* Research Center, Maisonneuve-Rosemont Hospital and
Department of Medicine, University of Montréal, Québec, Canada H1T 2M4
Correspondence: Dr. János G. Filep, Research Center, Maisonneuve-Rosemont Hospital, 5415 boulevard de l’Assomption, Montréal, Québec, Canada H1T 2M4. E-mail: filepj{at}ere.umontreal.ca
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-stimulated human
leukocytes in whole blood. The NO synthase inhibitors aminoguanidine
and NG-nitro-L-arginine methyl
ester blocked nuclear accumulation of activator protein-1 (AP-1) and
nuclear factor (NF)-
B in both polymorphonuclear (PMN) and
mononuclear leukocytes and inhibited IL-8 mRNA expression and IL-8
release by 
90% in response to IL-1ß and TNF-. Enhanced
ONOO- formation was detected in granulocytes, monocytes,
and lymphocytes after challenge with IL-1ß or TNF-. The addition
of ONOO- (0.2–80 µM) to whole blood increased nuclear
accumulation of AP-1 and NF-B in PMN and mononuclear leukocytes and
augmented IL-8 mRNA expression and IL-8 production in a
concentration-dependent fashion. Pyrrolidine dithiocarbamate, an
inhibitor of NF-B activation, attenuated 70% of IL-8 release
evoked by IL-1ß, TNF-, or ONOO-. These results
indicate that ONOO- formation may underlie the action of
cytokines towards IL-8 gene expression in human leukocytes.
Key Words: neutrophil granulocytes • mononuclear leukocytes • interleukin-1ß • tumor necrosis factor- • inflammation • nitric oxide
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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[5
, 6
], which are important
mediators of inflammation. IL-8 is a chemoattractant and potent
activator of neutrophils [2
, 7
8
9
10
],
regulates expression of the adhesion molecules L-selectin
and Mac-1 [11
], and promotes neutrophil adherence to the
endothelium and transendothelial migration into tissues
[11
12
13
]. Neutralizing antibodies against IL-8 attenuate
neutrophil accumulation and neutrophil-dependent tissue injury in
various experimental models [14
, 15
],
indicating a pivotal role for IL-8 in neutrophil recruitment. Recent
evidence indicates that IL-8 is also required for firm adhesion of
monocytes to the endothelium [16
]. Oxidative stress is an ubiquitous inducer of IL-8 gene expression. Hydrogen peroxide increases whereas antioxidants inhibit IL-8 expression in epithelial cell lines, fibroblasts, and human blood [17 18 19 ]. Hypoxia followed by reoxygenation induces IL-8 expression in monocytes [20 ] and in the myocardium in vivo [21 ]. Another oxidant, NO, has also been implicated in the regulation of IL-8 production [22 23 24 25 ]. Recently, we have shown that peroxynitrite (ONOO-) formed in the reaction of NO with superoxide [26 ], rather than NO, mediates lipopolysaccharide (LPS)-induced IL-8 gene expression in human leukocytes [27 ]. Thus, it is important to characterize the oxidant(s) involved to gain the insight needed to create new approaches to controlling these events.
IL-8 expression is regulated primarily at the level of gene
transcription [5
, 6
, 28
],
although postranscriptional control has also been reported
[29
, 30
]. Within the IL-8 promoter
sequence, there are binding sites for AP-1, NF-B, and NF-IL-6
[5
, 28
, 31
32
33
34
]. In human
leukocytes, the AP-1 binding complex, composed of Jun-Fos (in
particular c-Fos) dimers [35
] and NF-B dimers
consisting of p50 (NF-B1), p65 (Rel-A), and/or c-Rel are present in
human neutrophils [36
]. Although NF-B appears to be
critical for IL-8 gene expression, cooperation with either AP-1 or
NF-IL-6 is required for optimal IL-8 gene activation in several cell
types [28
, 32
33
34
].
In this study, we examined the impact of IL-1ß and TNF- on
ONOO- formation in human leukocytes in whole blood and
investigated whether ONOO- mediates IL-8 mRNA and protein
expression in response to IL-1ß and TNF- via activation of AP-1
and NF-B.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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were purchased from R&D
Systems (Minneapolis, MN). Aminoguanidine hemisulfate, pyrrolidine
dithiocarbamate (PDTC), and
NG-nitro-L-arginine methyl ester
(L-NAME) were obtained from Sigma (St. Louis, MO).
NG-nitro-D-arginine methyl ester
(D-NAME) was purchased from Research Biochemicals International
(Natick, MA). Dihydrorhodamine 123 (DHR 123) and rhodamine were
obtained from Molecular Probes (Eugene, OR).
