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Effects of Reactive Oxygen and Nitrogen Metabolites on RANTES. and IL-5-Induced Eosinophil Chemotactic Activity in vitro

1999; Elsevier BV; Volume: 155; Issue: 2 Linguagem: Inglês

10.1016/s0002-9440(10)65154-1

1525-2191

Etsuro Sato, Keith L. Simpson, Matthew B. Grisham, Sekiya Koyama, Richard Robbins,

Mast cells and histamine

Eosinophils and increased production of nitric oxide (NO) and superoxide, components of peroxynitrite, have been implicated in the pathogenesis of a number of allergic disorders including asthma. Peroxynitrite induced protein nitration may compromise enzyme and protein function. We hypothesized that peroxynitrite may modulate eosinophil migration by modulating chemotactic cytokines. To test this hypothesis, the eosinophil chemotactic responses of regulated on activation, normal T cell expressed and secreted (RANTES) and interleukin (IL)-5 incubated with and without peroxynitrite were evaluated. Peroxynitrite-attenuated RANTES and IL-5 induced eosinophil chemotactic activity (ECA) in a dose-dependent manner (P < 0.05) but did not attenuate leukotriene B4 or complement-activated serum ECA. The reducing agents deferoxamine and dithiothreitol reversed the ECA inhibition by peroxynitrite, and exogenous L-tyrosine abrogated the inhibition by peroxynitrite. PAPA-NONOate, a NO donor, or superoxide generated by lumazine or xanthine and xanthine oxidase, did not show an inhibitory effect on ECA. The peroxynitrite generator, 3-morpholinosydnonimine, caused a concentration-dependent inhibition of ECA. Peroxynitrite reduced RANTES and IL-5 binding to eosinophils and resulted in nitrotyrosine formation. These findings are consistent with nitration of tyrosine by peroxynitrite with subsequent inhibition of RANTES and IL-5 binding to eosinophils and suggest that peroxynitrite may play a role in regulation of eosinophil chemotaxis. Eosinophils and increased production of nitric oxide (NO) and superoxide, components of peroxynitrite, have been implicated in the pathogenesis of a number of allergic disorders including asthma. Peroxynitrite induced protein nitration may compromise enzyme and protein function. We hypothesized that peroxynitrite may modulate eosinophil migration by modulating chemotactic cytokines. To test this hypothesis, the eosinophil chemotactic responses of regulated on activation, normal T cell expressed and secreted (RANTES) and interleukin (IL)-5 incubated with and without peroxynitrite were evaluated. Peroxynitrite-attenuated RANTES and IL-5 induced eosinophil chemotactic activity (ECA) in a dose-dependent manner (P < 0.05) but did not attenuate leukotriene B4 or complement-activated serum ECA. The reducing agents deferoxamine and dithiothreitol reversed the ECA inhibition by peroxynitrite, and exogenous L-tyrosine abrogated the inhibition by peroxynitrite. PAPA-NONOate, a NO donor, or superoxide generated by lumazine or xanthine and xanthine oxidase, did not show an inhibitory effect on ECA. The peroxynitrite generator, 3-morpholinosydnonimine, caused a concentration-dependent inhibition of ECA. Peroxynitrite reduced RANTES and IL-5 binding to eosinophils and resulted in nitrotyrosine formation. These findings are consistent with nitration of tyrosine by peroxynitrite with subsequent inhibition of RANTES and IL-5 binding to eosinophils and suggest that peroxynitrite may play a role in regulation of eosinophil chemotaxis. One of the hallmarks of allergic inflammation is the infiltration of eosinophilic granulocytes into the affected tissues. The importance of eosinophils has been emphasized by the finding that the degree of eosinophilia in both blood and bronchoalveolar lavage fluid (BALF) is associated with the clinical severity of asthma as well as with the degree of bronchial hyperresponsiveness.1Bousquet J Chanez P Lacoste JY Barneon G Ghavanian N Enander I Venge P Ahlstedt S Simony-Lafontaine J Godard P Michel F-B Eosinophilic inflammation in asthma.N Engl J Med. 1990; 323: 1033-1039Crossref PubMed Scopus (2184) Google Scholar, 2Gleich GJ The eosinophil and bronchial asthma: current understanding.J Allergy Clin Immunol. 