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(Journal of Leukocyte Biology. 2001;69:815-824.)
© 2001 by Society for Leukocyte Biology

Peroxynitrite mediates cytokine-induced IL-8 gene expression and production by human leukocytes

Christine Zouki*, Levente József*, Sophie Ouellet*, Yves Paquette* and János G. Filep*

* Research Center, Maisonneuve-Rosemont Hospital and
{dagger} 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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Recent studies indicate that nitric oxide (NO) or related compounds may regulate the production of interleukin (IL)-8, a potent proinflammatory chemokine. Here we report that peroxynitrite (ONOO-) formed by a reaction of NO with superoxide mediates IL-8 gene expression and IL-8 production in IL-1ß- and TNF-{alpha}-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)-{kappa}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-{alpha}. Enhanced ONOO- formation was detected in granulocytes, monocytes, and lymphocytes after challenge with IL-1ß or TNF-{alpha}. The addition of ONOO- (0.2–80 µM) to whole blood increased nuclear accumulation of AP-1 and NF-{kappa}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-{kappa}B activation, attenuated ~70% of IL-8 release evoked by IL-1ß, TNF-{alpha}, 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-{alpha} • inflammation • nitric oxide


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The chemokine interleukin (IL)-8 has been implicated in a variety of inflammatory diseases, including endotoxin shock, arthritis, and acute respiratory distress syndrome [1 ]. IL-8 is secreted by a variety of cell types, including neutrophils, monocytes-macrophages, and T lymphocytes, in response to a wide range of proinflammatory stimuli [2 3 4 ]. The strongest activators of IL-8 expression in most cells are the cytokines IL-1ß and TNF-{alpha} [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-{kappa}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-{kappa}B dimers consisting of p50 (NF-{kappa}B1), p65 (Rel-A), and/or c-Rel are present in human neutrophils [36 ]. Although NF-{kappa}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-{alpha} 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-{alpha} via activation of AP-1 and NF-{kappa}B.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
Human recombinant IL-1ß and TNF-{alpha} 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-{alpha} (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-{kappa}B and AP-1
Intranuclear, DNA-bound NF-{kappa}B/p50, NF-{kappa}B/p65, and AP-1/c-Fos were measured with a flow-cytometric assay [40 ] and were used as an estimate of NF-{kappa}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-{kappa}B/p50, NF-{kappa}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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Aminoguanidine and L-NAME inhibit IL-1ß- and TNF-{alpha}-induced IL-8 production and IL-8 mRNA expression
Incubation of human whole blood with IL-1ß (50 ng/mL) or TNF-{alpha} (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-{alpha} 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).



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  		Figure 1. Inhibition of NO synthesis prevents IL-8 production in human whole blood challenged with IL-1ß or TNF-{alpha}. 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-{alpha}, 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-{alpha}, respectively).

 
To determine whether the reduction of cytokine-stimulated IL-8 release by NO synthase blockers occurs at the level of transcription or translation, RNase protection assays were performed on RNA extracted from whole blood samples incubated for 4 h. IL-1ß- or TNF-{alpha}-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) .



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  		Figure 2. Aminoguanidine and L-NAME inhibition of IL-1ß- and TNF-{alpha}-stimulated IL-8 mRNA expression. Blood samples were incubated with IL-1ß (50 ng/mL), TNF-{alpha} (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-{alpha}, as indicated.

 
IL-1ß- and TNF-{alpha}-induced nuclear accumulation of AP-1 and NF-{kappa}B
To assess nuclear binding of NF-{kappa}B 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-{kappa}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-{kappa}B/p65 or anti-c-Fos antibodies to nuclei) represent NF-{kappa}B/p65 or c-Fos bound to DNA. These histograms show that IL-1ß mobilized NF-{kappa}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-{alpha} in the absence or presence of L-NAME or aminoguanidine gave qualitatively similar results with respect to the extent of NF-{kappa}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-{kappa}B/p65 and c-Fos in response to these cytokines (data not shown).



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  		Figure 3. IL-1ß-induced nuclear accumulation of NF-{kappa}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-{kappa}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-{kappa}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.

 


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  		Figure 4. NO synthase blocker inhibition of nuclear binding of AP-1/c-Fos and NF-{kappa}B/p65 in PMN and mononuclear leukocytes challenged with IL-1ß or TNF-{alpha}. Blood samples were stimulated with 50 ng/mL of IL-1ß or 100 ng/mL of TNF-{alpha} 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-{kappa}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-{alpha}, respectively).

 
Detection of ONOO- formation in leukocytes challenged with IL-1ß or TNF-{alpha}
Incubation of blood samples with TNF-{alpha} (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-{alpha} (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-{alpha}. 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-{alpha}, 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) .



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  		Figure 5. TNF-{alpha}-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-{alpha} 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-{alpha}).