Experimental design
Venous blood (10 mL; anticoagulated with sodium heparin, 50
U/mL) was obtained from nonsmoking healthy volunteers (male and female,
24–47 years 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 4,500 and 9,500
cells/µL. Whole blood aliquots (400 µL) were transferred to sterile
microcentrifuge tubes, and aminoguanidine (10 mM), L-NAME (10 mM),
D-NAME (10 mM), PDTC (100 µM), IL-1ß (50 ng/mL), TNF- (100
ng/mL), and phorbol myristate acetate [PMA (500 nM)] 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% fetal bovine serum
(FBS), and stored at -20°C for later cytokine analysis. For samples
used in RNase protection assays, 50 µL of blood were mixed with an
equal volume of lysis/denaturation solution (Ambion, Austin, TX) and
stored at -20°C for later processing. For detection of intracellular
ONOO- formation, DHR 123 (20 µM) was added to the
samples for the last 60 min of incubation. Leukocyte viability was
assessed by flow cytometry after staining with propidium iodide (0.5
µg/mL).
ONOO- and control experiments
ONOO- synthesized by using nitrite and hydrogen
peroxide in a quenched flow reactor [37
] was obtained
from Alexis Corp. (San Diego, CA). A pH-neutralized negative control
solution (Alexis Corp.), which contains the same concentrations of
nitrite, H2O2, and NaCl as the
ONOO- stock solution, and NaOH at 10 µM (the final
concentration of NaOH in 80 µM ONOO- solution) were used
as negative controls. Four-microliter aliquots of one of these
solutions were added to some blood samples, and the effects of these
additions were investigated.
Measurement of intracellular rhodamine fluorescence
NO synthase blocker-inhibitable fluorescence of rhodamine, an
oxidation product of DHR 123, was used as a marker for exposure of DHR
123 to ONOO- [38
, 39
]. At the
end of the incubation period, erythrocytes were lysed, and leukocytes
were prepared for flow cytometry analysis as described previously
[27
, 39
]. Granulocytes, monocytes, and
lymphocytes were gated by their forward- and side-scatter
characteristics, and single-color fluorescence was analyzed with a flow
cytometer (FACScan; Becton Dickinson Immunocytometry Systems, San Jose,
CA) with Lysis II software.
Measurement of IL-8
The plasma concentrations of IL-8 were determined by a highly
selective enzyme-linked immunosorbent assay (BioSource International,
Camarillo, CA). The detection limit of the assay was 10 pg/mL. Intra-,
and interassay coefficients of variation were typically <3% and
<5%, respectively. There was no cross-reactivity with the NO synthase
inhibitors or PDTC in the assays.
IL-8 RNase protection assay
The cDNA templates for the human IL-8 and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probes were prepared
as described previously [27
]. 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]uridine triphosphate ([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 as previously described
[27
]. In brief, 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 cocktail, 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
autoradiographs were scanned with a digital image analysis system
(Alpha Innotech Corp., San Leandro, CA) to quantitate the relative
intensities of the bands corresponding to the IL-8 and GAPDH protected
fragments. For each autoradiograph, the results were normalized to
represent equivalent RNA loading in each lane based on the intensity of
the GAPDH bands.
NF-B and AP-1
Intranuclear, DNA-bound NF-B/p50, NF-B/p65, and AP-1/c-Fos
were measured with a flow-cytometric assay [40
] and were
used as an estimate of NF-B and AP-1 activity in the cell,
respectively. This assay allows simultaneous quantification of
DNA-bound transcription factors in polymorphonuclear (PMN) and
mononuclear leukocyte populations in whole blood. In brief, after
incubation of blood samples with or without stimulus, red blood cells
were lysed, leukocytes were washed, and then leukocyte nuclei were
prepared for immunostaining using the Cycletest Plus DNA reagent kit
(Becton Dickinson) in accordance with the manufacturer’s instructions.
Affinity purified, rabbit polyclonal anti-human NF-B/p50,
NF-B/p65, or c-Fos antibody or normal rabbit immunoglobulin G (IgG)
(to assess nonspecific binding of IgG to leukocyte nuclei) (Santa Cruz
Biotechnology, Santa Cruz, CA) was then added for 10 min at room
temperature, followed by a further 10-min incubation with fluorescein
isothiocyanate (FITC)-conjugated anti-rabbit IgG antibody (Santa Cruz
Biotechnology) and addition of propidium iodide solution for a further
10 min. As a negative control, the p50, p65, and c-Fos polyclonal
antibodies were incubated for 120 min with saturating concentrations of
appropriate blocking peptides (i.e., the peptides used for
immunization, all from Santa Cruz Biotechnology) before addition to
preparations of nuclei. The samples were then analyzed with a FACScan
flow cytometer equipped with an electronic doublet-discrimination
module. The flow cytometer was set up using the DNA Quality Control
Particles kit (Becton Dickinson) and CellFit software. Acquisition was
performed using the Lysis II software and the saved settings obtained
with CellFit. Singlet nuclei were gated by using the
doublet-discrimination module and were visualized on an FSC/SSC dot
plot. FITC fluorescence intensity measured in relative rhodamine
fluorescence units (RFUs) was determined for both PMN and mononuclear
cell nuclei. RFU was expressed as follows: RFU =
RFUexperimental - RFUcontrol, where
RFUexperimental and RFUcontrol are the
fluorescence intensities of nuclei incubated with one of the polyclonal
antibodies and normal rabbit IgG, respectively, followed by staining
with an FITC-labeled anti-rabbit IgG antibody.