1990; 85: 422-436Abstract Full Text PDF PubMed Scopus (602) Google Scholar, 3Holgate ST Roche WR Church MK The role of the eosinophil in asthma.Am Rev Respir Dis. 1991; 143: S66-S70Crossref PubMed Google Scholar In tissues, eosinophils releasing toxic proteins and oxidants that have been hypothesized to be responsible for the pathological changes observed in allergic diseases of the airways.4Wardlaw AJ Kay AB The role of the eosinophil in the pathogenesis of asthma.Allergy. 1987; 42: 321-335Crossref PubMed Scopus (87) Google Scholar Although the mechanism(s) accounting for the recruitment of eosinophils to the airway is not fully understood, it seems likely to involve several chemotactic cytokines. Nitric oxide (NO) is an important messenger molecule released from a variety of cell types in the airways and the concentration of NO in exhaled air of asthmatic patients is increased.5Kharitonov SA Yates D Robbins RA Logan-Sinclair R Shinebourne EA Barnes PJ Increased nitric oxide in exhaled air of asthmatic patients.Lancet. 1994; 343: 133-135Abstract PubMed Scopus (1297) Google Scholar High levels of superoxide anion have also been found in the BALF of asthmatic patients.6Jarjour NN Bussee WW Calhoun WJ Enahanced production of oxygen radicals in nocturnal asthma.Am Rev Respir Dis. 1992; 146: 905-911Crossref PubMed Scopus (103) Google Scholar, 7Joseph BZ Routes JM Borish L Activities of superoxide dismutases and NADPH oxidase in neutrophils obtained from asthmatic and normal donors.Inflammation. 1993; 17: 361-370Crossref PubMed Scopus (40) Google Scholar Moreover, superoxide dismutase activity is reduced in the leukocytes of asthmatics,7Joseph BZ Routes JM Borish L Activities of superoxide dismutases and NADPH oxidase in neutrophils obtained from asthmatic and normal donors.Inflammation. 1993; 17: 361-370Crossref PubMed Scopus (40) Google Scholar and increased formation of peroxynitrite, a potent oxidant, is found in the airways of asthmatic patients.8Saleh D Ernst P Lim S Barnes PJ Giaid A Increased formation of the potent oxidant peroxynitrite in the airways of asthmatic patients is associated with induction of nitric oxide synthase: effect of inhaled glucocorticoid.FASEB J. 1998; 12: 929-937Crossref PubMed Scopus (387) Google Scholar Peroxynitrite, an oxidant generated by the interaction between superoxide and NO, is known to nitrate several amino acids including cysteine,9Radi R Beckman JS Bush KM Freeman BA Peroxynitrite-induced membrane lipid peroxidation: the cytotoxic potential of superoxide and nitric oxide.Arch Biochem and Biophys. 1991; 288: 481-487Crossref PubMed Scopus (2024) Google Scholar methionine,10Pryor WA Jin X Squadrito GL One- and two-electron oxidations of methionine by peroxynitrite.Proc Natl Acad Sci USA. 1994; 91: 11173-11177Crossref PubMed Scopus (359) Google Scholar tryptophan,11Alvarez B Rubbo H Kirk M Barnes S Freeman BA Radi R Peroxynitrite-dependent tryptophan nitration.Chem Res Toxicol. 1996; 9: 390-396Crossref PubMed Scopus (229) Google Scholar and tyrosine.12Ischiropoulos H Zhu L Beckman JS Peroxynitrite-mediated tyrosine nitration catalyzed by superoxide dismutase.Arch Biochem Biophys. 1992; 298: 431-437Crossref PubMed Scopus (1417) Google Scholar Peroxynitrite nitrates free or protein-associated tyrosine to form the stable product nitrotyrosine by addition of a nitro group to the 3-position adjacent to the hydroxyl group of tyrosine. Several studies have shown that peroxynitrite-induced protein nitration may alter protein function. Peroxynitrite inactivates manganese superoxide dismutase13MacMillan-Crow LA Crow JP Kerby JD Beckman JS Thompson JA Nitration and inactivation of manganese superoxide dismutase in chronic rejection of human renal allografts.Proc Natl Acad Sci USA. 1996; 93: 11853-11858Crossref PubMed Scopus (715) Google Scholar and surfactant14Haddad IY Crow J Yoazu Y Beckman JS Matalon S Concurrent generation of nitric oxide and superoxide damages surfactant protein A (SP-A).Am J Physiol. 