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The Journal of Immunology, 1999, 162: 2922-2930.
Copyright © 1999 by The American Association of Immunologists

Effect of Nitric Oxide Donors on Oxygen-Dependent Cytotoxic Responses Mediated by Neutrophils1

G. Andonegui2,*,{dagger}, A. S. Trevani*,{dagger}, R. Gamberale*,{dagger}, M. C. Carreras{ddagger}, J. J. Poderoso{ddagger}, M. Giordano*,{dagger} and J. R. Geffner*,{dagger}

* Laboratory of Immunology, Institute of Hematologic Research, National Academy of Medicine, Buenos Aires, Argentina; and {dagger} Department of Microbiology and {ddagger} Laboratory of Oxygen Metabolism, University Hospital, Buenos Aires University School of Medicine, Buenos Aires, Argentina. The National Academy of Medicine is an organism independent from the Buenos Aires University and both the Department of Microbiology and the Laboratory of Oxygen Metabolism, University Hospital depend on Buenos Aires University School of Medicine.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We analyzed the effect of nitric oxide (NO) on oxygen-dependent cytotoxic responses mediated by neutrophils against unopsonized erythrocytes using three NO donors: S-nitrosoglutathione (GSNO), S-nitroso-N-acetylpenicillamine (SNAP), and sodium nitroprusside (SNP). Neutrophils were treated with these compounds for 1–2 min at 37°C and cytotoxicity was then triggered in the presence of NO donors by precipitating immune complexes, aggregated IgG, the chemotactic peptide FMLP, or opsonized zymosan. GSNO induced, in all cases, a marked increase in cytotoxic responses, while SNAP moderately increased cytotoxicity triggered by immune complexes, aggregated IgG, or Z, opsonized zymosen, without modifying those responses induced by FMLP. By contrast, SNP dramatically suppressed cytotoxicity triggered by all of the stimuli assessed. The enhancing effects mediated by GSNO and SNAP did not depend on the stimulation of guanylyl cyclase and were prevented by the NO scavengers hemoglobin and PTIO (2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl 3-oxide). The inhibitory activity of SNP, on the other hand, was not prevented by NO scavengers, suggesting that it cannot be ascribed to the release of NO. In another set of experiments, neutrophils were pretreated with GSNO or SNAP for different times. Then cells were washed to remove NO donors from the culture medium, and cytotoxicity was triggered by different stimuli. It was found that neutrophils must be pretreated with NO donors for at least 4 h to increase cytotoxic responses, and pretreatment for longer periods (i.e., 8 or 18 h) further increased cytotoxicity. Not only cytotoxic responses, but also the production of O2- and H2O2, and the release of myeloperoxidase were increased under these conditions.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Growing evidence suggests that nitric oxide (NO)3 inhibits neutrophil function. Adherence, chemotaxis, aggregation, and LTB4 synthesis are inhibited in neutrophils treated with NO or NO-releasing compounds 1, 2, 3, 4 . Contradictory results, on the other hand, have been published regarding the effect of NO on the production of oxygen-reactive intermediates (IRO) as well as on neutrophil degranulation 3, 4, 5, 6, 7, 8, 9 . Most of these modulatory actions of NO are believed to involve the activation of soluble guanylate cyclase. In addition, other mechanisms, such as the stimulation of the ADP-ribosylation of actin and the inactivation of the enzymes nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase and protein kinase C, have been proposed 1, 2, 3, 4, 6, 10, 11 .

In vivo studies support the notion that NO inhibits the inflammatory activity of neutrophils. In fact, the suppression of basal NO tone increases the attachment of neutrophils to postcapillary venules and their emigration into peripheral tissues, suggesting that the impairment of NO production by constitutive NO synthases results in a pattern of leukocyte adhesion and emigration that is characteristic of acute inflammation 12, 13, 14 . Administration of NO donors, on the other hand, has shown to inhibit the course of acute inflammatory processes. Andrews et al. 15 demonstrated that fewer neutrophils infiltrated the postischemic gastric mucosa after SNP administration, while Kurose et al. 16 showed that NO donors greatly inhibited leukocyte adhesion, emigration, and vascular dysfunction in postischemic mesentery.

Oxygen-dependent cytotoxic responses mediated by phagocytes play an important role in immune response against infectious agents 17, 18 . They involve a number of different mechanisms, all of which require the production of O2- by effector cells to induce target cell injury 17, 18 . While O2- can be directly cytotoxic, its chemical reactivity is, in general, limited compared with other free radicals. Cytotoxic potential of O2- appears to be dependent on its conversion to much stronger oxidants by secondary reactions 17, 18, 19, 20 . Since, at inflammatory sites, cells can produce not only O2-, H2O2, ·OH, and HOCl, but also NO 20, 21 , in this study we focused our attention on the analysis of the interplay between IRO and NO in regard to neutrophil cytotoxic ability. For this purpose, we employed three NO donors: GSNO, SNAP, and SNP 1, 2, 3, 4, 5, 6, 21 .


