO as measured by assessing nitrite/nitrate concentrations in cell supernatants (A and B), intracellular NO formation in cells loaded with DAF-DA (C), and intracellular cGMP accumulation (D). The data are the mean ± SE of four to six donors. ∗, p < 0.01 compared with aspirin. ∗∗, p < 0.01 compared with LPS. E and F, Incubating PBMC-derived monocytes with 1 mM ODQ, to inhibit the adenylate cyclase activity, prevents cGMP accumulation induced by SNAP and NCX-4016. The data are the mean ± SE of four donors. ∗, p < 0.01 compared with aspirin alone. ∗∗, p < 0.01 compared with SNAP or NCX-4016 alone.

NO-aspirin causes ICE S-nitrosylation

Because the S-nitrosylation is a well-established mechanism by which NO inhibits enzyme activity (16, 17, 19, 20), we biochemically analyzed whether inhibition of YVADase activity caused by NCX-4016 was due to enzyme nitrosylation. As shown in Fig. 9⇓A, incubating PBMC-derived monocytes with 100 μM SNAP and NCX-4016, but not aspirin, inhibited ICE-like cysteine protease activity, as measured by assessing YVAD.AMC peptide cleavage. Incubating monocyte lysates with 20 mM DTT to remove thiol-bound group from proteins reverted the inhibitory effect exerted by the two NO-releasing agents, resulting in ≈65% recover of protease activity. The finding that incubation with HgCl2, an agent that binds thiol group, caused a 90% loss of proteolytic activity and that inhibition induced by HgCl2 was partially reverted by DTT, 20 mM, and was not additive with NCX-4016 (Fig. 9⇓B), is further evidence that YVADase inactivation induced by NCX-4016 was due to enzyme S-nitrosylation. To further confirm this finding, we measured NO released by incubating monocyte lysates with HgCl2 in the presence of DAF-AM. In this experimental setting, lysates obtained from monocytes cultured with LPS in the presence of SNAP or NCX-4016, but not those incubated with aspirin (100 μM each), released detectable amounts of NO, further confirming that exposure to NCX-4016 results in cell-protein S-nitrosylation (Fig. 9⇓C).

FIGURE 9.
FIGURE 9.

NCX-4016 causes ICE-like proteases S-nitrosation/inhibition. A, Reversibility of inhibition of ICE-like cysteine proteases caused by SNAP and NCX-4016 by DTT. Lysates obtained from monocytes pretreated with LPS alone or in combination with SNAP, NCX-4016, or aspirin were incubated with 20 mM DTT and ICE activity measured. Data are mean ± SE of six determinations conducted in triplicate. ∗, p < 0.01 vs homogenates incubated with medium alone. ∗∗, p < 0.01 in comparison with homogenates incubated without DTT. B, Inhibition and reversibility of ICE-like cysteine protease activity by thiol-reactive agents. Cell lysates obtained from monocytes incubated with LPS and NCX-4016 were incubated with 5 mM HgCl2 and/or 20 mM DTT and ICE activity measured. Data are mean ± SE of six determinations conducted in triplicate. ∗, p < 0.01 in comparison with cells incubated with the medium alone. ∗∗, p < 0.01 in comparison with the cells incubated with LPS alone. C, Release of NO-derived compounds from monocyte lysates obtained from cells incubated with LPS, 1 μg/ml, alone or in combination with 200 μM SNAP, NCX-4016, and aspirin. Data are mean ± SE of six determinations conducted in triplicate. ∗, p < 0.01 vs homogenates incubated with medium alone.

Finally, incubating the cells with the cell-permeable cGMP analogue, 8Br-cGMP (1 mM), or with ODQ to inhibit cGMP accumulation, had no effect on YVADase activity or IL-1β release induced by LPS (data not shown), suggesting that inhibition of cytokine production by NCX-4016 is cGMP-independent.

