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Reactive nitrogen and oxygen species attenuate interleukin- 8-induced neutrophil chemotactic activity in vitro.

Peroxynitrite, formed by the reaction between nitric oxide and superoxide, has been shown to induce protein nitration, which compromises protein function. We hypothesized that peroxynitrite may regulate cytokine function during inflammation. To test this hypothesis, the neutrophil chemotactic activity (NCA) of interleukin-8 (IL-8) incubated with peroxynitrite was evaluated. Peroxynitrite attenuated IL-8 NCA in a dose-dependent manner (p < 0.01) but did not significantly reduce NCA induced by leukotriene B(4) or complement-activated serum. The reducing agents, dithionite, deferoxamine, and dithiothreitol, reversed and exogenous L-tyrosine abrogated the peroxynitrite-induced NCA inhibition. Papa-NONOate [N-(3-ammoniopropyl)-N-(n-propyl)amino]diazen-1-ium-1, 2-dialase or sodium nitroprusside, NO donors, or a combination of xanthine and xanthine oxidase to generate superoxide did not show an inhibitory effect on NCA induced by IL-8. In contrast, small amounts of SIN-1, a peroxynitrite generator, caused a concentration-dependent inhibition of NCA by IL-8. Consistent with its capacity to reduce NCA, peroxynitrite treatment reduced IL-8 binding to neutrophils. Nitrotyrosine was detected in the IL-8 incubated with peroxynitrite by enzyme-linked immunosorbent assay. These findings are consistent with nitration of tyrosine by peroxynitrite with subsequent inhibition of IL-8 binding to neutrophils and a reduction in NCA and suggest that oxidants may play an important role in regulation of IL-8-induced neutrophil chemotaxis.

Interleukin-8 (IL-8), 1 an 8400-dalton protein, is synthesized and secreted by monocytes, macrophages, and other cells, including epithelial and endothelial cells, lymphocytes, fibroblasts, and keratinocytes (1)(2)(3). Current concepts suggest an important role for IL-8 in the pathogenesis of neutrophil-mediated inflammation because of the potent neutrophil chemo-tactic activity (NCA) of IL-8 and its detection in affected tissues or body fluids taken from patients suffering from neutrophilic inflammatory lesions (3).
Peroxynitrite is produced by the rapid reaction of nitric oxide (NO) and superoxide (4,5). In addition to its role in oxidative reactions, peroxynitrite also 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 (6). Several studies have shown that peroxynitriteinduced protein nitration may compromise protein function. For example, nitration of tyrosine residues on human IgG abrogates C1q binding (7) and inhibits protein phosphorylation by tyrosine kinase (8).
Recently, the tyrosine residue at position 13 of IL-8 has been shown to be important in neutrophil chemotaxis (9). We hypothesized peroxynitrite might regulate NCA by nitrating tyrosine residues. To test this hypothesis, the chemotactic responses of human neutrophils to IL-8 incubated with peroxynitrite and other compounds were evaluated in vitro. We found that peroxynitrite and 3-morpholinosydnonimine (SIN-1), a donor of peroxynitrite, significantly attenuated IL-8-induced NCA. In contrast activated serum and LTB 4 -induced NCA was not inhibited by peroxynitrite significantly. These data suggest that peroxynitrite plays an important role in regulating NCA during inflammation.

EXPERIMENTAL PROCEDURES
Measurement of NCA-Human polymorphonuclear leukocytes were isolated from venous blood as described previously (10,11). Briefly, 15 ml of venous blood was obtained from healthy volunteers and then sedimented with 3% dextran in isotonic saline for 45 min to separate white blood cells from red blood cells. The leukocyte-rich upper layer was collected, and neutrophils were separated from mononuclear cells by Ficoll-Paque density centrifugation (Histopaque 1077, Sigma). Contaminating red blood cells were removed by hypotonic lysis (0.1% KHCO 3 and 0.83% NH 4 Cl). The suspension was then centrifuged at 400 ϫ g for 5 min and washed three times in HBSS (Biofluids, Rockville, MD). The resulting cell pellet, as determined by trypan blue and erythrosin exclusion, consisted of Ͼ96% neutrophils and Ͼ98% viable cells. The cells were suspended in Gey's balanced salt solution (Life Technologies, Inc.) containing 2% bovine serum albumin (Sigma) at a final concentration of 3 ϫ 10 6 cells/ml. NCA was assayed in 48-well microchemotaxis chambers (Neuroprobe, Inc., Cabin John, MD) as described previously (11). The bottom wells of the chamber were filled with 25 l of the chemotactic stimulus or media in duplicate. A 10-m thick polyvinylpyrrolidone-free polycarbonate filter with 3-m pores was placed over the samples. The silicon gasket and the upper pieces of the chamber were applied, and 50 l of the cell suspension was placed into the upper wells. The chambers were incubated in humidified air in 5% CO 2 at 37°C for 30 min. Nonmigrated cells were wiped away from the filter. The filter was immersed in methanol for 5 min, 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. NCA was expressed as * This work was supported by a Merit Review grant from the Department of Veterans Affairs and a grant from Rotary International. 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.