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

GSNO and SNP were obtained from Alexis (Leufelfingen Switzerland). SNAP, FMLP, zymosan, acridine orange, ethidium bromide, propidium iodide, catalase (from bovine liver, 50,000 U/mg protein), superoxide dismutase (SOD) (from bovine erythrocytes, 5,000 U/mg), sodium azide, sodium cyanide, N2, 2'-O-dibutyryl guanosine 3':5'-cyclic monophosphate (Bt2cGMP), 1H- 1, 2, 4 oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), cytochrome c, 4-aminoantipyrine, horseradish peroxidase, and 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl 3-oxide (PTIO) were from Sigma (St. Louis, MO). Precipitating immune complexes (IC) were prepared at the equivalence zone, as we previously described 22 , using human IgG (Sigma) as Ag and specific rabbit IgG Abs to human IgG. Equivalence points were determined by quantitative precipitin curves. In all cases, Ags and Abs were incubated for 1 h at 37°C and 18 h at 4°C. After this period, IC were centrifuged at 3000 x g for 10 min, the precipitate was recovered, and it was resuspended in culture medium. The IgG aggregates (aIgG) were prepared by heating human IgG at a concentration of 5 mg/ml for 12 min at 63°C. Then aIgG was centrifuged at 10,000 x g for 5 min and the precipitate was discarded. Opsonized zymosan (Z) was prepared by incubating 100 mg of zymosan with 10 ml of normal human serum for 30 min at 37°C. After washing, Z was resuspended in culture medium.

Blood samples

Blood samples were obtained from healthy donors who had taken no medication for at least 10 days before the day of sampling. Blood was obtained by venipuncture of the forearm vein, and it was drawn directly into heparinized plastic tubes.

Effector cells

Neutrophils were isolated by Ficoll-Hypaque gradient centrifugation (Ficoll Pharmacia, Uppsala, Sweden; Hypaque, Winthrop Products, Buenos Aires, Argentina) and dextran sedimentation, as described 23 . Contaminating erythrocytes were removed by hypotonic lysis. After washing, the cells (more than 96% of neutrophils on May Grünwald/Giemsa-stained cytopreps) were resuspended in RPMI 1640 (Life Technologies, Detroit, MI) supplemented with 1% of FCS (Life Technologies).

Treatment with NO donors

Neutrophils (3 x 106/ml, in RPMI 1640 supplemented with 1% FCS) were incubated with NO donors for 1–2 min, 30 min, 1 h, 2 h, 4 h, 8 h, or 18 h at 37°C, in 5% CO2-95% humidified air.

Cytotoxic assay

In a first set of experiments, neutrophils were cultured in the absence or presence of NO donors for 1–2 min and, after this time, cytotoxic responses were triggered by IC, aIgG, FMLP, or Z, without removal of NO donors from the culture medium. In a second set of experiments, neutrophils were pretreated with NO donors for different times (1–2 min, 30 min, 1 h, 2 h, 4 h, 8 h, and 18 h). At each time point, neutrophils were washed to remove NO donors from the culture medium, targets were added, and cytotoxicity was triggered by IC, aIgG, FMLP, or Z. Cytotoxic assays were performed as we previously described 24 . Briefly, neutrophils (2 x 105 in 0.1 ml of RPMI 1640 supplemented with 1% FCS) and unopsonized 51Cr-labeled chicken RBC (2 x 105) were incubated for 18 h at 37°C in 5% CO2-95% humidified air. After this time, the culture plate was centrifuged, the radioactivity of supernatants and pellets was measured in a gamma counter, and the percentage of lysis was determined. Spontaneous release was always less than 6%. In no cases, treatment with NO donors, in the absence of triggering stimuli, increased spontaneous 51Cr release.

Superoxide anion generation

Superoxide anion was recorded continuously by monitoring the superoxide dismutase-inhibitable reduction of cytochrome c at 550 nm, using a Hitachi U 1100 spectrophotometer at 37°C, as previously described 25 .

Hydrogen peroxide generation

Hydrogen peroxide was measured by the horseradish peroxidase/p-hydroxyphenyl acetic acid (4-HPA) assay at 37°C using a Hitachi F-2000 spectrofluorometer at 315–425 nm, as previously described 26 .

Myeloperoxidase release

MPO release to the extracellular medium was evaluated by using 4-aminoantipyrine as chromogen, as previously described 27 . Briefly, 5 x 106 neutrophils/ml in RPMI medium containing 1% FCS were treated with GSNO (100 µM) for 8 or 18 h. After washing, cells were triggered by IC, aIgG, FMLP, or Z for 30 min at 37°C. An aliquot of 100 µl of each supernatant was added to a 3-ml cuvette containing 1.3 ml of 2.5 mM 4- aminoantipyrine in 215 mM phenol (Merck, Darmstadt, Germany) and 1.5 ml of freshly prepared 1.7 mM H2O2. The increase in A510 was measured for 5 min with continuous stirring at 25°C in a Hewlett-Packard 8452 A spectrophotometer. The total content of MPO in neutrophils was determined in a similar manner after treating the cells with 0.1% Triton X-100, followed by sonication for 1 min. Results were expressed as percent release of total MPO content.

