Peroxynitrite is an essential component of cytokines production mechanism in human monocytes through modulation of nuclear factor-kappa B DNA binding activity.

The mechanism of release of proinflammatory cytokines by peripheral blood monocytes is unknown. Peroxynitrite (ONOO(-)) formed by the reaction of nitric oxide (NO) and superoxide is released predominantly by inflammatory cells at the site of injury in several inflammatory pathologies. Here we show that human monocytes treated with ONOO(-) at micromolar concentrations induce a dose-dependent release of proinflammatory cytokines. These effects were not antagonized by up to 100 microm epigallocatechin gallate, an inhibitor of protein nitration. However, the proteasome inhibitor Z-Ile-Glu(OtBu)-Ala-Leu-CHO and 5,10,15,20-tetrakis(4-sulfonatophenyl)prophyrinato iron (III) chloride, a cell-permeable scavenger of ONOO(-), almost completely inhibited the release of cytokines and the nuclear translocation of the nuclear factor (NF)-kappaB transcription factor. SDS-PAGE electrophoresis separation with Western blotting of cell extracts also indicated that phosphorylation and nitration of tyrosine residues in IkappaB-alpha molecules correlated with NF-kappaB translocation and cytokine release. In addition, the DNA binding activity of the NF-kappaB from the nuclear extracts also correlated with its nuclear translocation. These findings indicate ONOO(-) plays an essential role in the mechanism of proinflammatory cytokine release by monocytes and that Rel/NF-kappaB activation is the obligatory pathway.

Peroxynitrite (ONOO Ϫ ) formed by the reaction of nitric oxide (NO), and superoxide is released predominantly by inflammatory cells at the site of injury in several inflammatory pathologies (1)(2)(3). ONOO Ϫ is highly reactive toward all classes of biomolecules, including proteins, lipids, and nucleic acids and is a potential candidate for effecting the production of proinflammatory cytokines. In proteins, ONOO Ϫ modifies residues such as cysteine, methionine, tryptophan, and tyrosine (4). The irreversible modification of protein residues by ONOO Ϫ , such as nitration of critical tyrosine amino acids exhibits functional consequences like inactivation of a wide variety of enzymes (5)(6)(7) and may also affect structural proteins (8,9). It has also been reported that tyrosine nitration may interfere with signal transduction pathways involving tyrosine phosphorylation (10). The formation of 3,3Ј-dityrosine is known to result in inter-or intramolecular cross-linking of proteins such as membrane receptors consequently affecting the cell signaling pathways (11). Accumulating evidence suggests that oxidants may play a role as signaling intermediates required for receptor tyrosine kinase function and downstream activation of mitogen-activated protein kinases (12). The aim of the present studies was to examine the role of ONOO Ϫ in the regulation of cytokines production by human monocytes and to investigate the underlying mechanism.

EXPERIMENTAL PROCEDURES
Cell Preparations-Peripheral blood monocytes were isolated from whole blood taken from healthy donors by a density-gradient centrifugation method (Histopaque-1017® and 1119® mediums, Sigma, Poole, UK). Briefly, into 15-ml conical Falcon centrifuge tubes (Falcon, Roskilde, Denmark) 3 ml of Histopaque-1017® were layered on top of 3 ml of Histopaque-1119® medium. 6 ml of whole blood carefully layered onto the upper gradient of the tubes were then centrifuged at 700 ϫ g for 30 min at room temperature (18 -22°C). Distinct opaque layers of mononuclear cells were recovered from the top band and twice washed in Tris-buffered saline, pH 7.4. The cells were suspended in serum-free Hanks' balanced salt medium without phenol red, supplemented with Ham's nutrients F-12 and bovine serum albumin (0.25% w/v) at pH 7.4. 1 ml of cell suspension was added into 1.5-ml plastic tubes and incubated at 37°C for 90 min. The culture medium was then removed from tubes leaving monocytes adhering to the plastic surface. The monocytes were further washed with phosphate-buffered saline to remove any non-adherent cells. Fresh 1 ml of medium was then added at the start of experiments.
