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*
Dipartimento di Medicina Clinica e Sperimentale, Clinica di Gastroenterologia ed Epatologia; Università degli Studi di Perugia;
Dipartimento di Farmacologia, Universita’ di Napoli, Italy;
Dipartimento di Medicina Interna, Universita’ di Messina; and
§
NicOx, Sophia Antipolis, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, and TNF-
release with an
EC50 of 10–20 µM for IL-1ß 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-1ß,
IL-8, IFN-, and TNF- release from LPS-stimulated monocytes. Taken
together, these data indicate that incubating human monocytes with
NCX-4016 causes intracellular NO formation and suppresses IL-1ß and
IL-18 processing by inhibiting caspase-1 activity. Caspase-1 inhibition
is a new, cycloxygenase-independent antiinflammatory mechanism of
NO-aspirin. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IL-18, formerly termed IFN--inducing factor (IGIF) is a
proinflammatory cytokine structurally related to IL-1ß
(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- release and
confers protection against acute toxicity induced by LPS
(8). In addition to acting as a costimulus for
IFN- production, IL-18 stimulates IL-1ß, IL-2, IL-8, and TNF-
secretion from human PBMC, potentiates anti-CD3-induced T
cell proliferation, and increases Fas ligand expression on NK
cells (9). Similar to the IL-1ß 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- 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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 x 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 x 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 x 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 x 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 x
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-1ß 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-1ß, IL-18, and IFN- 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-1ß 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).
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IL-1ß, IL-8, IL-12, IL-18, TNF-, and IFN- 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 (1x107) 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 x
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 x
107) were incubated for a variable amount of time
with aspirin, SNAP, or NCX-4016 (see Fig. 8C), 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.
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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).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Incubation of PBMC-derived monocytes with LPS for 24 h
resulted in a concentration-dependent stimulation of IL-1ß 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-1ß and
IL-18 in response to 1 µg/ml LPS (Fig. 1B). 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-1ß
and IL-18 release induced by 1 µg/ml LPS. Inhibition of IL-1ß 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-1ß and IL-18 release.
Indeed, at concentrations of 100 and 200 µM it potentiated IL-1ß
(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-1ß 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-1ß, IL-8, IL-12, IL-18, IFN-, and TNF-)
released from by monocytes challenged with LPS. Exposure of
PBMC-derived monocytes to 1 µg/ml LPS for 24 h significantly
increased IL-1ß, IL-8, IL-12, IL-18, IFN-, and TNF-
concentrations in cell supernatants (Fig. 2, A–F). In this experimental
setting, NCX-4016, at the concentration of 100 µM, almost completely
prevented IL-1ß, IL-12, IL-18, and IFN- release
(p < 0.001), and caused an 40% reduction
of TNF- 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- release,
although it significantly potentiated the effect of LPS on IL-1ß and
TNF- release (Fig. 2, A–F).
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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.
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Because maturation of pro-lL-1ß 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-1ß and
IL-18 from LPS-challenged monocytes. As illustrated in Fig. 4A, 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-1ß, IL-18, and IFN- release
(Fig. 4B) 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- 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).
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As illustrated in Fig. 5A, 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. 5B). 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. 5C, we found that, in
contrast to aspirin, the NO-derivative significantly reduced the amount
of p20 subunit released upon LPS stimulation.
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, IL-8, IL-12, and
TNF- processing, but IL-1ß and IL-18 modulate IFN-, IL-8, and
TNF- release, experiments were performed to assess whether
inhibition of IL-1ß and/or IL-18 production accounts for the
inhibitory effect exerted by NCX-4016 on these cytokines. As
demonstrated in Fig. 6A,
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-1ß, IFN-, IL-8, and TNF- (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-1ß, IL-8, and
TNF- (but not IL-12) release from human monocytes (Fig. 6B), and potentiated IFN- 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- 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-1ß, IL-8, IL-18, IFN-, and
TNF-) and ICE-independent (IL-12) pathways.
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A). Although LPS
and Poly I:C individually stimulated IL-1ß release, Poly I:C did not
potentiate IL-1ß release induced by LPS (Fig. 7B).
Coincubating the cells with 10 µM L-NIL had no
effect on IL-1ß 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. 7C). Incubating the cells with NCX-4016, 100 µM, markedly
inhibited IL-1ß release induced by Poly I:C. Thus, driving human MDM
to produce endogenous NO results in partial ICE inhibition.
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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. 8B). 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. 8C). 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. 8D). 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.
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. 9A, 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. 9B), 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. 9C).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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-1ß 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-1ß 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-1ß
and IL-18 as well as IFN-, 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-1ß 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-1ß 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-1ß 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-, and TNF- 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-1ß 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-, and TNF- release from LPS-challenged monocytes.
Support for this concept comes from the observation that IL-18
immunoneutralization almost completely inhibited IL-1ß, IL-8, and
IFN- and caused an 
40% reduction of TNF- release induced by
LPS, whereas cotreating the cells with the IL-RA, to block IL-1ß
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-1ß (13). IL-18 induces T
lymphocyte proliferation, up-regulates IL-2R expression, promotes IL-1
ß, IL-8, IFN-, TNF-, 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- 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-1ß, IL-8, IL-12,
IL-18, and IFN- 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-1ß, IL-18, and IFN- 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-1ß and IL-18, pivotal in the proinflammatory cytokine hierarchy, present results may have important therapeutic implications for treatment of inflammatory disorders (6, 54).
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: ICE, IL-1ß 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 for publication December 30, 1999. Accepted for publication August 9, 2000.
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