Shape change assay

This assay was performed as described previously 28 . Briefly, neutrophils (1.5 x 105) suspended in 100 µl of culture medium supplemented with 1% FCS were cultured with or without 10-7 M FMLP for 15 min at 37°C in a shaking water bath. After washing, cells were suspended in PBS and fixed by the addition of an equal volume of 0.5% glutaraldehyde in PBS. Shape change was assayed in a FACS analyzer (Becton Dickinson, San Jose, CA). Results were expressed in mean forward light scatter units.

CD18 expression

Neutrophils (1.5 x 105) suspended in 100 µl of culture medium supplemented with 1% FCS were cultured with or without 10-7 M FMLP for 15 min at 37°C, fixed with 1% paraformaldehyde (1 h at room temperature), and washed with PBS. Then cells were stained with anti-CD18 mAb (IB4; Immunotech, Marseille, France) and FITC-conjugated goat anti-mouse IgG (Sigma). Flow cytometry was performed on a FACS analyzer (Becton Dickinson). Fluorescence intensity was determined on 10,000 cells from each sample. Results were expressed as the mean fluorescence intensity in arbitrary fluorescence units.

Adherence

Adherence was assessed as previously described 29 . Briefly, neutrophils were suspended in RPMI 1640 medium supplemented with 1% FCS and were labeled with 51CrO4Na2 (1 µCi/106 cells) for 1 h at 37°C. The cells were then washed four times with saline and resuspended in RPMI 1640 medium supplemented with 10% FCS, to a density of 4 x 106 cells/ml. One hundred microliters of this suspension were added to each well in 96-well flat-bottom polystyrene plates. Neutrophils were incubated in the presence or absence of NO donors for 1–2 min or 8 h. Then they were cultured with or without FMLP (10-7 M) for 15 min at 37°C in 5% CO2-95% humidified air. After this time, cells were washed three times with culture medium to remove nonadherent neutrophils. Adherent neutrophils were then lysed with 1 N NH4OH, and the radioactivity present in the lysates was measured. Cell adherence was expressed as the number of neutrophils that remained adherent to the plastic surface after washing.

Quantitation of cellular apoptosis and viability by fluorescence microscopy

Quantitation of cellular apoptosis and viability was performed as previously described, using the fluorescent DNA-binding dyes acridine orange (100 µg/ml), to determine the percentage of cells that had undergone apoptosis, and ethidium bromide (100 µg/ml), to differentiate between viable and nonviable cells 30 . With this method, nonapoptotic cell nuclei show variations in fluorescent intensity that reflect the distribution of euchromatin and heterochromatin. By contrast, apoptotic nuclei exhibit highly condensed chromatin that is uniformly stained by acridine orange. To assess the percentage of cells showing morphologic features of apoptosis, at least 200 cells were scored in each experiment.

Quantitation of neutrophil apoptosis by propidium iodide staining and flow cytometry

The proportion of neutrophils that displays a hypodiploid DNA peak, i.e., apoptotic cells, was determined using a modification of Nicoletti’s protocol 31 . Briefly, cell pellets containing 2.5 x 106 neutrophils were suspended in 400 µl of hypotonic fluorochrome solution (propidium iodide, 50 µg/ml in 0.1% sodium citrate plus 0.1% Triton X-100) and incubated for 2 h at 4°C. The red fluorescence of propidium iodide of individual nuclei was measured using a FACScan flow cytometer (Becton Dickinson). The forward scatter and side scatter of particles were simultaneously measured. Cell debris were excluded from analysis by appropriately raising the forward scatter threshold. The red fluorescence peak of neutrophils with normal (diploid) DNA content was set at channel 250 in the logarithmic mode. Apoptotic cell nuclei emitted fluorescence in channels 4–200.

Cyclic GMP assays

The assays were performed by RIA (Immunotech).

Statistical analysis

Results were compared using the Student’s t test, and p values <=0.05 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of NO donors on neutrophil cytotoxic responses triggered by IC, aIgG, FMLP, and Z

Neutrophils were incubated for 1–2 min with NO donors and, after this time, cytotoxic responses were triggered by the addition of different stimuli, without removing NO donors from the culture medium. As shown in Fig. 1Go, GSNO increased, in a concentration-dependent manner, cytotoxicity induced by all of the stimuli employed, the maximal effect being observed at 100 µM of GSNO. Higher concentrations did not further enhance cytotoxic responses (not shown). Data depicted in Fig. 2Go show the effects of SNAP and SNP on cytotoxicity. It was found that SNAP moderately increased cytotoxicity triggered by IC, aIgG, or Z without modifying those responses induced by FMLP. By contrast, SNP dramatically inhibited cytotoxicity triggered by all of the stimuli used. It is important to note that: 1) in the absence of triggering stimuli, NO donors did not induce cytotoxic responses, and 2) the enhancement of cytotoxicity induced by NO donors was not impaired by high concentrations of serum. In fact, when cytotoxic reactions were performed in culture medium supplemented with 25% of FCS, cytotoxic responses triggered by 10-7 M FMLP were increased from 6 ± 3 (untreated cells) to 31 ± 6 (GSNO 100 µM-treated cells) (X ± SEM, n = 6, p < 0.001). Similar results were obtained using IC as stimulus (not shown).



