help button home button
AJRCMB
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sato, E.
Right arrow Articles by Robbins, R. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sato, E.
Right arrow Articles by Robbins, R. A.
Am. J. Respir. Cell Mol. Biol., Volume 22, Number 1, January 2000 61-67

Effects of Reactive Oxygen and Nitrogen Metabolites on Eotaxin-Induced Eosinophil Chemotactic Activity In Vitro

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

Research Services, Tucson and Overton Brooks VA Medical Centers, and the Department of Medicine, University of Arizona, Tucson, Arizona; Department of Molecular and Cellular Physiology, LSU Medical Center, Shreveport, Louisiana; and The First Department of Internal Medicine, Shinshu University School of Medicine, Matsumoto, Japan


    Abstract
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Peroxynitrite, an oxidant generated by the interaction between superoxide and nitric oxide (NO), has been implicated in the etiology of numerous disease processes. Several studies have shown that peroxynitrite-induced protein nitration may compromise enzyme and protein function. We hypothesized that peroxynitrite may regulate cytokine function during inflammation. To test this hypothesis, the eosinophil chemotactic responses of eotaxin incubated with and without peroxynitrite were evaluated. Peroxynitrite attenuated eotaxin-induced eosinophil chemotactic activity (ECA) in a dose-dependent manner (P < 0.05). The inhibitory effects were not significant on ECA induced by leukotriene B4 or complement-activated serum incubated with peroxynitrite. The reducing agents deferoxamine and dithiothreitol reversed the ECA inhibition by peroxynitrite, and exogenous L-tyrosine abrogated the inhibition by peroxynitrite. PAPA-NONOate (an NO donor) or a combination of xanthine and xanthine oxidase to generate superoxide did not show an inhibitory effect on ECA induced by eotaxin. In contrast, 3-morpholinosydnonimine, a peroxynitrite generator, caused a concentration-dependent inhibition of ECA by eotaxin. Consistent with its capacity to reduce ECA, peroxynitrite treatment reduced eotaxin binding to eosinophils. Nitrotyrosine was detected in the eotaxin incubated with peroxynitrite. These findings are consistent with nitration of tyrosine by peroxynitrite with subsequent inhibition of eotaxin binding to eosinophils and a reduction in ECA. These data demonstrate that peroxynitrite modulates the eosinophil migration by eotaxin, and suggest that oxidants may play an important role in regulation of eotaxin-induced eosinophil chemotaxis.


    Introduction
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Eotaxin is a recently characterized chemokine that has selective chemotactic activity for eosinophils. Eotaxin increases the chemotaxis of eosinophils, but not of neutrophils, monocytes, or lymphocytes, in vitro (1). In guinea pigs, eotaxin causes the accumulation of eosinophils within the lung, and Northern blot analysis demonstrates constitutive expression of eotaxin messenger RNA (mRNA) in the airways that increases considerably after allergen challenge (2, 3). It has been suggested that eotaxin contributes to the pathogenesis of asthma by the specific recruitment of eosinophils into the airways (5).

Increased production of nitric oxide (NO) and superoxide, components of peroxynitrite, have been implicated in the pathogenesis of asthma (6, 7). Levels of NO are elevated in the air exhaled by persons with asthma (8), and may contribute to airway edema and mucus hypersecretion (6). High levels of superoxide anion have also been found in bronchoalveolar lavage fluid (BALF) of asthmatic patients (9). Moreover, superoxide dismutase activity is reduced in the leukocytes of asthmatic patients (10), and increased formation of peroxynitrite in the airways of asthmatic patients has been reported (11). In view of these findings, chemotactic factors, including eotaxin, are likely to encounter high local concentrations of NO, superoxide, and peroxynitrite in inflammatory sites.

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 (12). Several studies have shown that peroxynitrite-induced protein nitration may compromise protein function (13). We hypothesized that peroxynitrite might regulate eosinophil recruitment by modulating eotaxin. To test this hypothesis, the chemotactic responses of human eosinophils to eotaxin 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 eotaxin-induced eosinophil chemotactic activity (ECA). In contrast, activated serum and leukotriene B4 (LTB4)-induced ECA was not inhibited by peroxynitrite significantly. These data suggest that peroxynitrite plays an important role in regulating ECA during inflammation.

    Materials and Methods
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Measurement of ECA

Eosinophils were isolated with a modified method of Hansel and colleagues (14) with a magnetic cell-separation system. Briefly, venous blood anticoagulated with 130 mM trisodium citrate was obtained from normal human volunteers and diluted with phosphate-buffered saline (PBS) in a 1:1 ratio. Diluted blood was overlayered on an isotonic Percoll solution (density 1.082 g/ml; Sigma, St. Louis, MO), then centrifuged at 1,000 × g for 30 min 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 [1,4-piperazine-bis (ethane sulfonic acid)] (Pipes) buffer (25 mM Pipes, 50 mM NaCl, 5 mM KCl, 25 mM NaOH, and 5.4 mM glucose, pH 7.4) containing 1% defined calf serum (DCS) (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 a 60-min incubation on ice, 5 ml of Pipes buffer with 1% DCS 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% DCS. Purity of the eosinophils counted by Randolph's stain was > 94%. Viability was > 98%, assessed by eosin or trypan blue exclusion. 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) as previously described (15). The bottom wells of the chamber were filled with 25 µl of the chemotactic stimulus or media 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 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. ECA was expressed as the mean number of migrated cells per high-power field from duplicate wells.