Statistical analysis
Results are expressed as means ± SE.
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 values of <0.05 were
considered significant for all tests.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-induced IL-8
production and IL-8 mRNA expression
(100 ng/mL) evoked time-dependent increases in IL-8 production that
were markedly reduced by both L-NAME and aminoguanidine (Fig. 1A
and B). For instance, L-NAME and aminoguanidine at 10
mM suppressed 90 to 97% of IL-8 production evoked by IL-1ß and
TNF- at 24 h (Fig. 1B)
. The inhibitory action was concentration
dependent, with an apparent maximum inhibition achieved at 10 mM, as
assayed at 24 h post-cytokine administration (Fig. 1C)
. By
contrast, aminoguanidine and L-NAME did not inhibit IL-8 production
evoked by PMA (Fig. 1A
and 1B)
. Unlike L-NAME, D-NAME did not affect
cytokine-induced IL-8 release (data not shown). Neither L-NAME nor
aminoguanidine by itself affected IL-8 release (Fig. 1)
or leukocyte
viability (data not shown).
 View larger version (18K): [in a new window] |
Figure 1. Inhibition of NO synthesis prevents IL-8 production in human whole
blood challenged with IL-1ß or TNF-. Blood samples were left
unstimulated as control (columns C in panels A and B) or challenged
with 50 ng/mL of IL-1ß, 100 ng/mL of TNF-, or 500 nM PMA in the
presence of 10 mM aminoguanidine (AG) or L-NAME at 37°C for 4 h
(A) or 24 h (B). (C) Concentration-dependent inhibition of IL-8
production by L-NAME and aminoguanidine was assayed at 24 h
post-cytokine challenge. The plasma was analyzed for IL-8 production by
enzyme-linked immunosorbent assay. Values are means ±
SE of duplicate determinations for four to six experiments
with different donor cell preparations. **, P < 0.01
(compared with control); #, P < 0.05 (compared with
samples incubated with IL-1ß or TNF-, respectively).
|
-stimulated IL-8 mRNA levels were markedly reduced upon
incubation with either L-NAME or aminoguanidine (Fig. 2
). L-NAME or aminoguanidine alone had no detectable effect on IL-8
mRNA expression (Fig. 2) .
 View larger version (46K): [in a new window] |
Figure 2. Aminoguanidine and L-NAME inhibition of IL-1ß- and TNF--stimulated
IL-8 mRNA expression. Blood samples were incubated with IL-1ß (50
ng/mL), TNF- (100 ng/mL), 10 mM aminoguanidine (AG), or 10 mM L-NAME
as indicated for 4 h at 37°C. (A) Representative RNase
protection assay using probes for IL-8 and GAPDH. (B) Densitometric
analysis of autoradiographs of the samples probed for IL-8 and GAPDH.
The IL-8 results were adjusted based on the GAPDH values to represent
equivalent RNA loading in each lane, and the results were then
expressed as a percentage of the band obtained from unstimulated blood
[control (A, lane C; B, columns C)]. The results represent means ± SE of blots from five experiments with different blood
donors. *, P < 0.05 (compared with control); #,
P < 0.05 compared with samples incubated with IL-1ß
or TNF-, as indicated.
|
-induced nuclear accumulation of AP-1 and
NF-BB and AP-1, leukocyte nuclei
were prepared from whole blood, and the resulting PMN and mononuclear
fractions were analyzed by immunostaining. Figure 3
shows a representative gating for PMN and mononuclear cell nuclei
and representative histograms for staining PMN nuclei with
anti-NF-B/p65 or anti-AP-1/c-Fos antibodies after cell activation
with IL-1ß (50 ng/mL) for 30 min. The increases in fluorescence
(increased binding of anti-NF-B/p65 or anti-c-Fos antibodies to
nuclei) represent NF-B/p65 or c-Fos bound to DNA. These histograms
show that IL-1ß mobilized NF-B/p65 and c-Fos to the nucleus and
this mobilization was inhibited by L-NAME. Immunostaining was
completely blocked by preincubation of the antibodies with the
appropriate blocking peptides. Stimulation of leukocytes in whole blood
with either 50 ng/mL of IL-1ß or 100 ng/mL of TNF- in the absence
or presence of L-NAME or aminoguanidine gave qualitatively similar
results with respect to the extent of NF-B/p65 and c-Fos
translocation in PMN and mononuclear cells (Fig. 4
). Preincubation of blood samples with 35 µM cycloheximide had no
detectable effect on nuclear accumulation of NF-B/p65 and c-Fos in
response to these cytokines (data not shown).