Effects of Peroxynitrite on IL-8-induced NCA-Peroxynitrite was evaluated for its capacity to modulate IL-8-induced NCA in vitro. Human recombinant IL-8 (R & D Systems, Inc., Minneapolis, MN) was incubated for 2 h at 37°C with each concentration of peroxynitrite (Calbiochem) before the NCA assay. In control experiments, IL-8 was incubated with medium alone.
Effects of Peroxynitrite on LTB 4 and Activated Serum-induced NCA-The capacity of peroxynitrite to modulate LTB 4 and activated seruminduced NCA was evaluated for comparison with IL-8. LTB 4 (10 Ϫ6 M, Sigma) or complement-activated serum (12) (1:10 dilution) was incubated with peroxynitrite (10 Ϫ3 M) for 2 h at 37°C before performing the NCA assay. The 2-h time point was chosen to ensure that all residual peroxynitrite had spontaneously decomposed prior to the chemotaxis assay.
Effects of L-Tyrosine on Peroxynitrite-induced Attenuation of NCA by IL-8 -The capacity of L-tyrosine to reserve the attenuation of NCA induced by peroxynitrite was assessed by addition of L-tyrosine (10 Ϫ3 -10 Ϫ5 M, Sigma) to IL-8 (10 Ϫ7 g/ml) before exposure to peroxynitrite (10 Ϫ5 M).
Detection of Nitrotyrosine on IL-8 Incubated with Peroxynitrite-Nitrotyrosine on IL-8 incubated with peroxynitrite was evaluated because formation of peroxynitrite has been demonstrated to nitrate tyrosine residues (5). IL-8 (10 Ϫ7 g/ml) was incubated with peroxynitrite (10 Ϫ4 M) and frozen until assayed. Goat anti-human IL-8 IgG (R & D Systems, Inc.) was dissolved in Voller's buffer (1.59 g of sodium carbonate, 2.93 g of sodium bicarbonate, 0.2 g of sodium azide in 1 liter of distilled water, pH 9.6) at the final concentration 2 ϫ 10 Ϫ7 g/ml. Two hundred l was 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 (0.075% Tween 20, Sigma), 200 l of IL-8 with and without peroxynitrite incubation were added to the IL-8 antibody-coated plate and incubated for 60 min at room temperature. After washing three times with PBS/ Tween, 200 l of a 1:400 dilution of rabbit polyclonal anti-nitrotyrosine (Calbiochem) was added to the wells and incubated for 90 min. After again washing three times with PBS/Tween, 200 l of a 1:500 dilution of peroxidase-conjugated anti-rabbit IgG was added to the wells and incubated for 90 min. 200 l of o-phenylenediamine (100 g/ml, Sigma) in 0.003% H 2 O 2 was added and visually monitored. The reaction was terminated by addition of 25 l of 8 N H 2 SO 4 and the absorbance read at 490 nm. In separate experiments, plates were coated with IL-8 that had either been exposed or not exposed to peroxynitrite. Subsequently, anti-nitrotyrosine, peroxidase-conjugated goat anti-rabbit IgG, and ophenylenediamine were added sequentially with washing between each addition. The reaction was terminated with H 2 SO 4 and the absorbance read at 490 nm.