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  		FIGURE 1. Effect of GSNO on neutrophil cytotoxic responses triggered by IC, aIgG, FMLP, and Z. Neutrophils (3 x 106/ml in medium supplemented with 1% FCS) were incubated in the presence or absence of different concentrations of GSNO for 1–2 min at 37°C. After this time, cytotoxicity was triggered by the addition of different stimuli: IC (10 µg/ml), aIgG (50 µg/ml), FMLP (10-7 M), and Z (200 µg/ml), without removing GSNO from the culture medium. Data are expressed as the arithmetic mean ± SEM of triplicates from 10 donors. *, p < 0.001 vs cytotoxic responses assessed in the absence of GSNO.

 


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  		FIGURE 2. Effect of SNAP and SNP on neutrophil cytotoxic responses triggered by IC, aIgG, FMLP, and Z. Neutrophils (3 x 106/ml in medium supplemented with 1% FCS) were incubated in the presence or absence of SNAP or SNP (100 µM) for 1–2 min at 37°C. After this time, cytotoxicity was triggered by the addition of different stimuli: IC (10 µg/ml), aIgG (50 µg/ml), FMLP (10-7 M), and Z (200 µg/ml), without removing NO donors from the culture medium. Data are expressed as the arithmetic mean ± SEM of triplicates from eight donors. *, p < 0.001 vs cytotoxic responses assessed in the absence of SNAP or SNP.

 
To analyze whether NO accounts for the biological activity of NO donors, two NO scavengers were used, hemoglobin 20, 21 and PTIO 32 . Cells were incubated with GSNO or SNP (100 µM) in the presence of hemoglobin (500 µM) or PTIO (250 µM). After 1–2 min of incubation at 37°C, cytotoxicity was triggered by the addition of IC without removing NO donors or NO scavengers from the culture medium. It was found that NO scavengers prevented the increase in cytotoxicity induced by both, GSNO (Fig. 3Go) and SNAP (not shown), but did not modify the inhibition induced by SNP (Fig. 3Go), suggesting that it cannot be ascribed to the release of NO.



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  		FIGURE 3. Effect of hemoglobin and PTIO on the enhancement of IC-triggered cytotoxicity mediated by NO donors. Neutrophils (3 x 106/ml in medium supplemented with 1% FCS) were incubated with GSNO or SNP (100 µM) in the absence or presence of hemoglobin (500 µM) or PTIO (250 µM). After 1–2 min of incubation at 37°C, cytotoxicity was triggered by the addition of IC (10 µg/ml), without removing NO donors or NO scavengers from the culture medium. Data are expressed as the arithmetic mean ± SEM of triplicates from five to seven donors. *, p < 0.001 vs cytotoxic responses assessed in the absence of hemoglobin or PTIO.

 
The enhancing effect of NO donors on cytotoxicity does not involve the activation of soluble guanylate cyclase

The activation of soluble guanylate cyclase appears to be a cardinal mechanism of action of NO 21 . In agreement with previous observations 2, 3 , we found that neutrophil treatment with GSNO, SNAP, or SNP markedly enhanced cGMP production: fmol/107 cells = 215 ± 32, 1315 ± 215, 1468 ± 134, and 487 ± 79, for untreated and neutrophils treated with 100 µM of GSNO, SNAP, and SNP, respectively; mean ± SEM; n = 5; p < 0.05, untreated versus treated cells. To determine whether the enhancement exerted by GSNO and SNAP on neutrophil cytotoxic responses could be ascribed to the increase in intracellular concentrations of cGMP, we examined the effect of the cell-permeable analogue of cGMP, Bt2cGMP. It was found that treatment with Bt2cGMP, at concentrations of 10 µM (Fig. 4GoA) or 1 µM (not shown), did not modify neutrophil cytotoxic responses triggered by either IC, aIgG, FMLP, or Z. A possible role of cGMP in the enhancement of cytotoxicity induced by GSNO and SNAP was further examined using a selective inhibitor of the soluble form of the guanylyl cyclase enzyme, the compound ODQ 33 . At concentrations known to inhibit NO-stimulated guanylyl cyclase activity (10 µM) 33 , ODQ did not prevent the enhancing effect exerted by GSNO and SNAP on cytotoxic responses triggered by either IC, aIgG, FMLP, or Z (Fig. 4GoB).



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  		FIGURE 4. Role of the guanylyl cyclase enzyme in the enhancement of cytotoxicity mediated by NO donors. A, Effect of Bt2cGMP on neutrophil cytotoxic responses. Neutrophils (3 x 106/ml in medium supplemented with 1% FCS) were incubated in the presence or absence of Bt2cGMP (10 µM) for 1–2 min at 37°C. After this time, cytotoxic responses were assessed. B, Effect of ODQ, a selective inhibitor of the guanylyl cyclase enzyme, on the enhancement of cytotoxicity induced by GSNO. Neutrophils (3 x 106/ml) were incubated in the presence or absence of ODQ (10 µM) for 30 min at 37°C. Then cells were cultured with or without GSNO (100 µM) for 1–2 min at 37°C. After this time, cytotoxic responses were assessed. In all cases, cytotoxicity was triggered by the addition of IC (10 µg/ml), aIgG (50 µg/ml), FMLP (10-7 M), and Z (200 µg/ml). Bt2cGMP, ODQ, and/or GSNO were not removed from the culture medium and were present during the course of the cytotoxic reaction. Data are expressed as the arithmetic mean ± SEM of triplicates from five to seven donors.

