1 Egleston Pediatric
Subspecialists, Enhancing the clearance of neutrophils by
enhancing apoptotic cell death and macrophage recognition may be
beneficial in acute lung injury. Exogenous nitric oxide gas depresses
neutrophil oxidative functions and accelerates cell death (A. H. Daher,
J. D. Fortenberry, M. L. Owens, and L. A. Brown. Am.
J. Respir. Cell Mol. Biol. 16: 407-412, 1997). We
hypothesized that S-nitrosoglutathione
(GSNO), a physiologically relevant nitric oxide donor, could also
enhance neutrophil DNA fragmentation. Neutrophils were incubated for
2-24 h in the absence and presence of GSNO (dose range 0.1-5
mM) and evaluated for cell death by a fluorescent
viability/cytotoxicity assay. Neutrophil DNA fragmentation was assessed
by cell death detection ELISA and by terminal
deoxynucleotidyltransferase-mediated fluorescence-labeled dUTP nick end
labeling assay. Neutrophil oxidative function was also determined.
Incubation with GSNO increased cell death at 2, 4, and 24 h. GSNO
incubation for 24 h significantly increased DNA fragmentation in a
dose-dependent fashion at 0.5 (median 126% of control value;
P = 0.002) and 5 mM (185% of control value; P = 0.002) by
terminal deoxynucleotidyltransferase-mediated fluorescence-labeled dUTP
nick end labeling and at 0.5 mM by ELISA (164% of control
value; P = 0.03). The
apoptosis-to-total cell death ratio increased with increasing GSNO
concentration (P < 0.05). Effects
were mitigated by coincubation with superoxide dismutase. Five
millimolar GSNO decreased overall superoxide generation and
O2 consumption but not when
adjusted for dead neutrophils. GSNO significantly enhances cell death
and neutrophil DNA fragmentation in a dose-dependent fashion.
nitric oxide; apoptosis; S-nitrosothiol; glutathione
NEUTROPHILS play a primary role in the inflammatory
response to lung injury from infection or insult (42, 51). Timely and
effective clearance of neutrophils helps determine the extent and
persistence of pulmonary edema and pulmonary sequelae from acute
respiratory distress syndrome (8). The prolonged presence of activated
neutrophils in the injured lung may provide further fuel for
oxidant-induced alveolar injury and increase the risk for progression
to multiple organ system dysfunction (32).
Neutrophils are normally cleared from both the systemic circulation and
lung by apoptosis (19), a form of programmed cell death (54), and
subsequent phagocytosis by macrophages recognizing neutrophil membrane
changes (8, 40). The presence of cytokines such as
granulocyte-macrophage colony-stimulating factor or interleukin-1 can
inhibit neutrophil apoptosis and thus prolong cell survival in the
presence of such an inflammatory milieu (7).
Nitric oxide (NO), primarily regarded as an endogenous vasodilator (38,
55), has been discovered to serve a multitude of immunomodulatory
functions. NO inhibits platelet aggregation and basal neutrophil
adherence to endothelium (24, 36). At concentrations used with inhaled
NO therapy for respiratory failure and persistent pulmonary
hypertension of the newborn, exogenous NO can also inhibit neutrophil
oxidative function (9). NO may produce this effect, in part, by
inducing apoptosis, as seen with neutrophils and monocytes. A recent
study (11), however, suggested that NO gas, particularly with
superimposed hyperoxia, induces both apoptotic and necrotic cell death.
Necrotic cell death (41) brings the potential extrusion of large
quantities of neutrophil proteases and oxidants into an already damaged
lung (42). The role of apoptosis of either neutrophils or lung cells in
acute inflammatory lung injury is less certain. The effects of
apoptosis could range from mitigating to enhancing injury, dependent on the specific cell types affected, as well as the timing and nature of
the insult (30).
