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Research Center, Maisonneuve-Rosemont Hospital, and Department of Medicine, University of Montreal, Quebec, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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90% in response to LPS (1
µg/ml), but did not affect the production of IL-1ß or TNF-
. Both
aminoguanidine and L-NAME blocked the induction of IL-8 mRNA by LPS.
Authentic ONOO- (2.5–80 µM) augmented IL-8 mRNA
expression and stimulated IL-8 release in a concentration-dependent
manner, whereas the NO-releasing compounds,
S-nitroso-N-acetyl-DL-penicillamine
and sodium nitroprusside failed to induce cytokine production.
Combination of the NO-generating chemicals with a superoxide-generating
system (xanthine/xanthine oxidase) markedly increased IL-8 release.
Enhanced ONOO- formation was detected in granulocytes,
monocytes, lymphocytes, and plasma after challenge with LPS.
Furthermore, pyrrolidine dithiocarbamate, an inhibitor of activation of
nuclear factor-
B, markedly attenuated the induction of IL-8 mRNA
expression and IL-8 release by either LPS or ONOO-. Our
study identifies ONOO- as a novel signaling mechanism for
IL-8 gene expression and suggests that inhibition of ONOO-
formation or scavenging ONOO- may represent a novel
therapeutic approach to inhibit IL-8 production that could lead to
reduction of neutrophil accumulation and
activation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(1, 4). IL-8 induces neutrophil chemotaxis (1, 5), release of
lysosomal enzymes (5, 6), shedding of L-selectin, and up-regulation of
expression of Mac-1 (7) and promotes neutrophil adherence to
endothelial cells (8) and transendothelial migration (7, 9). The
presence of IL-8 has been detected in several disease states, including
endotoxin shock (10, 11), inflammatory arthritis (12), and skin injury
(13). Neutralizing Abs against IL-8 attenuate neutrophil accumulation
and neutrophil-dependent tissue injury in endotoxemia and other
experimental models (14, 15), indicating a pivotal role of IL-8
in recruitment and activation of neutrophils during acute inflammation. Recent studies have identified oxidative stress as a ubiquitous mechanism for the induction of IL-8. Hydroxyl radical scavengers markedly suppress (16, 17) whereas H2O2 or deprivation of cellular glutathione enhances LPS-induced expression of the IL-8 gene (18). Recently, another radical, nitric oxide (NO)3 has also been implicated in the regulation of IL-8 production (19, 20, 21, 22). Numerous pathophysiologic conditions, including endotoxin shock, are associated with simultaneously enhanced formation of oxygen radicals and NO. Superoxide and NO react together in a diffusion-limited manner to form peroxynitrite (ONOO-) (23), which might be responsible for some of the biologic actions attributed to NO. As a highly reactive oxidant, ONOO- has the potential to produce oxidant stress. Therefore, we have studied whether ONOO- mediates IL-8 gene expression and production in response to LPS in human whole blood.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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LPS (Escherichia coli O111:B4), sodium nitroprusside,
aminoguanidine hemisulfate, cycloheximide, pyrrolidine dithiocarbamate
(PDTC), xanthine, xanthine oxidase (from buttermilk) and
NG-nitro-L-arginine methyl ester
(L-NAME) were obtained from Sigma (St. Louis, MO).NG-nitro-D-arginine methyl ester (D-NAME),
S-nitroso-N-acetyl-DL-penicillamine
(SNAP), and 3-morpholinosydnonimine hydrochloride (SIN-1) were
purchased from Research Biochemicals International (Natick, MA).
Dihydrorhodamine 123 (DHR 123) and rhodamine were obtained from
Molecular Probes (Eugene, OR). Human recombinant IL-1ß and TNF-
were purchased from R&D Systems (Minneapolis, MN).