Discussion

NO-releasing NSAIDs are a recently described class of NSAID derivatives generated by adding a nitroxybuthyl moiety through an ether linkage to the parental NSAID (14, 15, 16, 17) These compounds are virtually devoid of gastrointestinal toxicity, while retaining antiinflammatory, antithrombotic, and antipyretic activity. Moreover, in some experimental models, these “NO-NSAIDs” exhibited significantly enhanced antiinflammatory and analgesic effects in comparison with their parental molecule (28, 29). In the present study, we provided the first evidence that the addition of an NO-releasing moiety to aspirin confers new pharmacological properties to this molecule. Indeed, we demonstrated that NCX-4016 inhibits proinflammatory cytokine release from LPS-challenged monocytes through a mechanism that involves the S-nitrosylation/inhibition of proteases required for cellular processing/maturation of IL-1β and IL-18. Support for this view comes from the following results: first, the effect exerted by NCX-4016 was unrelated to its ability to inhibit COX activity, as demonstrated by the finding that treating the cells with selective and nonselective COX-1 and/or COX-2 inhibitors caused an ≈90% inhibition of PGE2 generation but had no effect on cytokine generation; second, similar to the “conventional” NO-donor SNAP, NCX-4016 penetrates monocyte cell membranes to release free NO and/or NO-derived compounds as assessed by measuring nitrite/nitrate concentrations in cell supernatants (30, 31), [GMP]i, and intracellular NO formation in cells loaded with the NO-specific fluorochrome, DAF-AM (24, 25). The kinetic of NO generation from NCX-4016, however, is different from that of SNAP. In fact, the NO moiety of NCX-4016 dissociates slowly from aspirin, resulting in a lower, but sustained, release of NO; third, further supporting the view that the inhibitory activity of NCX-4016 on cytokine release was due to its NO moiety, we demonstrated that substituting the nitrooxymethyl-phenyl ester group, the NO moiety, with an hydroxymethyl-phenyl group resulted in a compound, the NCX-4017, highly cytotoxic, devoid of any pharmacological activity; fourth, NCX-4016 activates NO-dependent pathways as demonstrated by the finding that incubating the cells with ODQ, to prevent guanosilyl cyclase activation, almost completely inhibited changes in [cGMP]i, although it had no effect on cytokines release (17); fifth, incubating monocytes with LPS resulted in a time-dependent activation of ICE-like endoproteases, an effect that was almost completely prevented by cotreating cells with NCX-4016, but not with aspirin or NCX-4017; sixth, inhibition of LPS-induced YVAD cleaving activities by NCX-4016 was reverted by DTT, an agent that effectively removes thiol-bound NO groups from proteins (17, 19, 20, 27, 32); seventh, exposure to HgCl2, an agent that bind to thiol group, displaced NO from lysates obtained from NCX-4016-treated monocytes and resulted in a DTT-reversible inhibition of YVADase activity, indicating that the NO removed by HgCl2 was bound to a cysteine group (17, 19, 20, 27, 32); and, finally, incubating LPS-challenged monocytes with NCX-4016, but not with aspirin, prevented ICE activation as measured by assessing the release of the p20 subunit (23, 33). Taken together, these data demonstrated that incubating human monocytes with NCX-4016 results in intracellular NO formation and S-nitrosation/inhibition of ICE-like cysteine proteases involved in pro-IL-1β and pro-IL-18 processing (10, 11, 12).