1994; 267: L242-L249PubMed Google Scholar and inhibits protein phosphorylation by tyrosine kinases, thus interfering with signal transduction mechanisms.15Gow AJ Buerk DG Ischiropoulos H A novel reaction mechanism for the formation of S-nitrosothiol in vivo.J Biol Chem. 1997; 272: 2841-2845Crossref PubMed Scopus (276) Google Scholar It has been also suggested that myeloproxidase (MPO)-catalyzed nitration or reaction of MPO-generated HOCl with NO2− to form nitrating intermediates as an alternative mechanism of protein nitration independent of peroxynitrite.16Eiserich JP Hristova M Cross CE Jones AD Freeman BA Halliwell B van der Vliet A Formation of nitric oxide-derived inflammatory oxidants by myeloperoxidase in neutrophils.Nature. 1998; 391: 393-397Crossref PubMed Scopus (1345) Google Scholar In addition, asthmatic airways are rich in peroxidases that can not only nitrate, but also chlorinate and brominate, tyrosines.17Huber RE Edwards LA Carne TJ Studies on the mechanism of the iodination of tyrosine by lactoperoxidase.J Biol Chem. 1989; 264: 1381-1386Abstract Full Text PDF PubMed Google Scholar Current concepts suggest that chemokines lead to eosinophil locomotion by binding to receptors. Alteration of critical chemokine sequences may alter chemokine binding and inactivate chemotactic function. For example, mutation of amino acids 13–15 from the rabbit (His-Ser-Thr. to the human sequence (Tyr-Ser-Lys) confers the high affinity of human IL-8 for the human IL-8A receptor.18Schraufstatter IU Ma M Oades ZG Barritt DS Cochrane CG The role of Tyr13 and Lys15 of interleukin-8 in the high affinity interaction with the interleukin-8 receptor type A.J Biol Chem. 1995; 270: 10428-10431Crossref PubMed Scopus (51) Google Scholar Consistent with the concept that tyrosine is important in binding to receptor, point mutations of Tyr-13 greatly lowered monocyte chemoattractant protein-1 receptor binding and activity,19Steitz SA Hasegawa H Chiang SL Cobb RR Castro MA Lobl TJ Yamada M Lazarides E Cardarelli PM Mapping of MCP-1 functional domains by peptide analysis and site-directed mutagenesis.FEBS Lett. 1998; 430: 158-164Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar and changing Tyr-28 to aspartate essentially abolished the monocyte chemoattractant activity of monocyte chemoattractant protein-1.20Zhang YJ Rutledge BJ Rollins BJ Structure/activity analysis of human monocyte chemoattractant protein-1 (MCP-1) by mutagenesis: Identification of a mutated protein that inhibits MCP-1-mediated monocyte chemotaxis.J Biol Chem. 1994; 269: 15918-15924Abstract Full Text PDF PubMed Google Scholar We hypothesized that tyrosine nitration by reactive nitrogen species would inhibit cytokine binding and eosinophil migration. To test this hypothesis, the chemotactic responses of human eosinophils to two well known eosinophil chemotaxins, RANTES (regulated on activation, normal T cell expressed and secreted) and interleukin (IL)-5 incubated with peroxynitrite and other compounds were evaluated in vitro. We found that peroxynitrite and 3-morpholinosydnonimine (SIN-1), a peroxynitrite donor, significantly attenuated RANTES- and IL-5-induced eosinophil chemotactic activity (ECA). In contrast, activated serum and leukotriene B4 (LTB4)-induced ECA was not significantly inhibited by peroxynitrite. These data suggest that peroxynitrite may play a role in eosinophil recruitment and inflammation. Eosinophils were isolated with a modified method of Hansel et al.21Hansel TT De Vries IJ Iff T Rihs S Wandzilak M Betz S Blaser K Walker C An improved immunomagnetic procedure for the isolation of highly purified human blood eosinophils.J Immunol Methods. 