 View larger version (19K): [in a new window] |
Figure 3. IL-1ß-induced nuclear accumulation of NF-B/p65 and c-Fos in PMN
leukocytes. Whole blood samples were challenged with 50 ng/mL of
IL-1ß with or without L-NAME (10 mM) for 30 min at 37°C; then after
lysis of red blood cells, leukocyte nuclei were prepared for
immunostaining using the Cycletest Plus DNA reagent kit. Leukocyte
nuclei were first stained with a rabbit polyclonal anti-human c-Fos or
NF-B/p65 antibody followed by staining with an FITC-labeled
anti-rabbit IgG antibody and propidium iodide. (A) Acquisition of
leukocyte nuclei using CellFit software with the doublet-discrimination
module activated. A singlet gate was first set and then was visualized
on an FSC/SSC dot plot. (B) Representative FL1 (FITC) fluorescence
histograms of singlet nuclei of PMN leukocytes after staining with
c-Fos (left panel) or NF-B/p65 (right panel). Also shown is staining
of nuclei prepared from unstimulated blood samples as controls (curves
labeled C) and staining with normal rabbit IgG followed by FITC-labeled
anti-rabbit IgG antibody (curves labeled IgG). As a negative control,
antibodies were preincubated for 2 h with the appropriate blocking
peptide (curves labeled BP) before addition to nuclei prepared from
leukocytes challenged with IL-1ß. These results are representative of
five independent experiments.
|
 View larger version (36K): [in a new window] |
Figure 4. NO synthase blocker inhibition of nuclear binding of AP-1/c-Fos and
NF-B/p65 in PMN and mononuclear leukocytes challenged with IL-1ß
or TNF-. Blood samples were stimulated with 50 ng/mL of IL-1ß or
100 ng/mL of TNF- in the presence of aminoguanidine (AG; 10 mM),
L-NAME (10 mM), or their vehicle (control) for 30 min at 37°C. After
lysis of red blood cells, leukocyte nuclei were prepared for
immunostaining using the Cycletest Plus DNA reagent kit. Leukocyte
nuclei were first stained with a rabbit polyclonal anti-human
NF-B/p65 or c-Fos antibody followed by FITC-labeled anti-rabbit IgG
antibody and propidium iodide. Immunofluorescence of leukocyte nuclei
was analyzed with a flow cytometer using CellFit software with the
doublet-discrimination module activated. The results represent
means ± SE of four to six experiments with different
blood donors. *, P < 0.05 (compared with control); **,
P < 0.01 (compared with control); #, P < 0.05 (compared with samples challenged with IL-1ß or TNF-,
respectively).
|
(100 ng/mL) increased DHR
123 oxidation in neutrophils within 30 min (Fig. 5A
). The highest level of rhodamine was detected at 4 h after
TNF- (Fig. 5A) ; thereafter, the amount of DHR 123 oxidized per 60
min decreased. The increased rhodamine fluorescence was markedly
attenuated by L-NAME or aminoguanidine with an exception at 30 min,
when the inhibition was not statistically significant (Fig 5A)
. For
instance, aminoguanidine and L-NAME reduced DHR 123 oxidation on
average by 66 and 63%, respectively, at 4 h after TNF-. L-NAME
and aminoguanidine appeared to be equally potent inhibitors at all time
points studied. Similar trends were observed with monocytes and
lymphocytes (Fig. 5A)
. As with TNF-, IL-1ß-induced increases in
intracellular DHR 123 oxidation were partially prevented by
aminoguanidine and L-NAME (data not shown). By contrast, neither L-NAME
nor aminoguanidine inhibited PMA-induced DHR 123 oxidation (Fig. 5B)
.
 View larger version (28K): [in a new window] |
Figure 5. TNF--induced oxidation of DHR 123 to rhodamine in human leukocytes.