Experimental design
Venous blood (10 ml; anticoagulated with sodium heparin, 50 U/ml) was obtained from nonsmoking healthy volunteers (male and female, 23–46 yr old) who had not taken any drugs for at least 10 days before the experiments. Informed consent was obtained from each volunteer, and the protocol was approved by the clinical research committee. White blood cell counts were between 5000 and 9000 cells/µl. Whole blood aliquots (1 ml) were transferred to sterile 1.5-ml centrifuge tubes, and aminoguanidine (10 mM), L-NAME (10 mM), D-NAME (10 mM), cycloheximide (35.5 µM), PDTC (100 µM), and LPS (1 µg/ml) were added as required. The blood was then placed on a rotator and incubated at 37°C in 95% air/5% CO2. At the designated time point, the plasma was harvested, diluted 1/5 with RPMI 1640 medium (Life Technologies, Grand Island, NY) containing 1% FBS, and stored at -20°C for later cytokine analysis. For samples used in RNase protection assays, 100 µl of blood were mixed with an equal volume of lysis/denaturation solution (Ambion, Austin, TX) and stored at -20°C for later processing.
ONOO- and control experiments
ONOO- synthesized by using nitrite and hydrogen peroxide in a quenched flow reactor (24) was obtained from Alexis (San Diego, CA). Stock solutions of ONOO- (160 mmol/L) were stored at -70°C at pH 12.7. Since the ONOO- stock solution contains nitrite, hydrogen peroxide (3.4 mM), and NaCl, we performed control experiments using the following solutions: 1) a pH-neutralized negative control solution (Alexis) that contains the same concentrations of nitrite, H2O2, and NaCl as the ONOO- stock solution; 2) a solution of 3.4 mM H2O2 at pH 12.7, prepared and diluted 1/2000 (a dilution factor that achieved an 80 µM final ONOO- concentration); and 3) NaOH at 0.15 mM (the final concentration of NaOH in 80 µM ONOO- solution). A 10-µl aliquot of one of these solutions was added to some blood samples, and its effects were investigated.
Measurement of intracellular and plasma rhodamine levels
NO synthase blocker-inhibitable fluorescence of rhodamine, an oxidation product of DHR 123, was used as a marker of exposure of DHR 123 to ONOO- (25). DHR 123 (20 µM) was mixed with whole blood samples; incubated with LPS (1 µg/ml), L-NAME (10 mM), or aminoguanidine (10 mM) for 3 h; and then incubated for an additional 60 min. At the end of the incubation period, erythrocytes were lysed, and leukocytes were prepared for flow cytometric analysis as described previously (26). Granulocytes, monocytes, and lymphocytes were gated by their forward and side scatter characteristics, and single-color fluorescence was analyzed by a cytofluorometer (FACScan, Becton Dickinson Immunocytometry Systems, Mountain View, CA) with LYSIS II software. The plasma rhodamine level was measured in a Perkin-Elmer fluorometer (excitation, 500 nm; emission, 526 nm; slit widths, 3 nm; Perkin-Elmer, Norwalk, CT) (25). The amount of plasma rhodamine was quantified using a rhodamine standard curve (0.8–400 nM) prepared in untreated plasma. Background fluorescence was subtracted from all samples.
Leukocyte viability
At the indicated times, leukocytes were prepared as described above, and staining with propidium iodide (0.5 µg/ml) was analyzed with a FACScan cytofluorometer.
Measurement of IL-8, IL-1ß, and TNF-
The plasma concentrations of IL-8, IL-1ß, and TNF- were
determined by highly selective ELISAs (R & D Systems). The detection
limits of the IL-8, IL-1ß, and TNF- assays were 31, 4, and 15
pg/ml, respectively. Intra- and interassay coefficients of variation
were typically <5% and <8%, respectively, in these assays. There
was no cross-reactivity with the NO synthase inhibitors or PDTC in the
assays.