Free NO is exquisitely reactive, with a half-life of seconds (44). NO
reacts with ambient O2 and
superoxide radicals to produce more toxic species, including nitrogen
dioxide (NO2) and peroxynitrite
(OONO We therefore hypothesized that GSNO induces cell death and DNA
fragmentation, consistent with apoptosis, in human neutrophils and
concomitantly inhibits neutrophil oxidative function.
Neutrophil Isolation
ABSTRACT
Top
Abstract
Introduction
MATERIALS AND METHODS
RESULTS
DISCUSSION
References
INTRODUCTION
Top
Abstract
Introduction
MATERIALS AND METHODS
RESULTS
DISCUSSION
References
), which have been
associated with toxicity in several forms of lung injury (3, 28).
Alternatively, NO may react with endogenous or exogenous thiols to form
more stable compounds. One such adduct is
S-nitrosoglutathione (GSNO), an
S-nitrosothiol synthesized by
S-nitrosylation of glutathione (GSH), an abundant intracellular thiol
(45). GSNO is more stable and much more abundant in plasma than
reactive free NO (44). Gaston et al. (13) found endogenous bioactive
GSNO in nanomolar to micromolar concentrations in the airways of normal
human subjects and in patients with pneumonia or receiving inhaled NO
therapy. The authors speculated that nitrosothiol formation in the
lungs might serve a dual function both by stabilizing NO in bioactive
form and by decreasing cytotoxicity. Exogenous GSNO is also bioactive.
Pharmacological doses of GSNO potently inhibit platelet aggregation and
adhesion in humans with forearm ischemia or undergoing coronary
angioplasty (25), similar to the inhibitory effects of endogenous NO on
platelet adhesion in vivo (36).
MATERIALS AND METHODS
Top
Abstract
Introduction
MATERIALS AND METHODS
RESULTS
DISCUSSION
References
Neutrophil Exposure
Neutrophils were incubated in the absence and presence of GSNO (0.1-5 mM; Sigma) or the NO donor S-nitroso-N-acetylpenicillamine (SNAP; 1 mM; Sigma). Cells were incubated in Iscove's modified Dulbecco's medium (GIBCO BRL) for 2 or 24 h and then assessed for cell viability and DNA fragmentation. In another group of experiments, neutrophils were incubated with GSNO (0.5 mM) in the absence and presence of 80% O2 to determine whether hyperoxia produced synergistic effects with GSNO. In separate experiments, neutrophils were incubated for 24 h with GSH (Sigma) to determine whether GSNO effects were related to the presence of GSH as a thiol antioxidant.To determine the role of endogenous NO in intrinsic regulation of neutrophil cell death, neutrophil exposures were also performed in the absence and presence of the L-arginine analog NO synthase inhibitor NG-monomethyl-L-arginine (L-NMMA; 0.5 mM; Calbiochem, San Diego, CA) for 24 h.
To determine whether peroxynitrite produced by interactions between superoxide radicals and NO released from GSNO played a role in cell death, neutrophils were also coincubated in separate experiments with manganese superoxide dismutase (MnSOD; nonpyrogen free, final concentration 100 µg/ml; Sigma) during GSNO exposures.