 
ONOO--induced IL-8 release and IL-8 mRNA expression
Addition of ONOO- to blood samples stimulated IL-8 release in a concentration-dependent manner, with the IL-8 concentrations measured after challenge with 20 µM ONOO- being comparable to those in samples incubated with 50 ng/mL of IL-1ß or 100 ng/mL of TNF-{alpha} (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 ).



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  		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).

 


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  		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).

 
ONOO- induces nuclear accumulation of AP-1 and NF-{kappa}B
Figure 8 shows representative results illustrating the effects of ONOO- on mobilization of NF-{kappa}B and AP-1. Nuclear staining with anti-NF-{kappa}B/p65, anti-NF-{kappa}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-{kappa}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-{kappa}B/p65 and c-Fos (data not shown).



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  		Figure 8. ONOO--induced nuclear accumulation of NF-{kappa}B/p50, NF-{kappa}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-{kappa}B or c-Fos to nuclei from PMN leukocytes was analyzed using anti-NF-{kappa}B/p50, anti-NF-{kappa}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-{kappa}B/p50, NF-{kappa}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.

 


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  		Figure 9. ONOO--induced activation of NF-{kappa}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-{kappa}B/p65 or c-Fos to nuclei from PMN or mononuclear leukocytes was analyzed using anti-human NF-{kappa}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).

 
PDTC suppresses the production of IL-8 in response to IL-1ß, TNF-{alpha}, and ONOO-
Treatment of blood samples with PDTC, a specific inhibitor of NF-{kappa}B activation [41 ], reduced the basal as well as the IL-1ß-, TNF-{alpha}-, and ONOO--induced nuclear accumulation of NF-{kappa}B/p65 to a low level in both PMN and mononuclear cells (Fig. 10A ). The nuclear binding of NF-{kappa}B/p65 could not be completely suppressed by PDTC (Fig. 10A) . PDTC attenuated ~70% of IL-8 production elicited by IL-1ß, TNF-{alpha}, 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).



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  		Figure 10. PDTC suppresses IL-1ß-, TNF-{alpha}-, and ONOO--stimulated nuclear accumulation of NF-{kappa}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-{alpha} (100 ng/mL), or ONOO- (20 µM) for 30 min (nuclear accumulation of NF-{kappa}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-{kappa}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-{alpha}, or ONOO- without PDTC).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we report that ONOO- functions as a novel intracellular signaling mechanism mediating IL-1ß- or TNF-{alpha}-induced IL-8 gene expression in human leukocytes via activation of NF-{kappa}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-{kappa}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-{alpha}; and (3) authentic ONOO- induces nuclear accumulation of AP-1 and NF-{kappa}B, leading to IL-8 gene expression and IL-8 release.

IL-1ß and TNF-{alpha} 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-{alpha}-induced nuclear accumulation of NF-{kappa}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-{alpha}. 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-{alpha}, 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-{alpha} 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-{alpha} 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 >{tau}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-{alpha} challenge was also slightly attenuated by L-NAME, which also inhibits cNOS [50 , 54 ]. Thus, activation of cNOS by IL-1ß or TNF-{alpha} (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-{alpha}, 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-{kappa}B and AP-1 activation in response to IL-1ß and TNF-{alpha} in both PMN and mononuclear cells by using a flow-cytometric assay. This assay has previously been used to analyze NF-{kappa}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-{kappa}B/p50, NF-{kappa}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-{kappa}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-{kappa}B has been proposed to contribute to constitutive expression of transcripts encoding {kappa}B-dependent genes [63 ].

ONOO-, when added to human blood, promoted the nuclear accumulation of NF-{kappa}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-{alpha}, 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-{kappa}B/p50 and NF-{kappa}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-{kappa}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-{kappa}B in human leukocytes are suppressed by PDTC in the microenvironment of human whole blood. However, the nuclear presence of NF-{kappa}B could not be completely suppressed in unchallenged cells, indicating the existence of a mechanism distinct from that inducing NF-{kappa}B translocation after cell stimulation with cytokines. PDTC abrogated IL-1ß-, TNF-{alpha}-, 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-{kappa}B activation takes place after the phosphorylation of the inhibitory subunit I{kappa}B-{alpha} [68 69 70 ]. PDTC is thought to prevent activation of I{kappa}B-{alpha} kinase via inhibition of formation of reactive oxygen radicals or, alternatively, to interfere with the activation of a process involved in the phosphorylation of I{kappa}B-{alpha} [41 , 68 , 71 ]. PDTC appears to be a fairly specific inhibitor of NF-{kappa}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-{alpha} 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-{kappa}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-{kappa}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-{alpha} induce formation of ONOO-, leading to nuclear accumulation of NF-{kappa}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-{alpha} 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.


   ACKNOWLEDGEMENTS
 
This work was supported by a grant from the Medical Research Council of Canada (MT-12573) to J.G.F. C.Z. is in receipt of a Studentship Award from the Medical Research Council of Canada.

C.Z. and L.J. contributed equally to this work.

Received June 19, 2000; revised December 10, 2000; accepted December 12, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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