IL-8 RNase protection assay
Human IL-8 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers (Clontech Laboratories, Palo Alto, CA) were used to amplify by PCR the positive control cDNA fragments provided by the primers’ manufacturer. The amplified products were analyzed on a 1% agarose gel, and single bands of the expected m.w. of 289 and 982 bp were obtained for IL-8 and GAPDH, respectively. The fragments were cloned in the SmaI site of the pGEM3Zf vector (Promega, Madison, WI). The IL-8 cDNA clone was linearized with BamHI. The probe generated by the T7 RNA polymerase contains 337 nucleotides, of which 289 bases are protected by hybridization to the IL-8 mRNA. The GAPDH clone was digested with ApaI, which cuts once in the GAPDH cDNA sequence. The probe synthesized by the T7 RNA polymerase contains 228 bases, 188 of which are protected by the corresponding mRNA. The linearized DNA was extracted with phenol/chloroform and precipitated with ethanol. The antisense mRNA probes were synthesized from 0.5 µg of cDNA template with the MAXIscript T7 transcription kit (Ambion) using 50 µCi of [32P]UTP (ICN Pharmaceuticals, Costa Mesa, CA) for the IL-8 probe and 10 µCi of [32P]UTP plus 100 µM unlabeled UTP for the GAPDH probe.
The RNase protection assay was performed with the Direct Protect kit
(Ambion) on aliquots of whole blood. The radiolabeled probes (5 µl,
5 x 105 cpm) were added to the samples (final volume,
50 µl), and the mixture was incubated overnight at 37°C. The
unhybridized probe was then digested with the RNase mixture, followed
by proteinase K digestion. The protected fragments were precipitated
with isopropanol and separated by electrophoresis on a 6%
polyacrylamide/8 M urea gel and visualized by autoradiography. All
autoradiograms were scanned with an image analysis system (IS-1000
Digital Imaging System, Innotech Corp., San Leandro, CA) to
quantitate the relative intensities of the bands corresponding to the
IL-8- and GAPDH-protected fragments. For each autoradiogram, the
results were normalized to represent equivalent RNA loading in each
lane based on the intensities of the GAPDH bands.
Statistical analysis
Results are expressed as the mean ± SEM. Statistical comparisons were made by analysis of variance using ranks (Kruskal-Wallis test) followed by Dunn’s multiple contrast hypothesis test to identify differences between various treatments or by the Wilcoxon signed rank test and Mann-Whitney U test for paired and unpaired observations, respectively. p < 0.05 was considered significant for all tests.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Consistent with previous observations (16, 27), our study also
documents the biphasic pattern of IL-8 production (Fig. 1
A). Previous studies have
shown that the secondary phase of IL-8 production occurring between 12
and 24 h can be completely inhibited by neutralizing anti-IL-1
and anti-TNF Abs, whereas the early phase of IL-8 production (4–12
h) is unaffected by these Abs (16). Both L-NAME and aminoguanidine
markedly reduced IL-8 production over the entire time course,
indicating that their mechanism of inhibition is different from that of
the anti-TNF and anti-IL-1 Abs. For instance, L-NAME and
aminoguanidine at 10 mM suppressed IL-8 production, on the average, by
75 and 81% and by 90 and 87% (n = 3) at 4 and 24
h, respectively (Fig. 1A). Both NO synthase blockers exerted
a concentration-dependent inhibitory action with an apparent maximal
inhibition achieved at 10 mM, as assayed 24 h post-LPS (Fig. 1D). The inhibitory action of 10 mM L-NAME was completely
reversed by 100 mM L-arginine, and unlike L-NAME, D-NAME
did not affect LPS-induced IL-8 release (data not shown). The
inhibitory action of NO synthase inhibitors was highly specific for
IL-8, as neither L-NAME nor aminoguanidine had a significant effect on
the release of TNF- and IL-1ß (Fig. 1, B and
C). Leukocyte viability was unaffected by L-NAME or
aminoguanidine (data not shown).