Cell Death Assessment
Cell necrosis was evaluated by a fluorescent viability/cytotoxicity assay (Eukolight, Molecular Probes, Eugene, OR) (29). Briefly, exposed cells were placed on glass slide covers within polyethylene six-well dishes that allowed neutrophil adherence, then stained with a mixture of the fluorescent probes calcein AM and ethidium homodimer. After uptake, only viable cells containing functioning esterases can cleave the ester group on calcein AM to generate a characteristic green fluorescence under fluorescent microscopy. Ethidium homodimer penetrates the permeable membranes of nonviable cells and binds with nucleic acids, identifiable by red-orange fluorescence. For each experiment, ~100 neutrophils were counted from three high-power fields on each slide cover for green and red fluorescence and averaged. Values are expressed as the percentage of calcein (green)-positive cells divided by total calcein- and ethidium (red)-positive cells counted.DNA Fragmentation Assessment
Samples were evaluated for evidence of DNA fragmentation associated with apoptosis by the following techniques.ELISA. We used an apoptotic cell death detection ELISA (Boehringer Mannheim, Indianapolis, IN) to quantitatively determine cytoplasmic histone-associated DNA oligonucleosome fragments associated with apoptotic cell death. This ELISA has demonstrated correlation with gel electrophoresis and terminal deoxynucleotidyltransferase (TdT)-mediated dUTP nick end labeling (TUNEL) assays in measurement of apoptotic cell death in HL-60 cell lines treated with camptothecin (Boehringer Mannheim; data on file) and with histological evidence of apoptosis (4, 27). We did not repeat these studies for neutrophils. Briefly, neutrophil samples were sonicated to obtain cytoplasmic lysates. Samples were incubated with microtiter plates adsorbed with mouse anti-histone antibody (clone H11-4) to bind histone-associated DNA oligonucleosomes uncovered by endonuclease-mediated DNA nicking. Plates were washed, and nonspecific binding sites were saturated with blocking buffer. Bound samples were then reacted with anti-mouse DNA monoclonal antibody (MCA-33) and then conjugated with peroxidase. To determine the amount of retained peroxidase, 2,2'-azino-di(3-ethylbenzthiazoline-6-sulfonate) (ABTS) was added as a substrate, and the complex was measured by spectrophotometer at 405 nm (Anthos HTII, Anthos Labtec Instruments, Frederick, MD). Results are expressed as the ratio of sample absorbance to absorbance of room air control sample measured daily.
TUNEL assay. Specific 3'-hydroxyl ends of DNA fragments generated by endonuclease-mediated apoptosis are preferentially repaired by TdT (14). The TUNEL assay (Boehringer Mannheim) labels these strand breaks with fluorescent nucleotides and provides a sensitive measure of DNA fragmentation consistent with apoptosis in individual cells (14, 17). After exposure, cells were fixed in 4% paraformaldehyde and permeabilized with 1% Triton X-100 and 0.1% sodium citrate. Samples were then incubated for 60 min at 37°C in the absence and presence of exogenous TdT and incubated with fluorescein-conjugated dUTP for repair of nicked 3'-hydroxyl DNA ends. Mean cell fluorescence of 10,000 neutrophils and percentage of TUNEL-positive cells were assessed by flow cytometry (FACScan, Becton Dickinson, Bedford, MA) for each condition. To estimate the relative contribution of apoptotic cell death to overall cell death, an apoptosis-to-total cell death ratio was calculated by dividing the percentage of TUNEL-positive neutrophils in a sample by the percentage of dead neutrophils as determined by viability/cytotoxicity assay.
Microscopy. Exposed neutrophils were treated with Wright stain and examined by phase microscopy (×150) to qualitatively assess for histological findings consistent with apoptosis.
Assessment of Neutrophil Oxidative Function
Superoxide anion generation. Superoxide anion generation was determined by a standard assay (16) after 24 h of GSNO exposure. Briefly, phorbol 12-myristate 13-acetate (PMA; Sigma), a potent neutrophil stimulant, and horse cytochrome c (Sigma) were added to isolated neutrophil suspensions after GSNO exposure and washing with DPBS to remove residual NO donor compounds. Cytochrome c reduction by generated superoxide was then determined by spectrophotometric absorbance (Beckman DU 64 spectrophotometer, Fullerton, CA) at a 550-nm wavelength. Results are expressed as nanomoles of superoxide anion per 1 × 106 neutrophils. This reaction could be quenched by the addition of superoxide dismutase before PMA, confirming that cytochrome c reduction was mediated by superoxide anion (data not shown). The superoxide generation assay was then performed in room air conditions in the absence of NO or hyperoxia.O2 consumption. Neutrophil O2 consumption was measured as an indicator of overall respiratory burst. O2 consumption was determined with a Clark-type O2 membrane electrode (Yellow Springs Instrument, Yellow Springs, OH) as described originally by Chance and Williams (6) and used for neutrophil measurements by Rossi et al. (39). Briefly, neutrophils were exposed to the conditions in Superoxide anion generation and then washed in DPBS. Cells (1 × 107) were added to DPBS with constant stirring and maintained at 37°C in a glass chamber, with DPBS bathing the electrode membrane. After the addition of PMA to the chamber (final concentration 500 nM), the slope of the electrode response (O2 consumption/time) was recorded for 5-6 min on a strip-chart recorder and used to calculate O2 consumption, assuming an O2 solubility coefficient of 0.024 µmol/ml medium. Results are expressed as nanomoles of O2 per minute per 107 neutrophils.