Effects of Peroxynitrite on Eotaxin-Induced ECA

Peroxynitrite was evaluated for its capacity to modulate eotaxin-induced ECA in vitro. Eotaxin (R&D Systems, Minneapolis, MN) was incubated for 2 h at 37°C with each concentration of peroxynitrite (Calbiochem, La Jolla, CA) before the ECA assay. In control experiments, eotaxin was incubated with medium alone.

Effects of Peroxynitrite on LTB4 and Activated, Serum-Induced ECA

The capacity of peroxynitrite to modulate LTB4 and activated, serum-induced ECA was evaluated to compare with eotaxin. LTB4 (1 µm; Sigma) or complement-activated serum (16) (1:10 dilution) was incubated with peroxynitrite (100 µM) for 2 h at 37°C before performing the ECA assay.

Effects of PAPA-NONOate on Eotaxin-Induced ECA

To evaluate the ability of NO to modulate eotaxin-induced ECA, we utilized PAPA-NONOate (Alexis Corp., San Diego, CA) as a NO donor (17). Eotaxin (100 ng/ml) was incubated with PAPA-NONOate (10-6, 10-5, 10-4, and 10-3 M) for 2 h at 37°C before performing the ECA assay.

Effects of Xanthine/Xanthine Oxidase on Eotaxin-Induced ECA

To evaluate the effect of superoxide on eotaxin-induced ECA, xanthine (10-6, 10-5, 10-4, and 10-3 M; Sigma) and xanthine oxidase (3.4 × 10-6, 3.4 × 10-5, 3.4 × 10-4, and 3.4 × 10-3 U/ml; Sigma) were combined to produce superoxide (18). Eotaxin (100 ng/ml) was incubated with xanthine and xanthine oxidase for 2 h at 37°C before performing the ECA assay.

Effects of SIN-1 on Eotaxin-Induced ECA

The capacity of SIN-1 (Alexis Corp.), a peroxynitrite generator (19), to modulate eotaxin-induced ECA was evaluated by incubating eotaxin (100 ng/ml) with SIN-1 (10-4, 10-5, 10-6, and 10-7 M) for 2 h at 37°C before performing the ECA assay.

Effects of Reducing Agents on Peroxynitrite-Induced Attenuation of ECA by Eotaxin

The capacity of the reducing agents dithiothreitol (DTT) or deferoxamine (DEF) to attenuate the effect of peroxynitrite on eotaxin-induced ECA by peroxynitrite was assessed. DTT (10-3 M; Sigma), DEF (5 × 10-5 M; Sigma) and peroxynitrite (10-4 M) were added to eotaxin (100 ng/ ml) and incubated for 2 h at 37 °C before evaluation for ECA.

Effects of L-Tyrosine on Peroxynitrite-Induced Attenuation of ECA by Eotaxin

The capacity of L-tyrosine to reserve the attenuation of ECA induced by peroxynitrite was assessed by addition of L-tyrosine (10-3, and 10-4 M; Sigma) to eotaxin (100 ng/ml) before exposure to peroxynitrite (10-4 M).

Detection of Nitrotyrosine on Eotaxin Incubated with Peroxynitrite

Detection of nitrotyrosine on eotaxin incubated with peroxynitrite was evaluated using a modification of a previously described enzyme-linked immunosorbent assay (ELISA) (20). Eotaxin (100 ng/ml) was incubated with peroxynitrite (10-4 M) as described earlier and frozen until assayed. A total of 200 µl of eotaxin was added to flat-bottomed 96-well plates (Costar, Cambridge, MA) and allowed to adsorb to the plastic for 2 h at 4°C. After washing the flat-bottomed plate three times with PBS-Tween, 200 µl of a 1:400 dilution of rabbit polyclonal antinitrotyrosine (Calbiochem) was added to the wells and incubated for 90 min. After again washing three times with PBS-Tween, 200 µl of a 1:500 dilution of peroxidase-conjugated antirabbit immunoglobulin G was added to the wells and incubated for 90 min. A total of 200 µl of o-penylenediamine (100 µg/ml; Sigma) in 0.003% hydrogen peroxide was added and visually monitored. The reaction was terminated by addition of 25 µl of 8 N H2SO4 and the absorbance read at 490 nm.