 
Hydrogen peroxide plays a critical role in cytotoxicity mediated by either untreated or NO donor-treated neutrophils

To analyze the role of the respiratory burst in cytotoxic reactions, we employed neutrophils isolated from three patients with chronic granulomatous disease (CGD), which produced, in response to either FMLP or IC, O2- concentrations lesser than 1% compared with normal cells. CGD neutrophils were incubated for 1–2 min with GSNO or SNAP (100 µM) and, after this time, cytotoxic responses were triggered by the addition of FMLP or IC, without removing NO donors from the culture medium. It was observed that neither untreated nor GSNO or SNAP-treated CGD neutrophils mediated significant levels of cytotoxicity (percentage of cytotoxicity <7, n = 3 for each NO donor and stimulus).

The role of O2- in cytotoxicity was analyzed by performing cytotoxic reactions in the presence of SOD. Taking into account that O2- rapidly combines with NO to form ONOO- 34, 35 , high concentrations of SOD (2, 500 U/ml) were employed. It was found that the addition of SOD did not decrease cytotoxicity mediated by normal neutrophils triggered by either IC, aIgG, FMLP, or Z, assessed in the absence or presence of GSNO or SNAP (data not shown). By contrast, in all cases, the addition of catalase (500 U/ml) almost completely suppressed cytotoxicity (percentage of inhibition >88%, n = 3–6 for each of the stimuli employed, either in the presence or absence of GSNO or SNAP), indicating that cytotoxic responses were dependent on the release of H2O2 by effector cells. Additional experiments were performed to analyze whether cytotoxic reactions conducted in the presence of SNP were also dependent on H2O2 release. Since 100 µM of SNP markedly inhibited cytotoxicity, these reactions were performed in the presence of 50 µM of SNP, which decreased cytotoxic responses triggered by IC and Z by 48 ± 7% and 59 ± 6%, respectively (n = 4). It was found that cytotoxic responses triggered by either IC or Z, in the presence of 50 µM of SNP, were not modified by the addition of SOD (2500 U/ml), but were almost completely suppressed by catalase (500 U/ml) (percentage of inhibition >92, n = 3 for both stimuli).

Long-term incubation with NO donors enhances neutrophil ability to mediate oxygen-dependent cytotoxicity in response to triggering stimuli

In all of the above-described experiments, cytotoxic responses were assessed in the presence of NO donors, after pretreatment for 1–2 min. Additional studies were performed to determine whether removal of NO donors before the addition of triggering stimuli impaired the enhancement of cytotoxicity. Neutrophils were treated with GSNO (100 µM) for different times. At each time point, cells were washed to remove NO donors from the culture medium, targets were added, and cytotoxicity was triggered by IC (Fig. 5GoA) or FMLP (Fig. 5GoB). Our results show that neutrophils must be pretreated with GSNO for at least 4 h to increase cytotoxic responses. Similar results were obtained using SNAP (not shown). Pretreatment for longer periods (i.e., 8 or 18 h) further increased cytotoxicity (Fig. 6Go, A and B). Taken together, these results suggest that long-term treatment of neutrophils with NO donors induces a priming effect on neutrophil cytotoxic capacity. It was also observed that: 1) in all cases, the addition of catalase (500 U/ml) almost completely suppressed cytotoxicity (percentage of inhibition >91%, n = 4–5 for each stimulus), while SOD had no inhibitory effect, and 2) the enhancement of cytotoxicity induced by NO donors was impaired by hemoglobin (500 µM) and was not modified by ODQ (10 µM), an inhibitor of the guanylyl cyclase enzyme (not shown).



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  		FIGURE 5. Short-term exposure of neutrophils to GSNO or SNAP does not increase cytotoxic responses. Neutrophils (3 x 106/ml in medium supplemented with 1% FCS) were incubated in the presence or absence of GSNO (100 µM) for different times at 37°C, 1–2 min, 30 min, 60 min, 120 min, and 240 min. At each time point, neutrophils were washed to remove NO donors from the culture medium, and cytotoxicity was triggered by the addition of 10 µg/ml IC (A) or 10-7 M FMLP (B). Data are expressed as the arithmetic mean ± SEM of triplicates from five to six donors. *, p < 0.05, untreated vs NO donor-treated neutrophils.

 


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  		FIGURE 6. Long-term exposure of neutrophils to GSNO or SNAP increases cytotoxic responses. Neutrophils (3 x 106/ml in medium supplemented with 1% FCS) were incubated in the presence or absence of GSNO or SNAP (100 µM) for 8 h (A) or 18 h (B) at 37°C. After these times, cells were washed twice and cytotoxicity was triggered by the addition of different stimuli: IC (10 µg/ml), aIgG (50 µg/ml), FMLP (10-7 M), and Z (200 µg/ml). Data are expressed as the arithmetic mean ± SEM of triplicates from seven donors. *, p < 0.001 vs cytotoxic responses mediated by neutrophils cultured in the absence of NO donors.