Cell Treatments-Monocytes (1 ϫ 10 (6)) were treated with authentic ONOO Ϫ (CN Biosciences, Nottingham, UK) suspended in 4.7% NaOH at pH 12.0, in bolus doses. After 6 h of incubation at 37°C in an air, 5% CO 2 incubator, the effect of various concentrations of ONOO Ϫ on proinflammatory cytokine release was determined in the cell lysates. Cell lysates were prepared after rapid homogenization in the medium by using an Ultra-Turrax T25 homogenizer, with a dispersing tool (IKA Laboratories, Staufen, Germany) at 20,000 rpm. The crude lysates were then centrifuged for 5 min at 1000 ϫ g, and the supernatants were transferred into precooled tubes, snap-frozen in liquid nitrogen, and stored at Ϫ80°C until analysis. A control aliquot treated with decomposed ONOO Ϫ (suspended in phosphate-buffered saline at pH 7.4) was also included. To study the effect of tyrosine nitration on proinflammatory cytokine release, cells were treated with the nitration inhibitor EGCG, 1 at 0, 1, 10, and 100 M, with or without 10 M ONOO Ϫ for 6 h. Furthermore, the distinct role of ONOO Ϫ on cytokine release, was studied in monocytes by treatment with a ONOO Ϫ scavenger, 5,10,15,20-tetrakis(4-sulfonatophenyl)prophyrinato iron (III) chloride (FeTPPS) at 200 M in the presence and absence of EGCG (10 M).
* This work was supported in part by The Royal College of Surgeons of England and Wales and a personal donation from Professor M. Galiñ anes. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Transcriptional regulation of proinflammatory cytokine release was investigated by treating the cells with 10 M ONOO Ϫ in the presence and absence of proteasome inhibitor, Z-Ile-Glu(OtBu)-Ala-Leu-CHO, a known inhibitor of NF-B activity. In addition, the role of ONOO Ϫ , FeTPPS, and EGCG in the induction of NF-B activity in monocytes nuclear extracts was investigated.
Measurement of Cytokines-TNF-␣, IL-8, and IL-6 levels in cell lysates were determined by standard ELISA assays (Becton Dickinson, San Diego, CA).
Determination of Tyrosine Nitration-Nitration of protein tyrosine residues was determined by a highly sensitive competitive ELISA assay described previously (13) using rabbit anti-nitrotyrosine antibodies (Upstate Biotechnology, Lake Placid, NY).
Preparation of Cell Extracts-Nuclear and cytoplasmic extracts were prepared using a modification of a previously published method (14). Briefly, after experiments monocytes were washed in ice-cold phosphate-buffered saline and then 100 l of Dignam buffer A, composed of 10 mM HEPES pH 7.9, 1.5 mM MgCl 2 , 10 mM KCl, 0.5 mM dithiothreitol, 0.2% Nonidet P-40, 100 l of inhibitor mixture (Sigma)/ml of buffer A, was added, and the tubes were vortexed vigorously on the highest setting for 15 s. The tubes were then incubated on ice for 10 min. 11 l of 10% Nonidet P-40 solution was added to each tube followed by vortexing for 5 s on the highest setting and incubated on ice for 1 min. The tubes were then centrifuged at 16,000 ϫ g in a microcentrifuge for 5 min. The supernatant fractions were collected into clean prechilled tubes and stored on ice. The insoluble fractions (pellet), which contain nuclei, were then resuspended in 50 l of ice-cold buffer B (20 mM HEPES, pH 7.9, 25% glycerol, 0.42 mM NaCl, 1.5 mM MgCl 2, 0.5 mM dithiothreitol, 0.2 mM EDTA, and 100 l of inhibitor mixture/ml of buffer B), vortexed vigorously for 15 s, and incubated on ice for a total of 40 min with mixing at every 10 min. The tubes were then centrifuged at 16,000 ϫ g in a microcentrifuge, for 10 min. The supernatant (nuclear extract) fraction was immediately transferred into clean prechilled tubes and placed on ice. 5 l of all the extract fractions were used for protein quantification by a standard Bradford assay (15), and the rest was stored at Ϫ80°C until use.