Effects of Peroxynitrite on Eotaxin Binding to Eosinophils

To investigate the peroxynitrite effect on eotaxin binding to eosinophils, eotaxin was incubated with 100 µm of peroxynitrite for 2 h at 37°C. In control experiments, eotaxin was incubated with medium alone. Subsequently, eotaxin with or without peroxynitrite was incubated with eosinophils (106 cells) at 4°C for 30 min. Supernatants were then removed and eosinophils were washed three times in Hanks' balanced salt solution. Eosinophils were suspended in 1 ml PBS-Tween, sonicated for 20 s (MSE Soniprep, Crawley, UK), and then centrifuged at 20,000 × g for 30 min in a refrigerated microcentrifuge to obtain a supernatant (soluble) and particulate fraction. Eotaxin was then measured using a commercially available eotaxin ELISA (R&D Systems).

Statistics

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 means ± standard error of the mean.

    Results
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Effects of Peroxynitrite on ECA by Eotaxin

Various amounts of eotaxin were incubated with peroxynitrite (10-4 M). At each concentration, exposure to peroxynitrite caused a reduction in ECA (Figure 1; n = 6, P < 0.05). Incubation of eotaxin (100 ng/ml) with various amounts of peroxynitrite induced a significant, concentration-dependent attenuation of ECA (Figure 2; n = 6, P < 0.05). The lowest dose of peroxynitrite that significantly inhibited ECA was 10-5 M (P < 0.05). Peroxynitrite itself was not chemotactic for eosinophils and did not alter the pH of the eotaxin solution (data not shown). Incubation of peroxynitrite (10-4 M) with the eosinophils before the chemotaxis assay did not inhibit ECA to eotaxin (data not shown). The stable end products of peroxynitrite, nitrite, and nitrate (10-4 M) did not inhibit ECA.


View larger version (16K):
[in this window]
[in a new window]
  		Figure 1.   Inhibition of ECA by peroxynitrite. ECA is on the ordinate and eotaxin concentration is on the abscissa. Eotaxin was incubated for 2 h in the presence of medium alone or 100 µM peroxynitrite; n = 6 each condition. Open circles represent ECA by eotaxin incubated with 10-4 M peroxynitrite, and closed circles represent ECA by eotaxin without peroxynitrite incubation. *P < 0.05 compared with ECA by eotaxin incubated with medium alone.


View larger version (15K):
[in this window]
[in a new window]
  		Figure 2.   Dose-responsive inhibition of ECA by peroxynitrite. A total of 100 ng/ml of eotaxin was cultured for 2 h in the presence of medium alone or peroxynitrite at the concentrations indicated; n = 6 each condition. ECA is on the ordinate and the concentration of peroxynitrite is on the abscissa. *P < 0.05 compared with eotaxin incubated with medium alone.

Effects of Peroxynitrite on LTB4 and Activated, Serum-Induced ECA

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).


View larger version (18K):
[in this window]
[in a new window]
  		Figure 3.   Effect of peroxynitrite on ECA induced by eotaxin, activated serum, and LTB4. Eotaxin, activated serum, and LTB4 were cultured for 2 h in the presence of medium alone or 10-4 M peroxynitrite; n = 4 each condition. ECA is on the ordinate and the experimental groups are on the abscissa. *P < 0.05 compared with eotaxin incubated with medium alone.

Effects of PAPA-NONOate on Eotaxin-Induced ECA

To evaluate the capacity of NO to modulate eotaxin-induced ECA, the effect of the NO donor PAPA-NONOate was evaluated. PAPA-NONOate did not significantly change ECA induced by eotaxin (data not shown, P > 0.05, all comparisons).

Effects of Xanthine/Xanthine Oxidase on Eotaxin-Induced ECA

We investigated the effect of superoxide on ECA induced by eotaxin and eotaxin incubated with xanthine and xanthine oxidase. Xanthine and xanthine oxidase did not change ECA by eotaxin (data not shown, P > 0.05, all comparisons).

Effects of SIN-1 on Eotaxin-Induced ECA

SIN-1, a nitrovasodilator, spontaneously decomposes under aqueous conditions, generating first O2- and then NO at comparable rates, forming peroxynitrite (21). SIN-1 induced a significant, concentration-dependent attenuation of ECA by eotaxin (Figure 4; n = 6, P < 0.05). The lowest dose of SIN-1 to inhibit ECA was 10-5 M (P < 0.05). The quantity of 100 µM of SIN-1 at a concentration of 10-4 M induced 85% attenuation of ECA by eotaxin. SIN-1 itself was not chemotactic for eosinophils (data not shown).


View larger version (15K):
[in this window]
[in a new window]
  		Figure 4.   Effect of SIN-1 on ECA induced by eotaxin. Eotaxin was cultured for 2 h in the presence of medium alone or SIN-1 at the concentrations indicated; n = 6 each condition. ECA is on the ordinate and SIN-1 concentration is on the abscissa. *P < 0.05 compared with eotaxin incubated with medium alone.

Effects of Reducing Agents on Peroxynitrite-Induced Attenuation of ECA by Eotaxin

The reducing agents DTT and DEF were added to eotaxin before incubating with peroxynitrite. Each attenuated the inhibition of ECA induced by peroxynitrite (Figure 5; n = 4, P < 0.05). Neither DTT nor DEF alone was chemotactic for eosinophils (data not shown).