1991; 145: 105-110Crossref PubMed Scopus (563) Google Scholar Briefly, venous blood anticoagulated with 130 mmol/L trisodium citrate was obtained from normal human volunteers and diluted with phosphate-buffered saline (PBS) in a 1:1 ratio. Diluted blood was overlaid on an isotonic Percoll solution (density 1.082 g/ml; Sigma, St. Louis, MO), then centrifuged at 1000 × g for 30 minutes at 4°C with a Beckman TJ-6 centrifuge. The supernatant and mononuclear cells at the interface were carefully removed, and red blood cells in the sediment were lysed with two cycles of hypotonic lysis (0.1% KHCO3 and 0.83. NH4Cl). Isolated granulocytes were washed twice with PIPES buffer (25 mmol/L PIPES, 50 mmol/L NaCl, 5 mmol/L KCl, 25 mmol/L NaOH, and 5.4 mmol/L glucose, pH 7.4) containing 1% defined calf serum (Hyclone Laboratories, Logan, UT), and an approximately equal volume of anti-CD16 antibody conjugated with magnetic particles (Miltenyi Biotec, Bergisch Gladbach, Germany) was added to the cell pellet. After 60 minutes' incubation on ice, 5 ml of PIPES buffer with 1% defined calf serum were added to the cell-antibody mixture. The resuspended cells were loaded onto the separation column positioned in the magnetic cell separation system with a strong magnetic field. The cells were eluted three times with 5 ml of PIPES buffer with 1. defined calf serum. Purity of the eosinophils counted by Randolph's stain was >94%; viability was >98%. The eosinophils were resuspended in Gey's solution at 2.0 × 106 cells/ml and used for the chemotaxis assay. ECA was assayed in 48-well microchemotaxis chambers (Neuroprobe, Inc., Cabin John, MD).22Harvath L Falk W Leonard EJ Rapid quanitification of neutrophil chemotaxis: use of polyvinylpyrrolidone-free polycarbonate membrane in a multiwell assembly.J Immunol Methods. 1980; 37: 39-45Crossref PubMed Scopus (275) Google Scholar The bottom wells of the chamber were filled with 25 μl of the chemotactic stimulus or medium in duplicate. A 10-μm-thick polyvinylpyrrolidone-free polycarbonate filter with a pore size of 5 μm was placed over the samples. The silicon gasket and the upper pieces of the chamber were applied and 50 μl of the cell suspension was placed into the upper wells. The chambers were incubated in humidified air in 5% CO2 at 37°C for 90 minutes. Nonmigrated cells were wiped away from the filter. The filter was immersed in methanol for 5 minutes, stained with a modified Wright's stain, and mounted on a glass slide. Cells that had completely migrated through the filter were counted using light microscopy. ECA was expressed as the mean number of migrated cells per high-power field from duplicate wells. Peroxynitrite was evaluated for its capacity to modulate RANTES. and IL-5-induced ECA in vitro. RANTES or IL-5 (R&D Systems, Minneapolis, MN) was incubated for 2 hours at 37°C with each concentration of peroxynitrite (Calbiochem, La Jolla, CA) before the ECA assay. In control experiments, RANTES or IL-5 was incubated with medium alone. The capacity of peroxynitrite to modulate LTB4- and activated serum-induced ECA was compared to RANTES (10−7 g/ml) and IL-5 (10−8 g/ml). LTB4 (10−6 mol/L, Sigma) or complement-activated serum23Snyderman R Gewurz H Mergenhagen SE Interactions of the complement system with endotoxic lipopolysaccharide: generation of a factor chemotactic for polymorphonuclear leukocytes.J Exp Med. 1968; 128: 259-275Crossref PubMed Scopus (102) Google Scholar (1:10 dilution) were incubated with peroxynitrite (10−4 mol/L) for 2 hours at 37°C before performing the ECA assay. To evaluate the effect of NO to modulate RANTES and IL-5 induced ECA, we used PAPA-NONOate (Alexis, San Diego, CA) as a NO donor.24Wink DA Cook JA Christodoulou D Krishna MC Pacelli R Kim S DeGraff W Gamson J Vodovotz Y Russo A Mitchell JB Nitric oxide and some nitric oxide donor compounds enhance the cytotoxicity of cisplatin.Nitric Oxide. 1997; 1: 88-94Crossref PubMed Scopus (114) Google Scholar, 25Keefer LK Nims RW Davies KM Wink DA "NONOates" (1-substituted diazen-1-ium-1,2-diolates) as nitric oxide donors: convenient nitric oxide dosage forms.Methods Enzymol. 1996; 268: 281-293Crossref PubMed Google Scholar, 26Mooradian DL Hutsell TC Keefer LK Nitric oxide (NO) donor molecules: effect of NO release rate of vascular smooth muscle proliferation in vitro.J Cardiovasc Pharmacol. 