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and IL-1ß (16), we tested whether NO synthase inhibitors
could also suppress IL-8 release evoked by these cytokines. Both
aminoguanidine and L-NAME inhibited by >85% the amount of IL-8
released by TNF- (370 ng/ml) or IL-1ß (100 ng/ml; Fig. 2).
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To determine whether the reduction of LPS-stimulated IL-8 release
by NO synthase blockers is occurring at the level of transcription or
translation, RNase protection assays were performed on RNA extracted
from whole blood samples incubated for 4 h with LPS (1 µg/ml) in
the presence and the absence of aminoguanidine or L-NAME. A significant
reduction in LPS-stimulated IL-8 mRNA levels was detected upon
incubation with either L-NAME or aminoguanidine (Fig. 3). IL-8 mRNA was similarly suppressed by
both aminoguanidine and L-NAME in the absence and the presence of
cycloheximide (Fig. 3), indicating that these compounds do not induce
the synthesis of an intermediary protein; rather, they have a direct
inhibitory effect on transcription.
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Our studies have established that NO synthase inhibitors are
capable of inhibiting IL-8 production. We next wanted to determine
whether exposure of whole blood to chemically generated NO would induce
IL-8 release. Concentration-response studies were therefore performed
in which the NO donors, SNAP (0.4–2.4 mM) and sodium nitroprusside
(0.5–2 mM), were directly added to blood. None of these NO donors
produced significant increases in IL-8 release (Table I). Since LPS-stimulated neutrophils and
monocytes simultaneously produce both NO and superoxide anion, which
react to form the potent oxidant ONOO- (24), we tested
whether addition of either NO donor in combination with a
superoxide-generating system (xanthine/xanthine oxidase) or authentic
ONOO- to blood samples could enhance IL-8 production.
Incubation of blood samples with SNAP or sodium nitroprusside together
with xanthine/xanthine oxidase resulted in marked increases in IL-8
production, whereas IL-8 production was only slightly stimulated by
xanthine/xanthine oxidase (Table I). The compound SIN-1, which
simultaneously releases both NO and superoxide (28), caused a
concentration-dependent increase in IL-8 accumulation (Table I).
ONOO- stimulated IL-8 release in a concentration-dependent
manner, with the IL-8 concentrations measured after challenge with 80
µM ONOO- being comparable to those in samples incubated
with 1 µg/ml LPS (Table I). On the other hand, 80 µM
ONOO- produced only a slight increase in the TNF- level
(0.3 ± 0.1 ng/ml; n = 5), amounting to only 3%
of the samples challenged with LPS (11.1 ± 2.3 ng/ml;
n = 5). ONOO- up to a concentration of 80
µM did not affect leukocyte viability (data not shown). IL-8
concentrations in samples incubated with pH-neutralized (decomposed)
ONOO- solution or NaOH were similar to those in control
(unstimulated) samples (Table I). Furthermore,
H2O2 at a 1.7-µM concentration, which is
equivalent to its concentration in 80 µM ONOO- solution,
did not induce IL-8 release (data not shown).
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RNase protection assays performed on RNA extracted from whole
blood samples challenged for 4 h with an initial concentration of
80 µM ONOO- revealed a 2.4-fold increase in IL-8 mRNA
expression, whereas IL-8 mRNA in samples incubated with decomposed
ONOO- or NaOH (final concentration, 0.15 mM) did not
differ from that detected in control samples (Fig. 4).
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Since the IL-8 gene contains functional NF-B binding sites in
its promoter that are necessary for the transcriptional activation of
this gene (29, 30), we studied whether PDTC, a specific inhibitor of
the NF-B activation (31), would affect induction of IL-8 by LPS and
ONOO-. In the presence of PDTC, both IL-8 release (Fig. 5) and expression of IL-8 mRNA were
markedly suppressed in blood samples challenged with either LPS (Fig. 3
) or ONOO- (Fig. 4). PDTC at the concentration used did
not affect leukocyte viability (data not shown).