 
Long-term treatment with GSNO enhances neutrophil ability to produce O2- and H2O2 in response to triggering stimuli

To analyze the mechanisms underlying increased cytotoxicity by NO donors, we examined whether treatment with GSNO (100 µM) enhanced the ability of neutrophils to generate IRO. To this aim, neutrophils were incubated with GSNO for 8 or 18 h and, after these times, cells were washed to remove the NO donor from the culture medium. Then neutrophil production of O2- and H2O2 in response to FMLP (10-7 M) was evaluated. As shown in Fig. 7Go, treatment of neutrophils with GSNO induced a significant increase in the generation of either O2- (Fig. 7GoA) or H2O2 (Fig. 7GoB). Similar results were obtained employing IC as stimulus (not shown). These results suggest that the enhancing effect of NO donors on oxygen-dependent cytotoxic responses mediated by neutrophils could be due, at least in part, to an increased ability of effector cells to produce IRO.



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  		FIGURE 7. Long-term exposure of neutrophils to GSNO increases O2- and H2O2 production triggered by FMLP. Neutrophils (3 x 106/ml in medium supplemented with 1% FCS) were incubated in the presence or absence of GSNO (100 µM) for 8 or 18 h at 37°C. After these times, cells were washed twice and adjusted to 10 x 106/ml. Then the production of O2- (A) and H2O2 (B) triggered by FMLP (10-7 M) was evaluated. Data are expressed as the arithmetic mean ± SEM from six donors. *, p < 0.001 vs O2- or H2O2 produced by cells cultured in the absence of GSNO.

 
Treatment with GSNO switches the cytotoxic mechanisms triggered by aIgG and FMLP from a MPO-independent to a MPO-dependent pathway

As mentioned above, catalase almost completely suppressed cytotoxic responses conducted by untreated or NO donor-treated neutrophils, supporting a critical role for H2O2 in cytotoxicity. It is well known that H2O2 could directly mediate the lysis of the target cells or could serve as a component of the H2O2-halide-MPO system, which is capable of peroxiding Cl- to ClO-, a species with a strong cytotoxic potential 36 . To analyze the involvement of MPO in cytotoxicity, additional experiments were performed. Cells were cultured for 8 h with GSNO (100 µM) and, after this time, they were washed to remove the NO donor from the culture medium. Then azide or cyanide, two haem-enzyme inhibitors 36 , was added and cytotoxic responses were triggered by IC, aIgG, FMLP, or Z. Results depicted in Table IGo show that cytotoxicity mediated by neutrophils cultured in the absence of GSNO involved a MPO-independent mechanism for all of the stimuli employed. Interestingly, neutrophil exposure to GSNO switched the cytotoxic mechanisms triggered by aIgG and FMLP from a MPO-independent to a MPO-dependent pathway. By contrast, cytotoxic mechanisms triggered by IC and Z remained MPO independent, as seen with control cells. Similar results were obtained when neutrophils were treated with GSNO for 18 h, before the addition of triggering stimuli (not shown).


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  		Table I. Effect of azide and cyanide on neutrophil cytotoxic responses1

 
The switch in the cytotoxic mechanisms triggered by aIgG and FMLP from a MPO-independent to a MPO-dependent pathway, induced as a consequence of GSNO treatment, could be related to the ability of NO to increase MPO release from stimulated neutrophils, as previously described 6 . To analyze this point, neutrophils were treated with GSNO (100 µM) for 8 h and, after washing, the release of MPO was triggered. Treatment with GSNO significantly increased MPO release triggered not only by aIgG (percentage of MPO released: 1.6 ± 2.1 vs 7.5 ± 2.3) and FMLP (1.7 ± 0.9 vs 5.6 ± 1.6), but also by IC (17.6 ± 4.3 vs 28.5 ± 3.9) and Z (13.5 ± 2.9 vs 24.6 ± 3.4) (X ± SEM, n = 5–10, p < 0.05 untreated vs GSNO-treated neutrophils).

NO donors do not affect the rates of apoptosis or necrosis of cultured neutrophils

To rule out the possibility that the increase in neutrophil degranulation could be related to the induction of cytotoxic effects exerted by NO on neutrophils themselves, we evaluated neutrophil apoptosis and necrosis after 18 h of culture by fluorescence microscopy and flow cytometry. As shown in Fig. 8Go, there were no differences in apoptotic rates between untreated or NO donor-treated cells, the percentage of necrotic forms, analyzed by either exclusion of vital dyes trypan blue or ethidium bromide, being always less than 10%. Almost identical results were observed when apoptosis and necrosis were evaluated in FMLP-triggered neutrophils, cultured either in the presence or absence of NO donors (not shown).



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  		FIGURE 8. Effect of NO donors on neutrophil apoptosis. Neutrophils (3 x 106/ml in medium supplemented with 1% FCS) were cultured in the presence or absence of NO donors (100 µM) for 18 h at 37°C. Then the percentage of apoptotic cells was determined by fluorescence microscopy (A) and flow cytometry (B). A, Results are expressed as the mean ± SEM of 10 experiments. B, Histograms of a representative experiment (n = 10), showing the percentage (M1) of nuclei with hypodiploid DNA content.