1995; 25: 674-678Crossref PubMed Scopus (217) Google Scholar, 27Maragos CM Wang JM Hrabie JA Oppenheim JJ Keefer LK Nitric oxide/nucleophile complexes inhibit the in vitro proliferation of A375 melanoma cells via nitric oxide release.Cancer Res. 1993; 53: 564-568PubMed Google Scholar RANTES (10−7 g/ml) or IL-5 (10−8 g/ml) was incubated with PAPA-NONOate (10−3−10−6 M) for 2 hours at 37°C before performing the ECA assay. The samples were dialyzed overnight at 4°C against Hanks' balanced salt solution using tubing with a molecular weight cutoff of 3 kd. The half-life of PAPA-NONOate is 15 minutes in physiological buffer at 37°C and two moles of NO are released per mole of PAPA-NONOate. To evaluate the effect of superoxide on RANTES- and IL-5-induced ECA, lumazine (10−4 mol/L, Sigma) or xanthine (10−6, 10−5, 10−4, 10−3 mol/L, Sigma. was combined with xanthine oxidase (3.4 ×10−6, 3.4 × 10−5, 3.4 × 10−4, 3.4× 10−3 U/ml, Sigma) to produce superoxide.28Fridovich I Quantitative aspects of the production of superoxide anion radical by milk xanthine oxidase.J Biol Chem. 1970; 245: 4053-4057Abstract Full Text PDF PubMed Google Scholar, 29Nagano T Fridovich I Superoxide radical from xanthine oxidase acting upon lumazine.J Free Radic Biol Med. 1985; 1: 39-42Crossref PubMed Scopus (34) Google Scholar Xanthine was combined with xanthine oxidase in descending order of concentration. RANTES (10−7 g/ml) or IL-5 (10−8 g/ml) was incubated with lumazine and xanthine oxidase or xanthine and xanthine oxidase for 2 hours at 37°C before performing the ECA assay. To confirm the results with the peroxynitrite, 3-morpholinosydnonimine (SIN-1) (Alexis), a slow peroxynitrite generator,30Holm P Kankaanranta H Metsa-Ketela T Moilanen E Radical releasing properties of nitric oxide donors GEA 3162, SIN-1 and S-nitroso-N-acetylpenicillamine.Eur J Pharmacol. 1998; 346: 97-102Crossref PubMed Scopus (74) Google Scholar, 31Darley-Usmar VM Hogg N O'Leary VJ Wilson MT Moncada S The simultaneous generation of superoxide and nitric oxide can initiate lipid peroxidation in human low density lipoprotein.Free Radic Res Commun. 1992; 17: 9-20Crossref PubMed Scopus (438) Google Scholar was used. RANTES- and IL-5-induced ECA was evaluated by incubating RANTES (10−7 g/ml) or IL-5 (10−8 g/ml) with SIN-1 (10−4, 10−5, 10−6, 10−7 mol/L) for 2 hours at 37°C before performing the ECA assay. The capacity of the reducing agents dithiothreitol and deferoxamine to attenuate the effect of peroxynitrite on RANTES and IL-5 induced ECA by peroxynitrite was assessed. Dithiothreitol (1 mmol/L, Sigma), deferoxamine (50 μmol/L, Sigma), and peroxynitrite (10−4 mol/L) were added to RANTES (10−7 g/ml) or IL-5 (10−8 g/ml) and incubated for 2 hours at 37°C before evaluating for ECA. The capacity of L-tyrosine to reserve the attenuation of ECA induced by peroxynitrite was assessed by addition of L-tyrosine (10−3, 10−4, 10−5 mol/L, Sigma) to RANTES (10−7 g/ml) or IL-5 (10−8 g/ml) before exposed to peroxynitrite (10−5 mol/L). Nitrotyrosine on RANTES and IL-5 incubated with peroxynitrite was detected using modifications of previously described techniques.32Jones KL Bryan TW Jinkins PA Simpson KL Grisham MB Owens MW Milligan SA Markewitz BA Robbins RA Superoxide released from neutrophils cause a reduction in nitric oxide gas.Am J Physiol. 1998; 275: L1120-1126PubMed Google Scholar RANTES (10−7 g/ml) or IL-5 (10−8 g/ml) was incubated with peroxynitrite (100 μmol/L) or media as above and frozen until assayed. Goat anti-human RANTES or IL-5 IgG (R&D Systems) was dissolved in Voller's buffer (1.59 g sodium carbonate, 2.93 g sodium bicarbonate, 0.2 g sodium azide in 1 L distilled water, pH 9.6) at the final concentration of 200 ng/ml. Two hundred microliters were added to flat-bottomed 96-well plates (Costar, Cambridge, MA) and allowed to adsorb to the plastic overnight at 4°C. After washing the flat-bottomed plate 3 times with PBS-Tween, 200 μl of RANTES or IL-5 with and without peroxynitrite incubation were added to their respective antibody-coated plate and incubated for 60 minutes at room temperature. After washing 3 times with PBS-Tween, 200 μl of a 1:400 dilution of rabbit polyclonal anti-nitrotyrosine (Calbiochem) were added to the wells and incubated for 90 minutes. After again washing 3 times with PBS-Tween, 200 μl of a 1:500 dilution of peroxidase-conjugated anti-rabbit IgG were added to the wells and incubated