NO has previously been found to play a role in inflammation (18, 31, 34). In addition to the well established proinflammatory effect, it is now well recognized that NO acts as a double-edge sword being antiinflammatory at low (micromolar) concentrations, and cytotoxic and proinflammatory at high (millimolar) concentrations (see Ref. 34 for review). Although the precise mechanisms by which endogenous NO exerts these effects are only partially known, a growing body of evidence indicates that low levels NO cause the S-nitrosylation of thiol groups located in the catalytic core of cysteine proteases required for cellular processing of proinflammatory cytokine (17, 19, 20, 34). In line with this view, Kim et al. (20) have recently demonstrated that endogenous NO down-regulates IL-1β and IL-18 production in LPS-challenged RAW264.7 rat macrophages by causing the S-nitrosation of caspase-1. Together with the finding that, in this cell line, exposure to LPS induces a parallel increase of iNOS expression and ICE activity and that caspase-1 products, i.e., IL-1β and IL-18 as well as IFN-γ, participate in the up-regulation of iNOS expression (20), these data suggest that NO represents an endogenous regulator of caspase-1. Supporting these functional findings, previous studies conducted with purified subunits of caspase-1 have demonstrated that the p20 subunit is a selective target for NO, and that p20 S-nitrosylation leads to a concentration-dependent inhibition of enzyme activity (19). More generally, there is now evidence that the S-nitrosylation is a mechanism extensively involved in caspase regulation. A recent report from Mannick et al. (36) indicates that in resting human cell lines caspase-3 zymogens are S-nitrosylated and denitrosylated upon Fas/Fas ligand cross-linking, indicating that caspases activation requires both denitrosylation and zymogen cleavage (36). Confirming the role of endogenous NO in modulating ICE-like peptidases, we demonstrated that, although MDM were unable to release NO when incubated with LPS alone, they produced detectable amount of NO in response to costimulation with Poly I:C, a viral mimetic that stimulates NO production from LPS-primed MDM (≈5 nM/106 MDM/24 h). Incubating MDM with l-NIL significantly increased IL-1β release and caspase-1 activity only in cells incubated with Poly I:C, (22). The finding that incubating LPS/Poly I:C-treated MDM with NCX-4016 inhibited caspase-1 activity and IL-1β production is a further evidence that NO exerts a regulatory function on ICE-dependent cytokines.

Although the reversal of the NO-mediated inhibition by DTT is consistent with the S-nitrosation as a the main mechanism for inhibition of caspase-1 activity by NCX-4016 and our results demonstrated NO formation in monocytes incubated with this compound, it cannot be excluded that a reaction product with NO+ activity (N2O3, the reaction product of NO + O2, and even peroxynitrite formed from NO + O2−) carry out this chemistry (31). The failure of DTT treatment to fully recover all YVADase activity, however, raises the possibility that NO may also suppress caspase-1 activation (19, 20, 32, 35, 36). Indeed because caspase-1 activation is partially due to the autocalytic cleavage of the inactive proenzyme and pro-caspase-1 zymogens are themselves substrate for ICE (1, 2, 3, 4, 5, 21, 33), it cannot be excluded that S-nitrosation/inhibition of activated ICE will reduces the amount of active enzyme that is further generated through this pathway (3). Supporting this concept, NCX-4016 markedly reduced the amount of the p20 subunit released in the cytoplasm of monocytes incubated with LPS.