SDS-PAGE and Western Blotting-Nuclear and cytoplasmic extracts, respectively, were separated for NF-B p65/Rel A and IB-␣ by electrophoresis on a 10% SDS-polyacrylamide gel. The presence of NF-B p65/Rel A in nuclear extracts was determined by immunoblotting using rabbit polyclonal NF-B p65 (Rel A) antiserum. IB-␣ was determined in the corresponding cytoplasmic extracts after immunoblotting with a rabbit polyclonal antiserum or a monoclonal anti-phospho-IB-␣ that recognizes phosphorylated IB-␣ at amino acid residues serine 32 and serine 36 only. All the antibodies used in this part of the study were obtained from CN Biosciences, Nottingham, UK. The immunoblot signal was visualized through enhanced chemiluminescence and scanned with an Epson Expression 1600 Pro. The relative intensities of bands were determined using an NIH Image 1.62 package.
Electrophoretic Mobility Shift Assay (EMSA)-The assay is based on that DNA-protein complexes migrate slower than unbound DNA or double-stranded oligonucleotides in a native polyacrylamide or agarose gel, resulting in a "shift" in migration of the labeled DNA band. The detection of bands was by "The LightShift TM Chemiluminescent EMSA kit" (Pierce) that uses a non-isotopic method to detect DNA-protein interactions. Biotin end-labeled DNA duplex of sequence 5Ј-AGT TGA GGG GAC TTT CCC AGG C-3 and 3Ј-TCA ACT CCC CTG AAA GGG TCC G-5Ј containing a putative binding site for nuclear factor B was incubated with the nuclear extracts. After the reaction the DNA-protein complexes were subjected to a 6% native polyacrylamide gel electrophoresis and transferred to a nylon membrane (Biodyne B membrane also supplied by Pierce). As gel shift controls, lane 1 contained Epstein-Barr virus nuclear extracts (EBNA) and biotin-labeled control oligonucleotide duplex with a binding site of: 5Ј-..TAGCATATGCTA..3Ј and 3Ј-..ATCGTATACGAT-5Ј. Lane 2 contained EBNA extract, biotin-labeled control oligonucleotide and unlabeled control oligonucleotide for competitive binding inhibition. After transfer the membrane was immediately cross-linked for 15 min on a UV transilluminator equiped with 312 nm bulbs. A chemiluminescent detection method utilizing a luminol/enhancer solution and a stable peroxide solution (Pierce) was used as described by the manufacturer, and membranes were exposed to x-ray films for 2-5 min before developing. The bands were scanned with an Epson Expression 1600 Pro, and relative intensities were analyzed by using an NIH Image 1.62 package Cytotoxicity Assay-Cytotoxicity was measured by the MTT cell viability assay (Sigma) according to the manufacturer's protocol.
Statistical Analysis-The results were expressed as the mean Ϯ S.E. Statistical comparisons were made by a one-way analysis of variance analysis, and values of p Ͻ 0.05 were considered statistically significant.

Dose-dependent Effect of ONOO
Ϫ on Cytokine Production-ONOO Ϫ caused a bell-shaped dose-response curve on the production of TNF-␣ and IL-6 by monocytes (Fig. 1, A and B). Peak values were observed at ONOO Ϫ concentrations of 2-20 M followed by a reduction at higher concentrations. The reduction in cytokine production at 200 M ONOO Ϫ was not due to the loss of cell viability, since MTT values, an index of viability, were unaffected (data not shown). These results suggest that ONOO Ϫ is an important regulator of cytokine production by monocytes.
Role of Protein Tyrosine Nitration on Cytokine Production-The pattern of 3-nitrotyrosine formation resulting from treatment of monocytes with a range of ONOO Ϫ concentrations suggests that significant increase in protein nitration occur at 200 M ONOO Ϫ (Fig. 2A). To study the effect of tyrosine nitration on cytokine response, monocytes were treated with EGCG, an inhibitor of nitration, in the presence and absence of 10 M ONOO Ϫ . It was observed that a significant reduction in nitration of tyrosine residues occurs when mononuclear cells are treated with EGCG at 10 -100 M in the presence and absence of ONOO Ϫ (Fig. 2B). Fig. 3, A and B, show that ONOO Ϫ (10 M) and EGCG (10 M) increased TNF-␣ and IL-6 production and that the combination of the two did not have additive effect.