View larger version (21K):
[in this window]
[in a new window]
  		Figure 5.   Effect of DTT and DEF on peroxynitrite-induced attenuation of ECA by eotaxin. Eotaxin was cultured with peroxynitrite for 2 h in the presence of DTT and DEF at the concentrations indicated; n = 4 each condition. ECA is on the ordinate and the experimental groups are on the abscissa. *P < 0.05 compared with eotaxin incubated with peroxynitrite.

Effects of L-Tyrosine on Peroxynitrite-Induced Attenuation of ECA by Eotaxin

One mechanism of peroxynitrite inhibition may be through nitrating tyrosine residues. Therefore, the effect of addition of L-tyrosine to eotaxin before incubating with peroxynitrite was investigated. Addition of L-tyrosine to eotaxin abrogated the attenuation of ECA induced by peroxynitrite (Figure 6; n = 4, P < 0.05). The addition of 10-4 M of L-tyrosine prevented the inhibition of ECA induced by 10-4 M peroxynitrite. L-Tyrosine itself was not chemotactic for eosinophils (data not shown).


View larger version (17K):
[in this window]
[in a new window]
  		Figure 6.   Effect of L-tyrosine on peroxynitrite-induced attenuation of ECA by eotaxin. Eotaxin was cultured with 10-5 M of peroxynitrite for 2 h in the presence of medium alone or L-tyrosine at the concentrations indicated; n = 4 each condition. ECA is on the ordinate and the experimental groups are on the abscissa. *P < 0.05 compared with eotaxin incubated with peroxynitrite.

Detection of Nitrotyrosine on Eotaxin Incubated with Peroxynitrite

The optical density of eotaxin with peroxynitrite incubation was significantly higher than that of eotaxin without peroxynitrite incubation. Peroxynitrite resulted in nitrotyrosine formation on eotaxin (Figure 7; n = 6, P < 0.05). Peroxynitrite (10-4 M) incubated with Voller's buffer for 2 h did not affect binding in the ELISA.


View larger version (13K):
[in this window]
[in a new window]
  		Figure 7.   Detection of nitrotyrosine on eotaxin incubated with peroxynitrite. Eotaxin was incubated with 10-4 M of peroxynitrite for 2 h; n = 6 each condition. Optical density is on the ordinate and the experimental groups are on the abscissa. *P < 0.05 compared with eotaxin incubated without peroxynitrite.

Effects of Peroxynitrite on Eotaxin Binding to Eosinophils

Eotaxin induces chemotactic activity by binding to eosinophils. Addition of peroxynitrite to eotaxin resulted in an inhibition of eotaxin binding to eosinophils (Figure 8; n = 4, P < 0.05). Peroxynitrite had no significant effect on the eotaxin ELISA at the same concentration (data not shown).


View larger version (14K):
[in this window]
[in a new window]
  		Figure 8.   Effect of peroxynitrite on eotaxin binding to eosinophils. Eotaxin was incubated with and without 10-4 M of peroxynitrite for 2 h at 37°C; after that, eotaxin was incubated with eosinophils (10-6 cells) at 37°C for 30 min. Eotaxin binding to eosinophils was measured by ELISA; n = 4 each condition. Eotaxin concentration is on the ordinate and the experimental groups are on the abscissa. *P < 0.05 compared with eotaxin incubated with medium alone.

    Discussion
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

The results of this study show that the peroxynitrite significantly attenuated eotaxin-induced ECA in vitro. The inhibitory effects of peroxynitrite were not significant on ECA induced by LTB4 and activated serum. DEF, DTT, or L-tyrosine attenuated the inhibition. NO or superoxide did not cause the reduction in ECA because neither PAPA-NONOate nor xanthine/xanthine oxidase showed an inhibitory effect. Addition of peroxynitrite to eotaxin resulted in an inhibition of eotaxin binding to eosinophils. The peroxynitrite donor SIN-1 induced a significant, concentration-dependent inhibition of ECA by eotaxin. These data suggest that peroxynitrite plays an important role in regulating human eosinophil locomotion by eotaxin.

Eotaxin is a recently characterized chemokine that has selective chemotactic activity for eosinophils. Atopic asthmatic patients have high concentrations of eotaxin in BALF and an increased expression of eotaxin mRNA in the epithelium and submucosa of the airways (5). Challenge of the airways of sensitized guinea pigs with aerosolized ovalbumin resulted in eosinophil accumulation and increase of eotaxin in the airway tissue and BALF (22). The above data suggest that eotaxin may contribute to the pathogenesis of asthma by the specific recruitment of eosinophils into the airways.