(A) Blood samples were left unstimulated (control) or incubated with
100 ng/mL of TNF- for the indicated times at 37°C in the presence
of aminoguanidine (AG; 10 mM) or L-NAME (10 mM), and DHR 123 (20 µM)
was added during the last 60 min of the incubation period. (B) Blood
samples were challenged with PMA (500 nM) for 3 h at 37°C in the
presence of aminoguanidine (AG) or L-NAME and then DHR 123 (20 µM)
was added for an additional 60 min. Intracellular rhodamine
fluorescence is expressed as RFUs. Values are means ±
SE (n = 4–5). *, P < 0.05
(compared with control); #, P < 0.05 (compared with
TNF-).
|
(Fig. 6
). ONOO- (0.2 to 80 µM) did not affect leukocyte
viability (data not shown). IL-8 concentrations in samples incubated
with pH-neutralized (decomposed) ONOO- solution were
similar to those in control (unstimulated) samples (Fig. 6)
. RNase
protection assays performed on RNA extracted from whole blood samples
challenged for 4 h with an initial concentration of 0.2 to 80
µM ONOO- revealed concentration-dependent
increases in IL-8 mRNA expression, whereas pH-neutralized decomposed
ONOO- (initial ONOO- concentration of 20
µM) failed to induce IL-8 mRNA expression (Fig. 7
).
 View larger version (21K): [in a new window] |
Figure 6. ONOO- stimulates IL-8 production. Whole blood samples were
left unstimulated (column C, control) or incubated with
ONOO- or decomposed ONOO- (dP) for 4 h
at 37°C. The plasma was analyzed for IL-8 production by enzyme-linked
immunosorbent assay. Values are means ± SE for four
experiments with samples from different blood donors. *,
P < 0.05 (compared with control).
|
 View larger version (40K): [in a new window] |
Figure 7. ONOO- induces IL-8 mRNA expression. Whole blood samples
were left unstimulated or incubated with ONOO- or
decomposed ONOO- (dP) for 4 h at 37°C. (A)
Representative RNase protection assay using probes for IL-8 and GAPDH.
(B) Densitometric analysis of autoradiographs of the samples probed for
IL-8 and GAPDH. The IL-8 results were adjusted based on the GAPDH
values to represent equivalent RNA loading in each lane, and the
results were then expressed as a percentage of the band obtained from
unstimulated blood. The results represent means ± SE
of blots from four experiments with different blood donors. *,
P < 0.05 (compared with control).
|
BB and AP-1. Nuclear
staining with anti-NF-B/p65, anti-NF-B/p50, and anti-c-Fos
antibodies was completely prevented by preincubation of the antibodies
with the appropriate blocking peptide (Fig. 8)
. The effect of
ONOO- on the nuclear translocation of NF-B/p65 and
c-Fos proteins was rapid, as nearly maximum changes were detectable
within 10 min in both PMN and mononuclear cells (Fig. 9
). The maximum changes were observed at 30 min, and then staining
with the antibodies gradually decreased (Fig. 9)
. The actions of
ONOO- were concentration dependent (Fig. 9)
. Preincubation
of whole blood samples with 35 µM cycloheximide did not affect the
nuclear accumulation of NF-B/p65 and c-Fos (data not shown).
 View larger version (16K): [in a new window] |
Figure 8. ONOO--induced nuclear accumulation of NF-B/p50,
NF-B/p65, and c-Fos in PMN leukocytes. Whole blood samples were
challenged with 20 µM ONOO- for 30 min at 37°C, and
then leukocyte nuclei were prepared and binding of NF-B or c-Fos to
nuclei from PMN leukocytes was analyzed using anti-NF-B/p50,
anti-NF-B/p65, or anti-c-Fos antibodies with flow cytometry. Also
shown is staining of nuclei prepared from unstimulated leukocytes
(control; curves C) and staining with normal rabbit IgG followed by
FITC-labeled anti-rabbit IgG antibody (curves IgG). As a negative
control, the NF-B/p50, NF-B/p65, and c-Fos antibodies were
preincubated with the appropriate blocking peptides (curves BP) before
addition to nuclei prepared from ONOO--stimulated
leukocytes. These results are representative of five experiments with
samples from different blood donors.
|
 View larger version (24K): [in a new window] |
Figure 9. ONOO--induced activation of NF-B/p65 and AP-1/c-Fos.
(A) Whole blood aliquots were left unstimulated (control; curves C) or
incubated with 20 µM ONOO- or decomposed
ONOO- for the indicated times at 37°C, and then
leukocyte nuclei were prepared and binding of NF-B/p65 or c-Fos to
nuclei from PMN or mononuclear leukocytes was analyzed using anti-human
NF-B/p65 or c-Fos antibodies with flow cytometry. Values represent
the means ± SE for 3–5 experiments with samples from
different blood donors. (B) Concentration-dependent effects of
ONOO-. Blood samples were left unstimulated or incubated
with various concentrations of ONOO- for 30 min at 37°C.