To further elucidate the molecular mechanisms for ONOO Ϫinduced cytokine release, aliquots of cells were treated with 200 M FeTPPS, a ONOO Ϫ scavenger, with 10 M EGCG and with the combination of FeTPPS and EGCG (Fig. 4, A and B). As shown before, treating the cells with EGCG significantly increased the release of TNF-␣ and IL-6; however, FeTPPS alone and in combination with EGCG almost completely abolished the release of cytokines.

FIG. 1. The effect of various ONOO ؊ concentrations on TNF-␣ (A) and IL-6 (B) production by peripheral blood monocytes.
Monocytes were treated with different concentrations of ONOO Ϫ for 6 h at 37°C in air, 5% CO 2 incubator. The graphs show a dose-dependent relationship between ONOO Ϫ and cytokine production. TNF-␣ and IL-6 levels were determined by standard ELISA methods. Values are expressed as means Ϯ S.E. of 12 experiments. *, p Ͻ 0.05 versus the group without ONOO Ϫ .
Transcriptional Modulation of Cytokine Production by ONOO Ϫ -To determine whether ONOO Ϫ modulates proinflammatory cytokines at the transcriptional level, mononuclear cells were stimulated with 0, 10, and 200 M ONOO Ϫ concentrations in the presence and absence of proteasome inhibitor (20 M). The proteasome inhibitor blocks the ubiquitin-proteasome pathway and therefore suppresses activation of NF-B by stabilizing IB-␣. The results show that the proteasome inhibitor blocks the release of proinflammatory cytokines induced by ONOO Ϫ (Fig. 5, A and B), suggesting that this action is regulated via NF-B activation.
Further evidence of transcriptional modulation of NF-B by ONOO Ϫ was obtained by investigating the nuclear translocation of p65/RelA subunits of NF-B and DNA binding capacity. The proportion of phosphorylated versus unphosphorylated IB-␣ molecules in cytoplasmic cell extracts was also deter-  (Fig. 6, A and B), whereas they were decreased with 200 M ONOO Ϫ and with FeTPPS. EMSA shift assays also showed that the greatest DNA binding activity was with monocytes treated with 10 M ONOO Ϫ and EGCG (1-100 M), while no binding was detected in those treated with 200 M ONOO Ϫ and FeTPPS (Fig. 7, A and B). As shown in Fig. 8, A-C, IB-␣ was significantly dephosphorylated by ONOO Ϫ at all concentrations and also significantly phosphorylated at all EGCG concentrations, but the greatest degree of IB-␣ phosphorylation was induced by FeTPPS. DISCUSSION The detrimental effect of ONOO Ϫ has been widely described; however, its role in the modulation of cell signaling pathways that modulate the inflammatory reaction has only recently began to attract attention. In particular, the actions of ONOO Ϫ on leukocyte migration and accumulation at the site of inflammation remains contradictory. On one hand, nanomolar concentrations of ONOO Ϫ have been reported to inhibit adhesion of granulocytes to the endothelium of the rat mesenteric vascular bed, and this has been shown to be mediated via downregulation of P-selectin expression on the endothelial cell surface (16). On the other hand, scavenging of ONOO Ϫ by uric acid has been shown to decrease the invasion of inflammatory cells into the spinal cord of a mouse model of multiple sclerosis (17). Furthermore, recent studies (18) have shown that micromolar concentrations of ONOO Ϫ activates Erk in neutrophils via the Ras/Raf-1/MEK signal transduction pathway, which although leads to down-regulation of L-selectin, it also up-regulates surface expression of CD11b/CD18 and consequently increases neutrophil adhesion to endothelial cells. The demonstration that ONOO Ϫ mediates IL-8 gene expression in lipopolysaccharide-stimulated human whole blood (19) has led to the notion that ONOO Ϫ is involved in the regulation of inflammatory reaction by blood leukocytes; however, the mechanism of this action remains unclear.