NO and superoxide are both formed by endothelial cells, neutrophils, and macrophages. Recent evidence suggests that under certain conditions, NO synthase can simultaneously generate both superoxide and NO (23). When produced in proximity, superoxide and NO can react at an almost diffusion-limited rate to produce peroxynitrite (24). Peroxynitrite is an oxidizing and nitrating agent that reacts with a variety of molecules, including lipids, proteins, and deoxyribonucleic acid (25). Peroxynitrite, or one of its intermediate reactive species, can react with phenolic compounds such as tyrosine to form nitrated (3-nitrotyrosine) products (29, 30). Stable 3-nitrotyrosine products have been detected in tissues after asthma (11) and acute lung injury (31, 32). Thom and associates (33) reported that nitrotyrosine concentration in lung homogenates of 30 to 60 ng/mg protein. If peroxynitrite concentrations were of the same order, the peroxynitrite concentration in this experiment should be sufficient to modulate eotaxin function.

Coincubation of eotaxin with several peroxynitrite scavengers ameliorated ECA inhibition. The protective effect of DTT on eotaxin may suggest the involvement of cysteine residues nitrosylation, however DTT also prevented peroxynitrite-mediated nitration of tyrosine (34). The iron chelator DEF also inhibited peroxynitrite-induced inhibition of eotaxin ECA but is also scavenger of peroxynitrite reaction independent of iron chelation (35). In addition, L-tyrosine abrogated the peroxynitrite ECA inhibition. These results are consistent with tyrosine nitration by peroxynitrite or its intermediates as a mechanism for eotaxin inhibition.

Some studies reported that NO donors or NO synthase inhibitors have produced variable effects on phagocyte chemotaxis in vitro (36). The effect of peroxynitrite on eosinophils is negligible in our assay because the half-time of peroxynitrite is so short that eosinophils would not have a peroxynitrite effect after the 2-h incubation time. We also incubated eosinophils with peroxynitrite for 90 min at 37°C before the chemotaxis experiments and induced no significant cytotoxicity on eosinophils as assessed by trypan blue exclusion. Lastly, peroxynitrite had no significant effect when incubated with the eosinophils on ECA to eotaxin.

Chemokines induce leukocyte migration by binding to specific G protein-coupled, seven transmembrane-spanning, cell-surface receptors. Although the mechanism of binding eotaxin to eosinophil receptors has not been understood, the N-terminal and loop regions of the C-C chemokines have been thought to be important for receptor binding and signaling. Tyrosine residues have been reported to be important in the binding of monocyte chemoattractant protein-1 (MCP-1), another C-C chemokine, to its receptor. Steitz and coworkers (41) reported that point mutations of Tyr-13 greatly lowered MCP-1 receptor binding and activity. Zhang and colleagues (42) reported that changing Tyr-28 to aspartate essentially abolished MCP-1's monocyte chemoattractant activity. Interestingly, the amino acid sequence of eotaxin from 48 through 54 is identical to 27 through 33 on MCP-1 and both sequences contain tyrosine residues (41, 43). Our findings of nitrotyrosine formation on eotaxin after peroxynitrite incubation are consistent with these observations, and suggest that tyrosine nitration by peroxynitrite may be a mechanism that alters eotaxin binding and chemotactic function. However, peroxynitrite may potentially affect protein function by other mechanisms including methionine (44), tryptophan (45), or formation of s-nitroso-thiol groups on cysteines (46).

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 been demonstrated (47). Nitrogen dioxide (NO2) promotes tyrosine nitration through formation of nitryl chloride (Cl-NO2) and NO2 by reaction with the inflammatory mediators hypochlorous acid (HOCl) or myeloperoxidase. Peroxidases may potentially affect protein function by not only nitrating but also chlorinating or brominating tyrosine residues (48). Therefore, it cannot be stated definitively that the formation of nitrotyrosine in vivo is due to peroxynitrite.

In summary, we found that peroxynitrite modulates ECA in vitro, and it is suggested that nitration of a tyrosine residue is responsible for inhibition of eotaxin-induced ECA. These data suggest that peroxynitrite attenuates eotaxin-induced chemotactic activity and plays an important role in regulating human eosinophil locomotion during inflammation.

    Footnotes

Abbreviations: bronchoalveolar lavage fluid, BALF; defined calf serum, DCS; deferoxamine, DEF; dithiothreitol, DTT; eosinophil chemotactic activity, ECA; enzyme-linked immunosorbent assay, ELISA; leukotriene B4, LTB4; monocyte chemoattractant protein-1, MCP-1; nitric oxide, NO; phosphate-buffered saline, PBS; 3-morpholinosydnonimine, SIN-1.

(Received in original form December 14, 1998 and in revised form July 6, 1999).

Acknowledgments: This work was supported by a Merit Review grant from the Veterans' Administration, Department of Veterans Affairs, and a grant from Rotary International.
    References
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

1. Garcia-Zepeda, E. A., M. E. Rothenberg, R. T. Ownbey, J. Celestin, P. Leder, and A. D. Luster. 1996. Human eotaxin is a specific chemoattractant for eosinophil cells and provides a new mechanism to explain tissue eosinophilia. Nat. Med. 2: 449-456 [Medline].