Values are means ± SE (n = 3–4). *,
P < 0.05 (compared with control).
|
, and ONOO-B activation [41
], reduced the basal as well as
the IL-1ß-, TNF--, and ONOO--induced nuclear
accumulation of NF-B/p65 to a low level in both PMN and mononuclear
cells (Fig. 10A
). The nuclear binding of NF-B/p65 could not be completely
suppressed by PDTC (Fig. 10A)
. PDTC attenuated 70% of IL-8
production elicited by IL-1ß, TNF-, or ONOO- (Fig. 10B)
. Likewise, PDTC also attenuated PMA-induced IL-8 production from
5.1 ± 0.6 pg/mL to 0.7 ± 0.1 pg/mL (n = 5;
P < 0.01).
 View larger version (13K): [in a new window] |
Figure 10. PDTC suppresses IL-1ß-, TNF--, and ONOO--stimulated
nuclear accumulation of NF-B/p65 and IL-8 production. Blood samples
were pretreated or not with PDTC (100 µM) for 30 min at 37°C, left
unchallenged (control columns C), or challenged with IL-1ß (50
ng/mL), TNF- (100 ng/mL), or ONOO- (20 µM) for 30 min
(nuclear accumulation of NF-B) or 4 h (IL-8 production). (A)
After lysis of red blood cells, leukocyte nuclei were prepared using
the Cycletest Plus DNA reagent kit and were first stained with a rabbit
polyclonal anti-human NF-B/p65 antibody followed by FITC-labeled
anti-rabbit IgG antibody and propidium iodide. Immunofluorescence of
leukocyte nuclei (expressed as RFU) was analyzed with a flow cytometer
using CellFit software with the doublet-discrimination module
activated. (B) Plasma levels of IL-8 were determined by enzyme-linked
immunosorbent assay. Unstimulated blood plasma contained 0.32 ±
0.04 ng/mL of IL-8 (n = 5). Values are means ±
SE (n = 5). *, P < 0.05
(compared with IL-1ß, TNF-, or ONOO- without PDTC).
|
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-induced IL-8 gene expression in human leukocytes via activation
of NF-B and AP-1. Our study provides three lines of evidence to
support this notion: (1) inhibition of NO synthesis and consequently
formation of ONOO- prevents cytokine-induced nuclear
accumulation of NF-B and AP-1 and expression of IL-8 mRNA and
protein; (2) enhanced ONOO- formation was detected in
leukocytes in response to IL-1ß and TNF-; and (3) authentic
ONOO- induces nuclear accumulation of AP-1 and NF-B,
leading to IL-8 gene expression and IL-8 release.
IL-1ß and TNF- are the two most potent stimuli for IL-8 expression
in most cell types [5
, 6
], including human
leukocytes [42
]. A novel finding of this study is that
incubation of blood samples with either aminoguanidine or L-NAME almost
completely inhibited IL-1ß- and TNF--induced nuclear accumulation
of NF-B and AP-1, coinciding with inhibition of IL-8 mRNA expression
and IL-8 release. These results pointed to the involvement of NO in
mediating the actions of IL-1ß- and TNF-. However, although
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 human whole blood
[27
, 43
]. Indeed, while the NO donors
S-nitroso-N-acetyl-DL-penicillamine
and sodium nitroprusside alone do not produce significant increases in
IL-8 release in human whole blood [27
, 43
],
in the simultaneous presence of a superoxide-generating system, they do
stimulate IL-8 production [27
]. Similarly,
3-morpholinosydnonimine, which releases NO and superoxide
[37
], also induces marked increases in IL-8 production
[27
, 43
]. Confirming and extending our
previous findings [27
], here we have demonstrated that
ONOO- induced IL-8 mRNA expression and IL-8 production in
a concentration-dependent fashion. These actions were caused by
ONOO- per se and were not a result of residual
contaminants that were present in the ONOO- stock
solution, because neither the decomposed and pH-neutralized
ONOO- solution, which contains all residual contaminants,
nor NaOH at 10 µM (final concentration in ONOO-
dilutions) induced IL-8 mRNA expression and protein release. It should
be noted that conflicting results have been reported on IL-8 release in
response to NO or NO synthase inhibitors (iNOS). NO donors have been
found to increase IL-8 secretion from melanoma cells
[23
], ECV304 endothelial cells [24
,
44
], and keratinocytes [45
]. By contrast,
NO inhalation reduces bronchoalveolar fluid IL-8 levels in patients
with acute respiratory distress syndrome [25
].