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Incubation of blood samples with LPS for 4 h resulted in
increases in intracellular rhodamine fluorescence in neutrophils,
monocytes, and, to a lesser extent, lymphocytes (Fig. 6). DHR 123 oxidation was partially
prevented by inhibition of NO synthase with either aminoguanidine or
L-NAME (Fig. 6). Parallel with intracellular DHR 123 oxidation, an
increase in the plasma rhodamine concentration was observed at 4 h
post-LPS, which was prevented by both aminoguanidine and L-NAME (Fig. 6). By contrast, LPS-induced increases in intracellular DHR 123
oxidation were not affected significantly by L-NAME at 1 h
post-LPS. Rhodamine fluorescence in neutrophils increased from 6.2
± 0.4 to 14.8 ± 1.4 and 14.1 ± 1.4 relative fluorescence
units in response to LPS in the absence and the presence of L-NAME,
respectively (n = 4; both p < 0.05
compared with unstimulated); in monocytes from 6.0 ± 1.2 to
20.6 ± 2.1 and 19.6 ± 2.7 relative fluorescence units in
response to LPS and L-NAME plus LPS, respectively (n =
4; both p < 0.05 compared with unstimulated); and in
lymphocytes from 0.9 ± 0.1 to 1.6 ± 0.3 and 1.5 ± 0.2
relative fluorescence units in response to LPS and L-NAME plus LPS,
respectively (n = 4; both p < 0.05
compared with unstimulated).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(32),
platelet-derived growth factor (33), TGF-ß (34), and LPS (35). Our
study identifies ONOO- as a novel signaling mechanism
regulating IL-8 gene expression. Three lines of evidence support a role
for ONOO- in mediating LPS-induced IL-8 release in human
whole blood: 1) enhanced ONOO- formation can be detected
intracellularly in leukocytes as well as in plasma; 2) inhibition of NO
synthesis, and consequently ONOO- formation, prevents IL-8
mRNA expression and IL-8 release; and 3) authentic ONOO-
enhances IL-8 gene expression and release.
We monitored the NO-dependent oxidation of DHR 123 to rhodamine as a
marker of ONOO- formation (25) and detected increases in
rhodamine fluorescence in neutrophils, monocytes, lymphocytes, and
plasma in response to LPS. The enhanced oxidation of DHR 123 at 4
h post-LPS can presumably be attributed to ONOO-, because
it is dependent on an NO-related species, for it can be inhibited by
aminoguanidine or L-NAME, inhibitors of NO synthase (36, 37, 38), whereas
NO by itself does not increase oxidation of DHR 123 (25). It is
conceivable that ONOO- is formed by a reaction of
superoxide produced by activated leukocytes with NO produced by the
inducible form of NO synthase (iNOS), since LPS is a potent inducer of
iNOS expression (39, 40), and it can be inhibited by aminoguanidine, a
selective inhibitor of iNOS (37, 38). Since L-arginine
binding to the catalytic site of constitutive NO synthase (cNOS) is
preferential to aminoguanidine by a factor >1600 (41), a normal plasma
concentration of L-arginine (1 mM) would protect cNOS
against aminoguanidine-induced inactivation. Furthermore, intracellular
DHR 123 oxidation at 1 h post-LPS was not affected significantly
by L-NAME, indicating that activation of cNOS by LPS (which occurs
within minutes after addition of LPS) cannot reasonably account for the
increases in ONOO- formation. In addition, aminoguanidine
may also scavenge an intermediate derived from ONOO- (42).
Our results imply that neutrophils, monocytes, and lymphocytes are
potential sources of ONOO-. The low rhodamine fluorescence
intensity detected in lymphocytes may indicate their low capacity to
produce ONOO- or, alternatively, that ONOO-
produced and released by neutrophils or monocytes diffused into
lymphocytes and resulted in DHR 123 oxidation. ONOO- is
released into the plasma, as evidenced by increases in the plasma
rhodamine concentration. However, the amounts of ONOO-
accumulated in plasma during 4-h incubation with LPS cannot be
precisely calculated, because ONOO- could react with
molecules other than DHR 123 in plasma.