2. Griffiths-Johnson, D. A., P. D. Collins, A. G. Rossi, P. J. Jose, and T. J. Williams. 1993. The chemokine, eotaxin, activates guinea-pig eosinophils in vitro and causes their accumulation into the lung in vivo. Biochem. Biophys. Res. Commun. 197: 1167-1172 [Medline].

3. Jose, P. J., D. A. Griffiths-Johnson, P. D. Collins, D. T. Walsh, R. Moqbel, N. F. Totty, O. Truong, J. J. Hsuan, and T. J. Williams. 1994. Eotaxin: a potent eosinophil chemoattractant cytokine detected in a guinea pig model of allergic airways inflammation. J. Exp. Med. 179: 881-887 [Abstract/Free Full Text].

4. Ponath, P. D., S. Qin, D. J. Ringler, I. Clark-Lewis, J. Wang, N. Kassam, H. Smith, X. Shi, J. A. Gonzalo, W. Newman, J. C. Gutierrez-Ramos, and C. R. Mackayk. 1996. Cloning of the human eosinophil chemoattractant, eotaxin: expression, receptor binding, and functional properties suggest a mechanism for the selective recruitment of eosinophils. J. Clin. Invest. 97: 604-612 [Medline].

5. Lamkhioued, B., P. M. Renzi, S. Abi-Younes, E. A. Garcia-Zepada, Z. Allakhverdi, O. Ghaffar, M. D. Rothenberg, A. D. Luster, and Q. Hamid. 1997. Increased expression of eotaxin in bronchoalveolar lavage and airways of asthmatics contributes to the chemotaxis of eosinophils to the site of inflammation. J. Immunol. 159: 4593-4601 [Abstract].

6. Robbins, R. A. 1997. Nitric oxide. In Asthma. P. J. Barnes, M. M. Grunstein, A. R. Leff, and A. J. Woolcock, editors. Lippincott-Raven, Philadelphia. 695-705.

7. Calhoun, W. J., H. E. Reed, D. R. Moest, and C. A. Stevens. 1992. Enhanced superoxide production by alveolar macrophages and air-space cells, airway inflammation, and alveolar macrophage density changes after segmental antigen bronchoprovocation in allergic subjects. Am. Rev. Respir. Dis. 145: 317-325 [Medline].

8. Kharitonov, S. A., D. Yates, R. A. Robbins, R. Logan-Sinclair, E. A. Shinebourne, and P. J. Barnes. 1994. Increased nitric oxide in exhaled air of asthmatic patients. Lancet 343: 133-135 [Medline].

9. Jarjour, N. N., W. W. Bussee, and W. J. Calhoun. 1992. Enahanced production of oxygen radicals in nocturnal asthma. Am. Rev. Respir. Dis. 146: 905-911 [Medline].

10. Joseph, B. Z., J. M. Routes, and L. Borish. 1993. Activities of superoxide dismutases and NADPH oxidase in neutrophils obtained from asthmatic and normal donors. Inflammation 17: 361-370 [Medline].

11. Saleh, D., P. Ernst, S. Lim, P. J. Barnes, and A. Giaid. 1998. 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. 12: 929-937 [Abstract/Free Full Text].

12. Radi, R., T. P. Cosgrove, J. S. Beckman, and B. A. Freeman. 1993. Peroxynitrite-induced luminol chemiluminescence. Biochem. J. 290: 51-57 .

13. Ischiropoulos, H., and A. B. al-Mehdi. 1995. Peroxynitrite-mediated oxidative protein modifications. FEBS Lett. 364: 279-282 [Medline].

14. Hansel, T. T., I. J. De Vries, T. Iff, S. Rihs, M. Wandzilak, S. Betz, K. Blaser, and C. Walker. 1991. An improved immunomagnetic procedure for the isolation of highly purified human blood eosinophils. J. Immunol. Methods 145: 105-110 [Medline].

15. Harvath, L., W. Falk, and E. J. Leonard. 1980. Rapid quanitification of neutrophil chemotaxis: use of polyvinylpyrrolidone-free polycarbonate membrane in a multiwell assembly. J Immunol. Methods 37: 39-45 [Medline].

16. Snyderman, R., H. Gewurz, and S. E. Mergenhagen. 1968. Interactions of the complement system with endotoxic lipopolysaccharide: generation of a factor chemotactic for polymorphonuclear leukocytes. J. Exp. Med. 128: 259-275 [Abstract].

17. Wink, D. A., J. A. Cook, D. Christodoulou, M. C. Krishna, R. Pacelli, S. Kim, W. DeGraff, J. Gamson, Y. Vodovotz, A. Russo, and J. B. Mitchell. 1997. Nitric oxide and some nitric oxide donor compounds enhance the cytotoxicity of cisplatin. Nitric Oxide 1: 88-94 . [Medline]

18. Fridovich, I.. 1970. Quantitative aspects of the production of superoxide anion radical by milk xanthine oxidase. J. Biol. Chem. 245: 4053-4057 [Abstract/Free Full Text].