Furthermore, inhibition of NO synthesis with
NG-monomethyl-L-arginine potentiates
IL-1ß- or LPS-stimulated IL-8 production in human chondrocytes
[46
] and enhances IL-8 release from EA.hy926 human
endothelial cells [47
], whereas inhibition of NO
synthesis has no effect on the respiratory syncytial virus-induced
release of IL-8 from epithelial cells [48
]. These
discordant observations might be explained by differences in the
balance between NO and superoxide generation that appears to be a
critical determinant in the induction of IL-8 release. Since different
cell types may produce NO at different rates and may use different
pathways to activate transcription factors, the NO modulation of
cytokine expression may differ. In addition, the observations with NO
donors may not necessarily indicate that NO per se is responsible for
IL-8 release, because exogenously added NO might react with superoxide
produced by mitochondria [49
] to form
ONOO-. PMA did not generate ONOO-, as
evidenced by the lack of effect of L-NAME and aminoguanidine on
PMA-induced DHR 123 oxidation. In a consistent finding, PMA-induced
IL-8 production was not inhibited by L-NAME or aminoguanidine,
indicating a selective inhibitory action of the NO synthase blockers
used.
To ascertain that ONOO- was formed in response to IL-1ß
and TNF-, we have utilized the NO-dependent oxidation of DHR 123 to
rhodamine to assess intracellular ONOO- formation
[27
, 39
]. This method is specific, because,
although ONOO- oxidizes DHR 123, neither NO nor superoxide
causes DHR 123 oxidation [38
]. A significant portion of
the increases in rhodamine fluorescence in neutrophils, monocytes, and
lymphocytes challenged with IL-1ß or TNF- can be attributed to
ONOO-, because it depends on an NO-related species, and it
can be inhibited by aminoguanidine or L-NAME, inhibitors of NO synthase
[50
, 51
]. A reaction of superoxide with NO
produced by constitutive NO synthase (cNOS) or iNOS could lead to
ONOO- formation [52
]. However, it is
conceivable that ONOO- was predominantly formed by a
reaction of superoxide with NO produced by iNOS, because both IL-1ß
and TNF- are potent inducers of expression of this enzyme
[53
, 54
] and stimulated human neutrophils
and monocytes express iNOS [53
, 55
].
Furthermore, ONOO- formation can be inhibited by
aminoguanidine, which is thought to be a selective inhibitor of iNOS
[51
]. Since L-arginine binding to the
catalytic site of cNOS is preferential to aminoguanidine by a factor of
>
1,600 [56
], a normal plasma concentration of
L-arginine (1 mM) would protect cNOS against
aminoguanidine-induced inactivation. Intracellular DHR 123 oxidation at
30 min post-IL-1ß or TNF- challenge was also slightly attenuated
by L-NAME, which also inhibits cNOS [50
,
54
]. Thus, activation of cNOS by IL-1ß or TNF-
(which occurs within minutes after addition of these cytokines) might
account for the early increases in ONOO- formation.
Although the selectivity of aminoguanidine towards iNOS has been
questioned [57
], the slight inhibition of DHR 123
oxidation by aminoguanidine observed at 30 min might instead be
attributed to its ability to scavenge low amounts of ONOO-
or an intermediate derived from ONOO- [58
].
Our results imply that neutrophils, monocytes, and, to a lesser extent,
lymphocytes are potential sources of ONOO-.
Although ONOO- at 20 µM increased IL-8 mRNA expression
and IL-8 release to levels similar to those observed with 50 ng/mL of
IL-1ß or 100 ng/mL of TNF-, this concentration is much higher than
those detected in human plasma in vitro [27
]. This
result is not surprising, because the half-life of ONOO-
at pH 7.4 is on the order of seconds. Beckman et al.
[37
] suggest that the concentration of exogenous
ONOO- and the exposure time are critical determinants for
mimicking biological responses to endogenous ONOO-.
Accordingly, much higher concentrations of exogenous ONOO-
may be required to achieve biological responses similar to those
produced by much lower concentrations of continuously produced
endogenous ONOO-. It is uncertain whether induction of
IL-8 gene expression can be attributed to a direct effect of
ONOO- or of one of its more stable decomposition products.
Peroxynitrous acid has been suggested to undergo homolysis to form
hydroxyl radicals [26
]; other studies did not detect
hydroxyl radical formation after addition of ONOO-
[59
, 60
]. Indeed, a reactive form of
peroxynitrous acid, HOONO*, has been suggested to be the
proximate oxidant [59
, 60
].
Consistent with previous reports [5
, 36
],
we detected NF-B and AP-1 activation in response to IL-1ß and
TNF- in both PMN and mononuclear cells by using a flow-cytometric
assay. This assay has previously been used to analyze NF-B
activation in unseparated human monocytes and PMN cells
[40
] and in isolated epithelial cells
[61
]. While this assay allows simultaneous detection of
nucleus-bound transcription factors in both mononuclear and PMN
leukocytes, nuclei from monocytes and lymphocytes cannot be analyzed
separately. Therefore, the quantities of transcription factors present
in neutrophils and monocytes cannot be compared.