Effects of Peroxynitrite on IL-8 Binding to Neutrophils-To investigate the peroxynitrite effect on IL-8 binding to neutrophils, IL-8 was incubated with 10 Ϫ4 M of peroxynitrite for 2 h at 37°C. In control experiments, IL-8 was incubated with medium alone. Subsequently, IL-8 with or without peroxynitrite was incubated with neutrophils (10 5 or 10 6 cells) at 4°C for 30 min. Then supernatants were removed, and neutrophils were washed three times by HBSS. Neutrophils were suspended in 1 ml of PBS/Tween, sonicated for 20 s, and then centrifuged at 20,000 ϫ g for 30 min in a refrigerated microcentrifuge to obtain a supernatant (soluble) and cellular fraction. Then IL-8 was measured in the cellular fraction using a commercially available IL-8 ELISA (R & D Systems).
Statistics-The differences between groups were tested using Student's paired t test. In all cases, a p value of Ͻ0.05 was considered significant. Data in figures are expressed as mean Ϯ S.E.
Effects of Peroxynitrite on LTB 4 and Activated Serum-induced NCA-To ensure that the effect of peroxynitrite was not a nonspecific effect on neutrophil chemotaxis, the effect of peroxynitrite on NCA induced by LTB 4 and complement-activated serum was assessed. Peroxynitrite did not significantly inhibit the NCA of LTB 4 or complement-activated serum ( Effects of Dithionite, Dithiothreitol, and Deferoxamine on Peroxynitrite-induced Attenuation of NCA by IL-8 -Addition of the reducing agents, dithiothreitol, deferoxamine, and dithionite to IL-8 prior to incubation with peroxynitrite attenuated the inhibition of NCA induced by peroxynitrite ( Fig. 3B; n ϭ 4, p Ͻ 0.01).
Effects of L-Tyrosine on Peroxynitrite-induced Attenuation of NCA by IL-8 -Addition of L-tyrosine to IL-8 before incubating with peroxynitrite abrogated the attenuation of NCA induced by peroxynitrite ( Fig. 3C; n ϭ 4, p Ͻ 0.01). The addition of 10 Ϫ4 M of L-tyrosine prevented the inhibition of NCA induced by 10 Ϫ5 M peroxynitrite.
Detection of Nitrotyrosine on IL-8 Incubated with Peroxynitrite-Optical density of IL-8 with peroxynitrite incubation was significantly higher than IL-8 without peroxynitrite incubation. Peroxynitrite resulted in nitrotyrosine formation on IL-8 ( Fig. 4; n ϭ 6, p Ͻ 0.01). There was also an increase in the absorbance of IL-8 exposed to peroxynitrite at 428 nm consistent with the formation of 3-nitrotyrosine (16).
Effects of Peroxynitrite on IL-8 Binding to Neutrophils-Addition of peroxynitrite to IL-8 resulted in a reduction of IL-8 bound to the neutrophils ( Fig. 5; n ϭ 4, p Ͻ 0.01). DISCUSSION The results of this study show that the peroxynitrite significantly attenuated IL-8-induced NCA in vitro. This was not explained by an inhibition of chemotaxis by peroxynitrite. Incubation of peroxynitrite (10 Ϫ4 M) with the neutrophils prior to the chemotaxis assay did not inhibit NCA to IL-8 (data not shown), and the inhibitory effects of peroxynitrite were not significant on NCA induced by LTB 4 or complement-activated serum. Sodium dithionite, deferoxamine, dithiothreitol, or tyrosine attenuated the inhibition but did not affect chemotaxis alone (data not shown). NO or superoxide did not cause the reduction in IL-8 NCA because papa-NONOate and xanthine/ xanthine oxidase did not show an inhibitory effect. The peroxynitrite donor, SIN-1, induced a significant concentration-dependent inhibition of NCA by IL-8. Nitrotyrosine was detected in the IL-8 incubated with peroxynitrite by ELISA. These data suggest that peroxynitrite plays an important role in regulating human neutrophil locomotion by IL-8.
Neutrophils migrate from the bloodstream across vascular endothelium and smooth muscle to vascular sites of inflammation, play an important role in host defenses during tissue injury and inflammation, and also play an important role in tissue remodeling after injury (17,18). Current concepts suggest that neutrophils are recruited from the bloodstream by chemotactic factors, such as IL-8, generated and released locally in injured tissues that may also generate NO and superoxide. Therefore, IL-8 is likely to encounter high local concentrations of peroxynitrite. Neutrophils have been shown to respond to NO (19,20); however, NO donors or NO synthase inhibitors have produced variable effects on neutrophil chemotaxis in vitro (21)(22)(23)(24).