19. Holm, P., H. Kankaanranta, T. Metsa-Ketela, and E. Moilanen. 1998. Radical releasing properties of nitric oxide donors GEA 3162, SIN-1 and S-nitroso-N-acetylpenicillamine. Eur. J. Pharmacol. 346: 97-102 [Medline].

20. Jones, K. L., T. W. Bryan, P. A. Jinkins, K. L. Simpson, M. B. Grisham, M. W. Owens, S. A. Milligan, B. A. Markewitz, and R. A. Robbins. 1998. Superoxide released from neutrophils cause a reduction in nitric oxide gas. Am. J. Physiol. 275: L1120-L1126 [Abstract/Free Full Text].

21. Darley-Usmar, V. M., N. Hogg, V. J. O'Leary, M. T. Wilson, and S. Moncada. 1992. The simultaneous generation of superoxide and nitric oxide can initiate lipid peroxidation in human low density lipoprotein. Free Radic. Res. Commun. 17: 9-20 [Medline].

22. Humbles, A. A., D. M. Conroy, S. Marleau, S. M. Rankin, R. T. Palframan, A. E. Proudfoot, T. N. Wells, D. Li, P. K. Jeffery, D. A. Griffiths-Johnson, T. J. Williams, and P. J. Jose. 1997. Kinetics of eotaxin generation and its relationship to eosinophil accumulation in allergic airways disease: analysis in a guinea pig model in vivo. J. Exp. Med. 186: 601-612 [Abstract/Free Full Text].

23. Mayer, B., S. Pfeiffer, A. Schrammel, D. Koesling, K. Schmidt, and F. Brunner. 1998. A new pathway of nitric oxide/cyclic GMP signaling involving S-nitrosoglutathione. J. Biol. Chem. 273: 3264-3270 [Abstract/Free Full Text].

24. Huie, R. E., and S. Padmaja. 1993. The reaction of NO with superoxide. Free Radic. Res. Commun. 18: 195-199 [Medline].

25. Pryor, W. A., and G. L. Squadrito. 1995. The chemistry of peroxynitrite: a product from the reaction of nitric oxide with superoxide. Am. J. Physiol. 268: L699-L722 [Abstract/Free Full Text].

26. Salgo, M. G., E. Bermudez, G. L. Squadrito, and W. A. Pryor. 1995. Peroxynitrite causes DNA damage and oxidation of thiols in rat thymocytes. Arch. Biochem. Biophys. 322: 500-505 [Medline].

27. Rubbo, H., R. Radi, M. Trujillo, R. Telleri, B. Kalyanaraman, S. Barnes, M. Kirk, and B. A. Freeman. 1994. Nitric oxide regulation of superoxide and peroxynitrite-dependent lipid peroxidation. Formation of novel nitrogen-containing oxidized lipid derivatives. J. Biol. Chem. 269: 26066-26075 [Abstract/Free Full Text].

28. Beckman, J. S., T. W. Beckman, J. Chen, P. A. Marshall, and B. A. Freeman. 1990. Apparent hydroxyl radical production by peroxynitirite: implications for endothelial injury from nitric oxide and superoxide. Proc. Natl. Acad. Sci. USA 87: 1620-1624 [Abstract/Free Full Text].

29. Ishiropooulos, H., L. Zhu, J. Chen, M. Tsai, J.C. Martin, Smith, and J. S. Beckman. 1992. Peroxynitrite-mediated tyrosine nitration catalyzed by superoxide dismutase. Arch. Biochem. Biophys. 298: 431-437 [Medline].

30. van der Vliet, A., J. P. Eiserich, C. A. O'Neill, B. Halliwell, and C. E. Cross. 1995. Tyrosine modification by reactive nitrogen species: a closer look. Arch. Biochem. Biophys. 319: 341-349 [Medline].

31. Royall, J. A., N. W. Kooy, and J. S. Beckman. 1995. Nitric oxide-related oxidants in acute lung injury. New Horiz. 3: 113-122 [Medline].

32. Haddad, I. Y., G. Pataki, P. Hu, C. Galliani, J. S. Beckman, and S. Matalon. 1994. Quantitation of nitrotyrosine levels in lung sections of patients and animals with acute lung injury. J. Clin. Invest. 94: 2407-2413 .

33. Thom, S. R., S. T. Ohnishi, D. Fisher, Y. A. Xu, and H. Ischiropoulos. 1999. Pulmonary vascular stress from carbon monoxide. Toxicol. Appl. Pharmacol. 154: 12-19 [Medline].

34. Rohn, T. T., and M. T. Quinn. 1998. Inhibition of peroxynitrite-mediated tyrosine nitration by a novel pyrrolopyrimidine antioxidant. Eur. J. Pharmacol. 353: 329-336 [Medline].