for 90 minutes. Two hundred microliters of o-phenylenediamine (100 μg/ml, Sigma) in 0.003. H2O2 were added and visually monitored. The reaction was terminated by addition of 25 μl of 8N H2SO4 and the absorbance read at 490 nm. To investigate the peroxynitrite effect on RANTES and IL-5 binding to eosinophils, RANTES (10−7 g/ml) or IL-5 (10−8 g/ml) was incubated with 100 μmol/L of peroxynitrite for 2 hours at 37°C. In control experiments, RANTES or IL-5 was incubated with medium alone. Subsequently, RANTES or IL-5 with or without peroxynitrite was incubated with eosinophils (5 × 105 cells) at 4°C for 30 minutes. Then supernatants were removed and eosinophils were washed 3 times with Hanks' balanced salt solution. Eosinophils were suspended in 1 ml PBS-Tween, sonicated for 20 seconds (MSE Soniprep, Crawley, UK), and then centrifuged at 20,000 × g for 30 minutes in a refrigerated microcentrifuge to obtain a supernatant (soluble) and particulate fraction. RANTES and IL-5 were measured using a commercially available enzyme-linked immunosorbent assay (R & D Systems). In experiments, the differences between groups were tested using Student's paired t-test. In all cases, a P value of <0.05 was considered significant. Data in figures are expressed as mean ± SE. Various amounts of RANTES (Figure 1A) or IL-5 (Figure 1B) were incubated with peroxynitrite (100 μmol/L). At each concentration, exposure to peroxynitrite caused a reduction in ECA (Figure 1) (n = 4, P < 0.05). Incubation of RANTES (Figure 2A, 10 −7 g/ml) or IL-5 (Figure 2B, 10 −8 g/ml) with various amounts of peroxynitrite induced a significant, concentration-dependent attenuation of ECA (n = 4, P < 0.05). The lowest dose of peroxynitrite tested, 10−6 mol/L, significantly inhibited ECA induced by RANTES and 10−5 mol/L inhibited ECA induced by. IL-5. Peroxynitrite itself was not chemotactic for eosinophils (data not shown). Similarly, incubation of peroxynitrite (100 μmol/L) with the eosinophils before the chemotaxis assay did not inhibit ECA to RANTES or IL-5 (data not shown).Figure 2Dose-responsive inhibition of ECA by peroxynitrite. RANTES (A; 10−7g/ml) or IL-5 (B; 10−8g/ml) was incubated for 2 hours in the presence with medium alone or peroxynitrite at the concentration indicated; n = 4 each condition. ECA is on the ordinate and the concentration of peroxynitrite is on the abscissa. *P < 0.05 compared with RANTES or IL-5 incubated with medium alone.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 10Effects of peroxynitrite on RANTES (A) or IL-5 (B. binding to eosinophils. RANTES or IL-5 were incubated with and without 100 μmol/L of peroxynitrite for 2 hours at 37°C. Subsequently, the treated RANTES or IL-5 was incubated with eosinophils (5 × 105 cells) at 37°C for 30 minutes. RANTES or IL-5 binding to eosinophils was measured by ELISA. RANTES or IL-5 concentration is on the ordinate and the experimental groups are on the abscissa. *P < 0.05 compared with RANTES or IL-5 incubated with medium alone.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To ensure that the effect of peroxynitrite was not a nonspecific effect on eosinophil chemotaxis, the effect of peroxynitrite on ECA induced by LTB4 and complement-activated serum was assessed. Peroxynitrite did not significantly inhibit the ECA of LTB4 or complement-activated serum (Figure 3) (n = 4, P < 0.05). To investigate the capacity of NO to modulate ECA induced by RANTES (Figure 4A) and IL-5 (Figure 4B), the effect of the NO donor, PAPA-NONOate, was evaluated. PAPA-NONOate did not significantly change ECA induced by RANTES or IL-5 (Figure 4) (n = 4). To evaluate the effect of superoxide on ECA, RANTES (Figure 5A) or IL-5 (Figure 5B) were incubated with lumazine or xanthine and xanthine oxidase (Figure 5) (n = 4). None significantly altered ECA to RANTES or IL-5. SIN-1, a nitrovasodilator, spontaneously decomposes under aqueous conditions, generating first O2− and then NO at comparable rates. SIN-1 induced a significant, concentration-dependent attenuation of ECA by RANTES (Figure 6A) (n = 4, P < 0.05) and IL-5 (Figure 6B) (n = 4, P < 0.05). The