The present study shows that incubation of blood samples with either aminoguanidine or L-NAME selectively inhibited IL-8 release. These observations are consistent with those obtained with L-NAME (43). Although scavenging superoxide with superoxide dismutase could also be expected to reduce ONOO- formation, superoxide dismutase does not affect IL-8 production (16). This may reflect the inability of this charged, high m.w. protein to cross cell membranes (44). The NO donors, SNAP and sodium nitroprusside, did not produce significant increases in IL-8 release, indicating that albeit formation of NO is a prerequisite for induction of IL-8 release, NO by itself is not a potent inducer of IL-8 release in whole blood. However, in the simultaneous presence of superoxide, NO-generating chemicals can stimulate IL-8 production. Previous studies using chemically generated NO or NO gas resulted in conflicting results. The NO donors, SNAP, SIN-1, S-nitrosoglutathione, and 2,2'-(hydroxynitrosohydrazino)bis-ethanamine, increased IL-8 secretion from melanoma cells (20) and ECV304 endothelial cells (21), whereas NO inhalation reduced the bronchoalveolar IL-8 levels in patients with acute respiratory distress syndrome (22). Furthermore, NOS inhibitors had no effect on the respiratory syncytial virus-induced release of IL-8 from epithelial cells (45). These discordant observations might be attributed to differences in the balance between NO and superoxide generation that appears to be a critical determinant in the induction of IL-8 release. Furthermore, the observations with NO donors may not necessarily indicate that NO per se is responsible for IL-8 release. For instance, SIN-1 simultaneously releases both superoxide and NO (28), and even exogenously added NO might react with superoxide produced by mitochondria (46), leading to the formation of ONOO-. Recently, the hydroxyl radical has also been implicated in mediating 2,2'-(hydroxynitrosohydrazino)bis-ethanamine-induced IL-8 release in blood (43), although the mechanism by which this NO donor might generate hydroxyl radical is unknown at present.
Unlike the NO donors, authentic ONOO- produced concentration-dependent IL-8 release, with maximal increases similar to those observed with 1 µg/ml LPS. Furthermore, ONOO- (80 µM) increased IL-8 mRNA expression by 2.4-fold. This action of ONOO- was due to ONOO- per se and was not a result of residual contaminants that are present in the ONOO- stock solution, because neither the decomposed and pH-neutralized ONOO- solution that contains all residual contaminants nor NaOH at 0.15 mM (final concentration in ONOO- dilutions) induced IL-8 gene expression and protein release. Since ONOO- was used in the range of 2.5–80 µM, one should consider its rapid decomposition. Thus, much higher concentrations of exogenous ONOO- may be required to achieve biologic responses similar to those produced by much lower concentrations of continuously produced endogenous ONOO- (24).
Since the half-life of ONOO- at pH 7.4 is on the order of seconds, we cannot conclude at present whether induction of the IL-8 gene expression was a direct effect of ONOO- or one of its more stable decomposition products. These decomposition products are still a subject of debate and appear to depend on the cellular and chemical environment (23, 47, 48, 49). One theory assumes that peroxynitrous acid undergoes homolysis to form hydroxyl radicals (23, 47). Although this would explain the effectiveness of hydroxyl radical scavengers to inhibit LPS-stimulated IL-8 release (16), the selectivity of such scavengers can be questioned. For instance, thiourea and dimethylthiourea have been found to protect against ONOO--mediated damage, indicating their capability of scavenging ONOO- (50). The second hypothesis proposes that a reactive form of peroxynitrous acid, HOONO*, is the proximate oxidant (48, 49). In the present experiments, ONOO- was added to whole blood. The presence of metal ions, amino acids, and proteins in the plasma might have either scavenged ONOO- or influenced its decomposition characteristics, thereby decreasing its apparent potency or concentration. This may explain why much higher concentrations of exogenous ONOO- were required to induce IL-8 release than those detected in plasma. Furthermore, the plasma levels of ONOO- may not correctly reflect ONOO- formation, because significant portions of ONOO- formed by leukocytes might be retained and decomposed intracellularly.