Despite their predominantly cytoplasmic localization in resting cells,
detectable amounts of NF-B/p50, NF-B/p65, and c-Fos DNA-binding
activities were consistently observed in the nuclei of unstimulated PMN
and mononuclear cells, and this was not attributable to cytosolic
contamination of the nuclei. NF-B/p50 DNA-binding activities
previously have been detected in nuclear extracts of unstimulated human
neutrophils [36
], of peripheral blood monocytes
[62
, 63
], and of various monocytic cell
lines [64
]. Although the significance of these
observations is unclear, constitutive nuclear NF-B has been proposed
to contribute to constitutive expression of transcripts encoding
B-dependent genes [63
].
ONOO-, when added to human blood, promoted the nuclear
accumulation of NF-B and AP-1 in PMN and mononuclear leukocytes.
Nearly maximum effects were already evident by 10 min after
ONOO- addition. This time course of action was similar to
that reported for LPS, TNF-, or IL-1ß in isolated human
neutrophils, but it differed from the time courses of
formyl-Met-Leu-Phe or PMA action, which require at least 30 min to
exert a similar action [36
]. The similar changes in
nuclear staining with NF-B/p50 and NF-B/p65 in response to
ONOO- suggested that ONOO- promotes nuclear
translocation of a p50/p65 heterodimer. These actions of
ONOO- resembled those of ozone in epithelial cells
[65
] and LPS in astrocytoma cells [66
]
but differed from those of H2O2, which
activates AP-1 but does not induce nuclear accumulation of NF-B in
endothelial and epithelial cell lines [17
]. Therefore,
it is possible that oxidant stress can differentially induce
transcription factors in a stimulus-specific and probably a cell
type-specific manner.
Our results showed that cytokine- and ONOO--induced
nuclear accumulations of NF-B in human leukocytes are suppressed by
PDTC in the microenvironment of human whole blood. However, the nuclear
presence of NF-B could not be completely suppressed in unchallenged
cells, indicating the existence of a mechanism distinct from that
inducing NF-B translocation after cell stimulation with cytokines.
PDTC abrogated IL-1ß-, TNF--, and ONOO--induced IL-8
production to a similar degree. Likewise, PDTC also attenuated
PMA-induced IL-8 production. Previous studies showed that PDTC
effectively attenuates LPS-induced transcription of the IL-8 gene in
endothelial cells [67
] and in human leukocytes
[27
]. NF-B activation takes place after the
phosphorylation of the inhibitory subunit IB-
[68
69
70
]. PDTC is thought to prevent activation of
IB- kinase via inhibition of formation of reactive oxygen
radicals or, alternatively, to interfere with the activation of a
process involved in the phosphorylation of IB- [41
,
68
, 71
]. PDTC appears to be a fairly
specific inhibitor of NF-B activation, for it has no significant
influence on the binding activity of AP-1, Oct-1, and proteins binding
to a cyclic-AMP response element and the GC-rich binding motif of sp1
[41
]. These findings coupled with the observations that
PDTC neither affects TNF- binding to its receptor nor impairs the
activity of protein kinase C [41
] argue against a
generalized PDTC effect on signal transduction and receptors. It is not
known at present whether PDTC functions as a scavenger of
ONOO-. The inhibition observed with PDTC did not exceed
75%, indicating that transcription factors other than NF-B are
required for mediating the optimal response to ONOO-.
Since ONOO- increased nuclear binding of AP-1 in PMN and
mononuclear cells, it is tempting to speculate that AP-1 might act in
concert with NF-B to induce IL-8 production. However, it remains to
be investigated whether ONOO- could activate other
transcription factors.
In summary, our study demonstrated that IL-1ß and TNF- induce
formation of ONOO-, leading to nuclear accumulation of
NF-B and AP-1 and subsequent induction of IL-8 mRNA and protein
expression in human leukocytes in whole blood. To our knowledge, this
constitutes the first report that ONOO- formation may
underlie the action of the proinflammatory cytokines IL-1ß and
TNF- towards human leukocyte IL-8 gene expression, and, as such,
this possibility adds a new facet to our understanding of leukocyte
biology in inflammation. These observations raise the possibility that
inhibition of the formation and/or actions of ONOO- may
represent a novel therapeutic approach to attenuation of
cytokine-induced IL-8 production and consequently
recruitment/activation of leukocytes at sites of inflammation.
C.Z. and L.J. contributed equally to this work.
Received June 19, 2000; revised December 10, 2000; accepted December 12, 2000.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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