lowest dose of SIN-1 to inhibit ECA was 10−5 mol/L. SIN-1 itself was not chemotactic for eosinophils (data not shown). The reducing agents dithiothreitol and deferoxamine were added to RANTES (Figure 7A) or IL-5 (Figure 7B) before incubating with peroxynitrite. Each attenuated the inhibition of ECA induced by peroxynitrite (Figure 7) (n = 4, P < 0.05). Dithiothreitol or deferoxamine alone was not chemotactic for eosinophils (data not shown). One mechanism of peroxynitrite inhibition may be through nitrating tyrosine residues. Therefore, the effect of L-tyrosine addition to RANTES (Figure 8A) and IL-5 (Figure 8B) before incubating with peroxynitrite was investigated. Addition of L-tyrosine to RANTES or IL-5 abrogated the attenuation of ECA induced by peroxynitrite (Figure 8) (n = 4, P < 0.05). L-tyrosine itself was not chemotactic for eosinophils (data not shown). Optical density of RANTES (Figure 9A) or IL-5 (Figure 9B) with peroxynitrite incubation was significantly higher than RANTES or IL-5 without peroxynitrite incubation. Peroxynitrite resulted in nitrotyrosine formation on RANTES and IL-5 (Figure 9) (n = 6, P < 0.05). RANTES (Figure 10A) and IL-5 (Figure 10B) induce chemotactic activity by binding to eosinophils. Addition of peroxynitrite to RANTES and IL-5 resulted in an inhibition of their binding to eosinophils. (Figure 10) (n = 4, P < 0.05). The results of this study show that the peroxynitrite significantly attenuated RANTES- and IL-5-induced ECA in vitro. The inhibitory effects of peroxynitrite were not significant on ECA induced by LTB4 and activated serum. Deferoxamine, dithiothreitol, or tyrosine attenuated the inhibition. NO or superoxide did not cause the reduction in RANTES- and IL-5-induced ECA, because PAPA-NONOate and lumazine/xanthine oxidase did not show an inhibitory effect. The peroxynitrite donor, SIN-1, induced a significant, concentration-dependent inhibition of ECA by RANTES and IL-5. Nitrotyrosine was detected on RANTES or IL-5 incubated with peroxynitrite by ELISA. Furthermore, peroxynitrite reduced RANTES and IL-5 binding to eosinophils. These data suggest that peroxynitrite plays an important role in regulating human eosinophil locomotion by modulating chemotactic cytokines. in vitro, NO production has been demonstrated from a variety of cell types, and various inflammatory mediators are reported to enhance production of NO and superoxide, leading to the in vivo formation of peroxynitrite. Inflammatory cell chemotactic factors, including RANTES and IL-5, are likely to encounter high local concentrations of NO, superoxide, and peroxynitrite in inflammatory sites. Several studies have examined the effects of NO on neutrophil, monocyte, and eosinophil chemotaxis,33Bath PM The effect of nitric oxide-donating vasodilators on monocyte chemotaxis and intracellular cGMP concentrations in vitro.Eur J Clin Pharmacol. 1993; 45: 53-58Crossref PubMed Scopus (63) Google Scholar, 34Moilanen E Vuorinen P Kankaanranta H Metsa-Ketela T Vapaatalo H Inhibition by nitric oxide-donors of human polymorphonuclear leucocyte functions.Br J Pharmacol. 1993; 109: 852-858Crossref PubMed Scopus (146) Google Scholar, 35Kaplan SS Billiar T Curran RD Zdziarski UE Simmons RL Basford RE Attenuation of chemotaxis with NG-monomethyl-L-arginine: a role for cyclic GMP.Blood. 1989; 74: 1885-1887Crossref PubMed Google Scholar, 36Belenky SN Robbins RA Rennard SI Gossman GL Nelson KJ Rubinstein I Inhibitors of nitric oxide synthase attenuate neutrophil chemotaxis in vitro.J Lab Clin Med. 1993; 122: 388-394PubMed Google Scholar, 37Belenky SN Robbins RA Rubinstein I Nitric oxide synthase inhibitors attenuate human monocyte chmotaxis in vitro.J Leukocyte Biol. 1993; 53: 498-503Crossref PubMed Scopus (83) Google Scholar, 38Ferreira HH Medeiros MV Lima CS Flores CA Sannomiya P Autunes E De Nucci G Inhibition of eosinophil chemotaxis by chronic blockade of nitric oxide biosynthesis.Eur J Pharmacol. 