In the present study, we demonstrated that exposure to NCX-4016, but not to aspirin, not only inhibited the release of ICE-regulated cytokines, IL-1β and IL-18, but, more impressively, resulted in an extensive down-regulation of a wide array of proinflammatory chemokines and cytokines. However, because IL-8, IL-12, IFN-γ, and TNF-α do not require an ICE-like peptidases for their maturation, it is likely that the inhibitory effect exerted by NCX-4016 was due to the inhibition of IL-1β and/or IL-18 production (6, 7, 8, 9, 10, 37, 38, 39, 40, 41, 42, 43, 44). In particular, our results indicate that inhibition of IL-18 maturation was the main mechanism by which NCX-4016 inhibited IL-8, IFN-γ, and TNF-α release from LPS-challenged monocytes. Support for this concept comes from the observation that IL-18 immunoneutralization almost completely inhibited IL-1β, IL-8, and IFN-γ and caused an ∼40% reduction of TNF-α release induced by LPS, whereas cotreating the cells with the IL-RA, to block IL-1β receptors, reduced IL-8 generation but had no effect on cytokines release (45). IL-18 is a recently cloned cytokine that exhibits powerful Th1-promoting activities (7, 8, 9, 10, 13). Pro-IL-18 is cleaved by ICE or ICE-like peptidases (caspase-4, -5, and -11) to release an active 18-kDa glycoprotein with significant structural similarity to IL-1β (13). IL-18 induces T lymphocyte proliferation, up-regulates IL-2R expression, promotes IL-1 β, IL-8, IFN-γ, TNF-α, and GM-CSF production by Th1 clones and enhances T cell and NK-cell cytotoxicity (see Ref. 7 for review). The finding of Fantuzzi et al. (46), demonstrating that IL-18 immunoneutralization inhibits IFN-γ release from splenocytes challenged with IL-12, strongly support the concept that IL-18 plays a major role in modulating proinflammatory response and is consistent with the ability of this cytokine to activate the nuclear translocation of NF kB in T cells (34, 47). In this context, the ability of NCX-4016 to suppress IL-1β, IL-8, IL-12, IL-18, and IFN-γ production suggests that the NO-aspirin derivative may be effective in treating Th1-sustained diseases (48, 49, 50, 51, 52, 53). Supporting this view, we have recently demonstrated that in vivo administration of NCX-4016, but not aspirin, to mice challenged with Con A protects from liver damage induced by this mitogen by inhibiting IL-1β, IL-18, and IFN-γ release, as well as Fas/Fas L up-regulation in circulating lymphocytes and liver cells (54).

In addition to its ability to inhibit ICE-regulated cytokines, our results demonstrated that NCX-4016 inhibited IL-12 release induced by LPS. IL-12 is a heterodimeric cytokine mostly produced by phagocytic cells. Functionally active IL-12 is a 70-kDa molecule formed by two covalently linked glycosylated chains of ∼40 (p40) and 35 (p35) kDa (40). Although aspirin itself had no effect on IL-12 regulation, it has been demonstrated that NO induces transcription of the IL-12 p40 gene, but not the IL-12 p 35 (40) Because the IL-12 (p40)2 homodimer is an antagonist for IL-12 production, and inhibits IL-12 synthesis, it cannot be excluded that IL-12 inhibition exerted by NCX-4016 was due to an impairment of IL-12 assembly.

To summarize, we demonstrated that, in contrast to aspirin, NCX-4016, an NO-aspirin derivative, that spares the gastric mucosa (17) causes the S-nitrosylation/inhibition of caspase-1 in human monocyte/macrophage challenged with bacterial endotoxin. Because ICE activation is a limiting step in the process of maturation and secretion of cytokines, IL-1β and IL-18, pivotal in the proinflammatory cytokine hierarchy, present results may have important therapeutic implications for treatment of inflammatory disorders (6, 54).

Acknowledgments

We thank Barbara Federici and Barbara Palazzetti for their technical support.

Footnotes

  • ↵1 Address correspondence and reprint requests to Dr. Stefano Fiorucci, Clinica di Gastroenterologia ed Endoscopia Digestiva, Policlinico Monteluce, Via E. Dal Pozzo, 06100 Perugia, Italy. E-mail address: fiorucci{at}unipg.it

  • ↵2 Abbreviations used in this paper: ICE, IL-1β converting enzyme; AMC, 7-amino-4-coumarin; l-NIL, l-N6-(1-iminoethyl)-lysine; SNAP, S-nitroso-N-acetyl-d-l-penicillamine; NCX-Ac-YVAD-CHO, acetyl Tyr-Val-Ala-Asp aldehyde; NSAID, nonsteroidal antiinflammatory drug; NO-NSAID, NO-releasing NSAID; MDM, monocyte-derived macrophage; COX, cyclooxygenase; Poly I:C, polyriboinosinic polyribocytidylic acid.

  • Received December 30, 1999.
  • Accepted August 9, 2000.

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