The present studies have demonstrated that the production of cytokines TNF-␣ and IL-6 by human mononuclear cells is predominantly regulated by ONOO Ϫ in a concentration-dependent manner. This action is mediated via the transcription factor NF-B, by a mechanism that may involve nitration or dephosphorylation of its inhibitor IB-␣. We have shown that ONOO Ϫ affects NF-B activity in a concentration-dependent  (Fig. 6, A and B) and completely dephosphorylates IB-␣ at concentrations of 10 -200 M (Fig. 7, A and B). In addition, ONOO Ϫ at high concentrations nitrates tyrosine residues in cell lysates ( Fig. 2A) that in turn block phosphorylation of these amino acids in IB-␣ molecules and possibly promotes IB-␣ degradation. This thesis is supported by previous reports showing that tyrosine nitration interferes with the process of phosphorylation and that nitrated IB-␣ molecules are themselves targets for rapid degradation by intracellular enzymes (10). Singh et al. (20) have shown that tyrosine residue at site 42 (Tyr-42) on IB-␣ is the specific acceptor site for phosphorylation, which prevents IB-␣ degradation. This implies that ONOO Ϫ may activate NF-B simply by nitrating critical residues in IB-␣ that would normally be phosphorylated. Taken together, it is possible to speculate that ONOO Ϫ at concentrations Յ20 M may nitrate tyrosine residues, including the critical site Tyr-42, a reaction that blocks its phosphorylation. However, IB-␣ molecules containing nitrated tyrosine residues are themselves targets for degradation that would result in increased NF-B activity. In contrast, 200 M ONOO Ϫ inhibits NF-B activation, because this concentration causes extensive nitration and dephosphorylation of amino acid residues in both NF-B subunits and IB-␣. This extensive nitration step prevents NF-B nuclear translocation probably in two ways. The first is by causing extensive nitration of tyrosine residues in NF-B and IB-␣, which renders both molecules targets for degradation by proteases, thus resulting in very little nuclear translocation of NF-B subunits (Fig. 6A) TNF-␣ and IL-6 release. Western blots of nuclear extracts separated by SDS-PAGE showed that EGCG at concentrations of 1-100 M increases p65 nuclear translocation and DNA binding in the presence and absence of ONOO Ϫ , suggesting that this is the mechanism by which EGCG promotes NF-B activity and cytokine release in mononuclear cells. From our results it may be possible to hypothesize that EGCG protects tyrosine residues from nitration ( Fig. 2) without preventing phosphorylation. Thus, although EGCG may effectively protect tyrosine residues from nitration, it may not prevent the phosphorylation of serines 32 and 36, a phenomenon previously described as the signal for IB-␣ degradation by the 26 S proteasome (10). Therefore, in the absence of nitration, phosphorylation of IB-␣ leads to the observed NF-B activation that in turn increases cytokine production.
The removal of ONOO Ϫ by FeTPPS protects tyrosine residues from nitration but not phosphorylation. The absence of nitration resulted in an extensive phosphorylation of IB-␣ molecule (Fig. 8A), probably of serine residues 32 and 36 and critical Tyr-42 residues. The phosphorylation of a large proportion of Tyr-42 residues would prevent the degradation of IB-␣ molecules, as observed by Singh et al. (20), that remain trapped within the cytoplasm. However, it cannot be ruled out that an increase in protein synthesis may also play a part in the total elevation of cytoplasmic IB-␣ (Fig. 8B).
It has been shown in the literature that ONOO Ϫ exhibits diverse effects in vivo dependent on the tissue affected and its environment (16,21,22). Our studies have disclosed that ONOO Ϫ modulates the production of proinflammatory cytokines by mononuclear leukocytes in a concentration-dependent manner via the transcription factor NF-B activation. Furthermore, the interplay between nitration and phosphorylation of amino acid residues involved in the intracellular signaling pathways appears to be an important regulatory mechanism.