Consistent with previous studies on LPS-induced transcription of IL-8
gene in endothelial cells (51), the present study also documents the
ability of PDTC to abrogate both LPS- and ONOO--induced
IL-8 production in human whole blood to a similar degree. NF-B
activation takes place following phosphorylation of the inhibitory
subunit IB- (52, 53, 54). The mechanism of action of PDTC has not yet
been fully defined, but probably involves the inhibition of formation
of reactive oxygen radicals that would result in the activation of an
IB- kinase or, alternatively, in the activation of a process
involved in phosphorylation of the inhibitor of NF-B (31, 52, 55).
This would suggest that NF-B activation can be attributed to
decomposition products of ONOO-, rather than to the parent
molecule itself. We cannot exclude the possibility that PDTC may also
function as a scavenger of ONOO- or one of the
intermediates derived from ONOO-. The 50–60% inhibition
observed with PDTC suggests a role for NF-B in the induction of IL-8
by ONOO-. In contrast to IL-8, ONOO- did not
induce expression of the TNF- gene, which also features B binding
motifs in its 5'-regulatory region (56). This suggests that NF-B may
be necessary, but not sufficient, for the induction of TNF-. Indeed,
deletion of B binding sites from the human TNF- promoter had
little influence on the induction of the gene by LPS (57). It is not
known at present whether NF-B by itself may be sufficient for the
induction of IL-8. The transcription factors activating protein-1 (51)
and NF-IL-6 (58) have been reported to act synergistically with NF-B
to activate IL-8 transcription. Therefore, it is plausible to assume
that ONOO- might have also activated these or other
transcription factors. Clearly, further studies are required to assess
the multiple promoter regions and factors that may act in an
inducer-specific manner.
In summary, the present study provides evidence that endogenously
produced ONOO- directly induces the transcription and
translation of IL-8 in human whole blood in response to LPS. This
occurs in part through activation of NF-B. The results suggest that
inhibition of the formation and/or actions of ONOO- may
represent a novel therapeutic approach to attenuate IL-8 production
that could lead to a reduction in neutrophil recruitment/activation and
consequently to inhibition of inflammatory responses.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. János G. Filep, Research Center, Maisonneuve-Rosemont Hospital, 5415 boulevard de l’Assomption, Montreal, Quebec, Canada H1T 2 M4. E-mail address:
3 Abbreviations used in this paper: NO, nitric oxide; ONOO-, peroxynitrite; PDTC, pyrrolidine dithiocarbamate; L-NAME, NG-nitro-L-arginine methyl ester; D-NAME, NG-nitro-D-arginine methyl ester; SNAP, S-nitroso-N-acetyl-DL-penicillamine; SIN-1, 3-morpholinosydnonimine hydrochloride; DHR 123, dihydrorhodamine 123; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; iNOS, inducible nitric oxide synthase; cNOS, constitutive nitric oxide synthase.
Received for publication April 16, 1998. Accepted for publication July 13, 1998.