1996; 310: 201-207Crossref PubMed Scopus (46) Google Scholar but few have examined the effects on chemotactic factors. The concentration of peroxynitrite in vivo is not known. Peroxynitrite is a transient intermediate in free radical chemistry and is highly reactive at physiological pH. For this reason, peroxynitrite cannot be considered as a pharmacological drug that has steady-state concentrations. Thom et al39Thom SR Ohnishi ST Fisher D Xu YA Ischiropoulos H Pulmonary vascular stress from carbon monoxide.Toxicol Appl Pharmacol. 1999; 154: 12-19Crossref PubMed Scopus (36) Google Scholar reported finding concentrations of nitrotyrosine, an end product of peroxynitrite, of 30 to 60 ng/mg protein in lung homogenates. Assuming that peroxynitrite nitrated tyrosine residues one-to-one, these levels would seem comparable to doses we used to affect chemokine function as reported in this manuscript. 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Our findings with RANTES and IL-5 after peroxynitrite incubation are consistent with these observations and suggest that tyrosine nitration by peroxynitrite on RANTES and IL-5 may be a mechanism altering their binding and chemotactic function. However, peroxynitrite may potentially affect protein function by other mechanisms, including nitration of methionine50Vogt W Oxidation of methionyl residues in proteins: tools, targets, and reversal.Free Radic Biol Med. 1995; 18: 93-105Crossref PubMed Scopus (770) Google Scholar and tryptophan11Alvarez B Rubbo H Kirk M Barnes S Freeman BA Radi R Peroxynitrite-dependent tryptophan nitration.Chem Res Toxicol. 1996; 9: 390-396Crossref PubMed Scopus (229) Google Scholar or formation of s-nitroso-thiol groups on cysteines.15Gow AJ Buerk DG Ischiropoulos H A novel reaction mechanism for the formation of S-nitrosothiol in vivo.J Biol Chem. 1997; 272: 2841-2845Crossref PubMed Scopus (276) Google Scholar Although NO and peroxynitrite are physiological regulators, they have been shown to alter respiration51Bolanos JP Heales SJ Peuchen S Barker JE Land JM Clark JB Nitric oxide-mediated mitochondrial damage: a potential neuroprotective role for glutathione.Free Radic Biol Med. 1996; 21: 995-1001Crossref PubMed Scopus (254) Google Scholar, 52Shen W Hintze TH Wolin MS Nitric oxide: an important signaling mechanism between vascular endothelium and parenchymal cells in the regulation of oxygen consumption.Circulation. 1995; 92: 3505-3512Crossref PubMed Scopus (226) Google Scholar and induce cell death.53Burney S Tamir S Gal A Tannenbaum SR A mechanistic analysis of nitric oxide-induced cellular toxicity.Nitric Oxide. 1997; 1: 130-144Crossref PubMed Scopus (89) Google Scholar To estimate the effect of peroxynitrite on eosinophils, we incubated eosinophils with peroxynitrite for 90 minutes at 37°C before chemotaxis experiments. It induced no significant cytotoxicity as assessed by trypan blue exclusion in comparison to medium alone, and it had no significant effect on ECA by RANTES and IL-5. The evidence for a role of peroxynitrite in vivo is based on detection of 3-nitrotyrosine in injured tissues; however, an additional mechanism of tyrosine nitration independent of peroxynitrite has lately been demonstrated.16Eiserich JP Hristova M Cross CE Jones AD Freeman BA Halliwell B van der Vliet A Formation of nitric oxide-derived inflammatory oxidants by myeloperoxidase in neutrophils.Nature. 1998; 391: 393-397Crossref PubMed Scopus (1345) Google Scholar NO2− promotes tyrosine nitration through formation of nitryl chloride (Cl-NO2) and nitrogen dioxide (·NO2) by reaction with the inflammatory mediators hypochlorous acid (HOCl) or MPO. Peroxidases may potentially affect protein function by not only nitrating but also chlorinating or brominating tyrosine residues.17Huber RE Edwards LA Carne TJ Studies on the mechanism of the iodination of tyrosine by lactoperoxidase.J Biol Chem. 1989; 264: 1381-1386Abstract Full Text PDF PubMed Google Scholar Therefore, it cannot be stated definitively that the formation of nitrotyrosine in vivo is due to peroxynitrite. In summary, we found that peroxynitrite nitrates tyrosine residue and modulates RANTES- and IL-5-induced ECA in vitro. These data suggest a role for peroxynitrite in regulating human eosinophil locomotion during inflammation.