 
NO donors do not affect neutrophil shape change nor the enhancement in CD18 expression and adherence triggered by FMLP

Finally, to establish whether variations in cytotoxicity induced by NO donors were associated with other functional changes, we also studied the impact of NO on three additional responses: shape change, CD18 expression, and adherence. Neutrophil treatment with NO donors for either 1–2 min (Table IIGo) or 8 h (not shown) did not exert any effect on these responses, assessed either in the absence or presence of FMLP.


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  		Table II. Effect of NO donors on different neutrophil-mediated responses1

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results presented in this study show that oxygen-dependent cytotoxic responses mediated by neutrophils can be modulated by NO donors. Stimulatory or inhibitory effects were observed depending on the NO donor employed. Thus, cytotoxic responses were increased by GSNO and SNAP, while they were dramatically suppressed by SNP. The enhancement mediated by GSNO or SNAP was prevented by hemoglobin and PTIO, two NO scavengers, which did not modify the inhibition induced by SNP, suggesting that it cannot be ascribed to the release of NO. The molecular identity of the species responsible for this inhibitory effect remains to be established. It is well known that SNP attacks thiols and releases cyanide 34 . This reaction could explain contrasting effects of SNP and other NO donors, such as GSNO and SNAP, on lymphocyte proliferation 37 . However, cyanide does not seem to account for the suppression of cytotoxicity reported in this study since, as we have shown, haem-enzyme inhibitors, including cyanide, do not inhibit neutrophil cytotoxicity triggered by IC and Z (Table IGo and 38 .

The mechanisms by which GSNO and SNAP enhance cytotoxic responses do not depend on the stimulation of guanylyl cyclase since: 1) treatment of neutrophils with the cell-permeable analogue of cGMP, Bt2cGMP, has no effect on cytotoxicity, and 2) ODQ, a selective inhibitor of the soluble form of the guanylyl cyclase enzyme 33 , does not impair the enhancing effect of GSNO and SNAP on cytotoxicity. These findings contrast with our previous observations regarding the modulatory action of NO donors on Ab-dependent cellular cytotoxicity mediated by neutrophils. We found that NO donors modulate Ab-dependent cellular cytotoxicity through mechanisms dependent on the increase in intracellular concentrations of cGMP 39 . Taken together, these observations suggest that NO could modulate different cytotoxic responses mediated by neutrophils through guanylyl cyclase-dependent and independent pathways.

The role of NO in cytotoxic reactions is still unclear. It has been proposed that NO can directly cause cell death. However, primary cell cultures of various types exposed to high concentrations of NO (1 mM) for long periods show no adverse effects 35, 40 . Taking into account that cells involved in inflammatory processes can produce not only O2-, H2O2, ·OH, and HOCl, but also NO, several studies have focused their attention on the analysis of the interplay between IRO and NO in the mediation of cytotoxicity 20, 21, 34, 35 . They showed that peroxinitrite (ONOO-), the product of the reaction of O2- with NO, has a strong oxidative potential 34, 35 . However, the combination of NO with O2- does not always increase cytotoxicity. Instead, in some models it may be H2O2 that synergizes more powerful with NO. In this regard, it has been shown recently that NO exacerbates H2O2-mediated killing of Escherichia coli as well as the increase in endothelial permeability induced by H2O2 41, 42 . While the above-mentioned observations suggest that NO potentiates oxidative injury, recent findings point out a more complex scenario. In fact, NO can also attenuate cell injury mediated by H2O2, as shown in different models 43, 44, 45 . Considering that all of these studies have been performed in host cell-free systems 41, 42, 43, 44, 45 , assumptions and extrapolations from them, regarding the interplay between IRO and NO and their effects on inflammation, should be reexamined in biological systems in which effector cells are included.

Our results showed that when neutrophils were treated with NO donors for 1–2 min, and cytotoxic responses were then triggered in their presence, there was a marked increase in cytotoxicity. Under these conditions, the enhancement of cytotoxicity induced by GSNO was higher compared with SNAP. This observation could be due to differences in the NO generation kinetics. In fact, it has been shown that SNAP discomposes more slowly than GSNO 46, 47 . It was also observed that the removal of NO donors after 1–2 min of pretreatment, before the addition of triggering stimuli, completely impaired the enhancement of cytotoxicity, indicating that a brief exposure of neutrophils to NO donors is unable to prime their cytotoxic capacity. The enhancement of cytotoxicity conducted in the presence of NO donors does not appear to involve the participation of ONOO- since high concentrations of SOD did not exert any inhibitory effect. A critical role for H2O2, on the other hand, was suggested by the fact that catalase almost completely abrogated cytotoxicity. However, the possibility that enhanced cytotoxicity may involve a direct effect of both NO and H2O2, acting together on target cells, should be ruled out since neither NO donors (100–1000 µM), H2O2 (10–10,000 µM), nor NO donors plus H2O2 induced cytotoxic effects on target cells when cultured in the absence of neutrophils (unpublished results).