Co-incubation of IL-8 with several peroxynitrite scavengers ameliorated peroxynitrite-induced NCA inhibition. The protective effect of dithiothreitol on IL-8 may suggest the involvement of cysteine residues; however, dithiothreitol has also been shown to prevent peroxynitrite-mediated nitration of tyrosine (25). The iron chelator, deferoxamine, also inhibited peroxynitrite-induced inhibition of IL-8 NCA but is also a scavenger of peroxynitrite reaction independent of iron chelation (26). Dithionite, which has been proposed to modify 3-nitrotyrosine by substituting an amine group (27), ameliorated the peroxynitrite inhibition. In addition, L-tyrosine abrogated the peroxynitrite NCA inhibition. These results are consistent with tyrosine nitration by peroxynitrite as a mechanism for IL-8 inhibition.
IL-8 is a stimulatory peptide for neutrophils that appears to play an important role in the inflammatory process (1-3). Local injection or genetic overexpression of IL-8 in experimental animals causes specific neutrophil migration to the targeted site (28). Current concepts suggest that the mechanisms that lead to neutrophil locomotion in response to IL-8 are by binding to receptors on neutrophils (29 -31). Consistent with this concept, our results demonstrate that peroxynitrite-treated IL-8 exhibited decreased binding to neutrophils.
Binding of IL-8 to neutrophil receptors has recently been studied by Schraufstatter et al. (9) who reported the importance of Tyr 13 and Lys 15 . Mutation of amino acids 13-15 of the rabbit (His-Ser-Thr) to the human sequence (Tyr-Ser-Lys) confers the high affinity for the human IL-8 receptor to this mutated form of rabbit IL-8. Consistent with the concept that tyrosine is important in binding of IL-8 to its receptor, Clark-Lewis et al. (32) reported a 6-fold decrease in affinity for a human IL-8 molecule in which Tyr 13 was replaced with Thr. Our findings of nitrotyrosine formation on IL-8 after peroxynitrite treatment are consistent with these observations and suggest that tyrosine nitration by peroxynitrite on IL-8 is a likely mechanism altering IL-8 binding and chemotactic function. However, peroxynitrite may potentially affect protein function by other mechanisms including nitration of methionine (33) or tryptophan (34) or formation of S-nitroso-thiol groups on cysteines (35). IL-8 does not contain methionine, but cysteines are present and form disulfide bonds.
Although NO and peroxynitrite are physiological regulators, they have been shown to alter cell respiration (36,37) and induce cell death (38). In the studies with SIN-1, neutrophils may have been exposed to peroxynitrite during their migration across the polycarbonate filter. However, neutrophils had no significant cytotoxicity as assessed by trypan blue exclusion after incubation with peroxynitrite. Furthermore, incubation of neutrophils with peroxynitrite had no significant effect on NCA induced by IL-8.
In summary, we found that peroxynitrite modulates IL-8 FIG. 4. Detection of nitrotyrosine on IL-8 incubated with peroxynitrite. IL-8 (10 Ϫ7 g/ml) was incubated with 10 Ϫ4 M of peroxynitrite for 2 h. IL-8 with and without peroxynitrite incubation was added to the IL-8 antibody coated (A) or allowed to adsorb to an uncoated (B) plates (see "Experimental Procedures" for details). Optical density is shown on the ordinate, and the experimental groups are shown on the abscissa. *, p Ͻ 0.01 compared with IL-8 not incubated with peroxynitrite.
FIG. 5. Effects of peroxynitrite on IL-8 binding to neutrophils. IL-8 (10 Ϫ7 g/ml) was incubated with and without peroxynitrite (OONO Ϫ , 10 Ϫ4 M) for 2 h at 37°C, and subsequently, IL-8 was incubated with neutrophils (10 5 cells and 10 6 cells) at 37°C for 30 min. IL-8 binding to neutrophils was measured by ELISA. IL-8 concentration is shown on the ordinate, and the experimental groups are shown on the abscissa. *, p Ͻ 0.01 compared with IL-8 incubated with medium alone.