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 T. Khreiss, L. Jozsef, L. A. Potempa, and J. G. Filep Loss of Pentameric Symmetry in C-Reactive Protein Induces Interleukin-8 Secretion Through Peroxynitrite Signaling in Human Neutrophils Circ. Res., September 30, 2005; 97(7): 690 - 697. [Abstract] [Full Text] [PDF]
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 S. Iho, Y. Tanaka, R. Takauji, C. Kobayashi, I. Muramatsu, H. Iwasaki, K. Nakamura, Y. Sasaki, K. Nakao, and T. Takahashi Nicotine induces human neutrophils to produce IL-8 through the generation of peroxynitrite and subsequent activation of NF-{kappa}B J. Leukoc. Biol., November 1, 2003; 74(5): 942 - 951. [Abstract] [Full Text] [PDF]
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C.-W. Chow, M. T. Herrera Abreu, T. Suzuki, and G. P. Downey Oxidative Stress and Acute Lung Injury Am. J. Respir. Cell Mol. Biol., October 1, 2003; 29(4): 427 - 431. [Full Text] [PDF]
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 S. Xiong, H. She, H. Takeuchi, B. Han, J. F. Engelhardt, C. H. Barton, E. Zandi, C. Giulivi, and H. Tsukamoto Signaling Role of Intracellular Iron in NF-kappa B Activation J. Biol. Chem., May 9, 2003; 278(20): 17646 - 17654. [Abstract] [Full Text] [PDF]
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H Sugiura, M Ichinose, S Yamagata, A Koarai, K Shirato, and T Hattori Correlation between change in pulmonary function and suppression of reactive nitrogen species production following steroid treatment in COPD Thorax, April 1, 2003; 58(4): 299 - 305. [Abstract] [Full Text] [PDF]
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 L. Jozsef, C. Zouki, N. A. Petasis, C. N. Serhan, and J. G. Filep Lipoxin A4 and aspirin-triggered 15-epi-lipoxin A4 inhibit peroxynitrite formation, NF-kappa B and AP-1 activation, and IL-8 gene expression in human leukocytes PNAS, October 1, 2002; 99(20): 13266 - 13271. [Abstract] [Full Text] [PDF]
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S. Yang, A. Panoskaltsis-Mortari, M. Shukla, B. R. Blazar, and I. Y. Haddad Exuberant Inflammation in Nicotinamide Adenine Dinucleotide Phosphate-Oxidase-Deficient Mice After Allogeneic Marrow Transplantation J. Immunol., June 1, 2002; 168(11): 5840 - 5847. [Abstract] [Full Text] [PDF]
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 S. Kastenbauer, U. Koedel, B. F. Becker, and H. W. Pfister Oxidative stress in bacterial meningitis in humans Neurology, January 22, 2002; 58(2): 186 - 191. [Abstract] [Full Text] [PDF]
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 E. W. Petersen, K. Ostrowski, T. Ibfelt, M. Richelle, E. Offord, J. Halkjar-Kristensen, and B. K. Pedersen Effect of vitamin supplementation on cytokine response and on muscle damage after strenuous exercise Am J Physiol Cell Physiol, June 1, 2001; 280(6): C1570 - C1575. [Abstract] [Full Text] [PDF]
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C. Zouki, L. József, S. Ouellet, Y. Paquette, and J. G. Filep Peroxynitrite mediates cytokine-induced IL-8 gene expression and production by human leukocytes J. Leukoc. Biol., May 1, 2001; 69(5): 815 - 824. [Abstract] [Full Text]
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R. Pawliczak, M. J. Cowan, X. Huang, U. B. Nanavaty, S. Alsaaty, C. Logun, and J. H. Shelhamer p11 Expression in Human Bronchial Epithelial Cells Is Increased by Nitric Oxide in a cGMP-dependent Pathway Involving Protein Kinase G Activation J. Biol. Chem., November 21, 2001; 276(48): 44613 - 44621. [Abstract] [Full Text] [PDF]
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B. M. Matata and M. Galinanes Peroxynitrite Is an Essential Component of Cytokines Production Mechanism in Human Monocytes through Modulation of Nuclear Factor-kappa B DNA Binding Activity J. Biol. Chem., January 11, 2002; 277(3): 2330 - 2335. [Abstract] [Full Text] [PDF]
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C.-L. M. Cooke and S. T. Davidge Peroxynitrite increases iNOS through NF-kappa B and decreases prostacyclin synthase in endothelial cells Am J Physiol Cell Physiol, February 1, 2002; 282(2): C395 - C402. [Abstract] [Full Text] [PDF]
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