35. Denicola, A., J. M. Souza, R. M. Gatti, O. Augusto, and R. Radi. 1995. Desferrioxamine inhibition of the hydroxyl radical-like reactivity of peroxynitrite: role of the hydroxamic groups. Free Radic. Biol. Med. 19: 11-19 [Medline].

36. Bath, P. M.. 1993. The effect of nitric oxide-donating vasodilators on monocyte chemotaxis and intracellular cGMP concentrations in vitro. Eur. J. Clin. Pharmacol. 45: 53-58 [Medline].

37. Moilanen, E., P. Vuorinen, H. Kankaanranta, T. Metsa-Ketela, and H. Vapaatalo. 1993. Inhibition by nitric oxide-donors of human polymorphonuclear leucocyte functions. Br. J. Pharmacol. 109: 852-858 [Medline].

38. Kaplan, S. S., T. Billiar, R. D. Curran, U. E. Zdziarski, R. L. Simmons, and R. E. Basford. 1989. Attenuation of chemotaxis with NG-monomethyl-L-arginine: a role for cyclic GMP. Blood 74: 1885-1887 [Abstract/Free Full Text].

39. Belenky, S. N., R. A. Robbins, S. I. Rennard, G. L. Gossman, K. J. Nelson, and I. Rubinstein. 1993. Inhibitors of nitric oxide synthase attenuate neutrophil chemotaxis in vitro. J. Lab. Clin. Med. 122: 388-394 [Medline].

40. Belenky, S. N., R. A. Robbins, and I. Rubinstein. 1993. Nitric oxide synthase inhibitors attenuate human monocyte chemotaxis in vitro. J. Leukoc. Biol. 53: 498-503 [Abstract].

41. Steitz, S. A., H. Hasegawa, S. L. Chiang, R. R. Cobb, M. A. Castro, T. J. Lobl, M. Yamada, E. Lazarides, and P. M. Cardarelli. 1998. Mapping of MCP-1 functional domains by peptide analysis and site-directed mutagenesis. FEBS Lett. 430: 158-164 [Medline].

42. Zhang, Y. J., B. J. Rutledge, and B. J. Rollins. 1994. 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. 269: 15918-15924 [Abstract/Free Full Text].

43. Crump, M. P., K. Rajarathnam, K. S. Kim, I. Clark-Lewis, and B. D. Sykes. 1998. Solution structure of eotaxin, a chemokine that selectively recruits eosinophils in allergic inflammation. J. Biol. Chem. 273: 22471-22479 [Abstract/Free Full Text].

44. Vogt, W.. 1995. Oxidation of methionyl residues in proteins: tools, targets, and reversal. Free Radic. Biol. Med. 18: 93-105 [Medline].

45. Alvarez, B., H. Rubbo, M. Kirk, S. Barnes, B. A. Freeman, and R. Radi. 1996. Peroxynitrite-dependent tryptophan nitration. Chem. Res. Toxicol. 9: 390-396 [Medline].

46. Gow, A. J., D. G. Buerk, and H. Ischiropoulos. 1997. A novel reaction mechanism for the formation of S-nitrosothiol in vivo. J. Biol. Chem. 272: 2841-2845 [Abstract/Free Full Text].

47. Eiserich, J. P., M. Hristova, C. E. Cross, A. D. Jones, B. A. Freeman, B. Halliwell, and A. van der Vliet. 1998. Formation of nitric oxide-derived inflammatory oxidants by myeloperoxidase in neutrophils. Nature 391: 393-397 [Medline].

48. Huber, R. E., L. A. Edwards, and T. J. Carne. 1989. Studies on the mechanism of the iodination of tyrosine by lactoperoxidase. J. Biol. Chem. 264: 1381-1386 [Abstract/Free Full Text].




This article has been cited by other articles:


Home page
 J. Immunol.Home page
J. L. Freels, D. K. Nelson, J. C. Hoyt, M. Habib, H. Numanami, R. C. Lantz, and R. A. Robbins
Enhanced Activity of Human IL-10 After Nitration in Reducing Human IL-1 Production by Stimulated Peripheral Blood Mononuclear Cells
J. Immunol., October 15, 2002; 169(8): 4568 - 4571.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
A. DUGUET, H. IIJIMA, S.-Y. EUM, Q. HAMID, and D. H. EIDELMAN
Eosinophil Peroxidase Mediates Protein Nitration in Allergic Airway Inflammation in Mice
Am. J. Respir. Crit. Care Med., October 1, 2001; 164(7): 1119 - 1126.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
P. J. Kingham, W. G. McLean, D. A. Sawatzky, M. T. Walsh, and R. W. Costello
Adhesion-dependent interactions between eosinophils and cholinergic nerves
Am J Physiol Lung Cell Mol Physiol, June 1, 2002; 282(6): L1229 - L1238.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sato, E.
Right arrow Articles by Robbins, R. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sato, E.
Right arrow Articles by Robbins, R. A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Proc. Am. Thorac. Soc. Am. J. Respir. Crit. Care Med.
Copyright © 2000 American Thoracic Society.