Different mechanisms could account for the enhancement in cytotoxicity observed for reactions performed in the presence of NO donors. First, NO may enhance neutrophil ability to produce O2- and H2O2 by acting in the course of the cytotoxic reaction, which involves long-term coincubation of triggered neutrophils and target cells (18 h at 37°C). Second, NO may interact with H2O2 and/or other free radicals derived from H2O2 to form a new agent with stronger oxidative potential, as previously proposed 48 . Third, considering the ability of NO and IRO to inhibit antioxidant enzymes such as glutathione peroxidase, catalase, and/or MPO 40, 48, 49, 50, 51, 52, 53, 54 , H2O2 could act in synergy with NO by depleting target and/or effector cell antioxidant defenses. Inhibition of antioxidant defenses in target cells may result in enhanced susceptibility to the cytotoxic action of IRO. On the other hand, inhibition of MPO and catalase is associated with a marked increase in both O2 consumption and H2O2 release during neutrophil activation 49, 50 . Nevertheless, since NO donors were present during the course of the cytotoxic reaction, it was very difficult to ascertain which of the above-mentioned mechanisms account for the enhancement in cytotoxicity.

To further analyze the mechanisms by which NO donors enhance oxygen-dependent cytotoxic responses, we treated neutrophils with GSNO or SNAP (100 µM) for different times. At each time point, cells were washed to remove NO donors from the culture medium before triggering cytotoxicity with IC, aIgG, FMLP, or Z. It was observed that neutrophils must be pretreated with NO donors for at least 4 h to increase cytotoxic responses. Pretreatment for longer periods (i.e., 8 or 18 h) further increased cytotoxicity. Since these cytotoxic reactions were performed after removal of NO donors from the culture medium, the enhancement of cytotoxicity can be attributable to the induction of a priming effect on the neutrophil itself. In this regard, we have also observed that pretreatment with GSNO for either 8 or 18 h markedly increased the ability of neutrophils to produce O2- and H2O2 as well as to release MPO to the extracellular medium in response to triggering stimuli. Both effects could explain, at least in part, the enhancement of cytotoxicity induced by NO donors. It should be pointed out that the increase in MPO release triggered by aIgG and FMLP was associated with a switch in the cytotoxic mechanism from a MPO-independent to a MPO-dependent pathway. Since aIgG and FMLP were unable to induce MPO release from control cells, we speculate that the switch in the cytotoxic mechanism, observed in GSNO-treated neutrophils, was due to the induction of MPO release. Regarding IC and Z, it should be noted that they were able to trigger MPO release not only from GSNO-treated cells, but also from untreated cells. In spite of this, cytotoxic responses were in all cases MPO independent. We speculate that this could be due, at least in part, to the scavenging of ClO- by these agonists. In fact, this scavenging activity has been shown for Z 55 , but remains to be evaluated for IC.

The physiologic relevance of our results can be only speculative at this time. It should be noted, however, that the concentrations of NO donors used in this work appear to be consistent with the levels of NO expected in vivo. GSNO and SNAP, at concentrations from 10–100 µM, produce 0.10–5.60 µM NO/min 45, 56, 57, 58 , while intraluminal rates of NO production in small blood vessels could be as high as 8 µM/min 59 . Stimulated macrophages and endothelial cells, on the other hand, can synthesize NO at rates of 0.02–0.2 µM/min/107 cells 60, 61, 62 . These observations support the possibility that, at inflammatory areas, NO may modulate oxygen-dependent cytotoxic responses mediated by neutrophils. This modulatory effect could involve an autocrine and/or a paracrine mechanism. The former possibility, however, is unlikely since cytotoxic responses triggered by IC, aIgG, FMLP, or Z, mediated by untreated or LPS-primed neutrophils, were not modified when conducted in the presence of NO synthase inhibitors or in L-arg-free culture medium (unpublished data).

In summary, we have shown that the physiologic NO donor GSNO, as well as SNAP exacerbate oxygen-dependent cytotoxic responses mediated by neutrophils. This effect appears to be related, at least in part, to the induction of a priming effect on neutrophils themselves, which requires long-term exposure of neutrophils to NO donors.


   Acknowledgments
 
We thank Fundación de la Hemofilia and Academia Nacional de Medicina for the use of the FACScan flow cytometer.


   Footnotes
 
1 This work was supported by grants from the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires University School of Medicine, Fundación Alberto J. Roemmers, and Fundación Antorchas, Buenos Aires, Argentina. Back

2 Address correspondence and reprint requests to Dr. Graciela Andonegui, Laboratorio de Inmunología, Instituto de Investigaciones Hematológicas, Academia Nacional de Medicina, Pacheco de Melo 3081, 1425 Buenos Aires, Argentina. E-mail address: Back

3 Abbreviations used in this paper: NO, nitric oxide; aIgG, human aggregated immunoglobulin G; Bt2cGMP, N2, 2'-O-dibutyrylguanosine 3':5'-cyclic monophosphate; CGD, chronic granulomatous disease; cGMP, cyclic GMP; GSNO, S-nitrosoglutathione; IC, precipitating immune complexes; IRO, oxygen-reactive intermediates; MPO, myeloperoxidase; ODQ, 1H-(1, 2, 4)oxadiazolo[4,3-a]quinoxalin-1-one; PTIO, 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl 3-oxide; SNAP, S-nitroso-N-acetylpenicillamine; SNP, sodium nitroprusside; SOD, superoxide dismutase; Z, opsonized zymosan. Back

Received for publication January 15, 1998. Accepted for publication November 11, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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