Statistics
Assays were performed in triplicate for each sample exposure, and an average value was determined. Results are expressed as means ± SE. Statistical analysis was performed with one-way analysis of variance (ANOVA) and Student-Newman-Keuls comparison for parametric data sets. Data not meeting parametric characteristics were analyzed with Kruskal-Wallis one-way ANOVA on ranks and Student-Newman-Keuls or Dunn's test for comparisons. P < 0.05 was considered significant. ![]() |
RESULTS |
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Effects of Exogenous NO Donor and Inhibitor Exposure on Cell Death and DNA Fragmentation
GSNO decreased neutrophil viability in a time-dependent and concentration-dependent manner at 2, 4, 12, and 24 h as assessed by viability/cytotoxicity assay (Fig. 1). GSNO exposure increased neutrophil DNA fragmentation in a concentration-dependent fashion. GSNO effects on DNA fragmentation were seen by ELISA at 12 (P < 0.05) and 24 h (P < 0.03; Fig. 2) but not at 2 or 4 h compared with control cells (data not shown). GSH exposure alone did not enhance cell death or DNA fragmentation at any concentration (data not shown).
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TUNEL staining also demonstrated GSNO effects on DNA fragmentation.
Mean fluorescence intensity from dUTP binding was increased at 24 h,
but not at 12 h, in neutrophils incubated in GSNO (Figs. 3 and 4).
GSNO produced a dose-dependent DNA fragmentation response; 0.5 and 5 mM
GSNO increased fragmentation above that in 24-h control cells (P < 0.05 by Kruskal-Wallis
ANOVA on ranks, n = 4-10
experiments/group) and SNAP-treated cells
(P = 0.002;
n = 6 experiments). GSNO exposure increased the total percentage of TUNEL-positive neutrophils at 24 h
compared with control cells (Fig.
5A). The
apoptosis-to-total cell death ratio also increased with increasing GSNO
concentration, from 1.7 ± 0.3 (control) to 30.7 ± 4.0% (5 mM
GSNO) after 24 h of exposure (Fig.
5B). By light microscopy,
neutrophils exposed to GSNO showed histological evidence of apoptosis,
demonstrated by nuclear chromatin condensation and cell involution at
12 h (Fig. 6).
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Incubation of neutrophils with SNAP did not increase cell death compared with those in room air at 24 h as assessed by cytotoxicity assay (Fig. 1). SNAP-exposed cells were also evaluated by TUNEL to rule out an early effect on DNA fragmentation without secondary necrosis. SNAP did not enhance apoptosis compared with control value as measured by TUNEL (105% of control value, range 99-126%; P = 0.28; n = 5 experiments) or by ELISA (P > 0.05; n = 5 experiments). SNAP exposure with hyperoxic conditions also did not increase DNA fragmentation (median 120%, range 78-160%; n = 5 experiments).
L-NMMA exposure for 24 h did not decrease cell viability (Fig. 1). Likewise, L-NMMA pretreatment did not halt the development of apoptosis at 2 or 24 h. At 24 h, neutrophil DNA fragmentation as measured by ELISA was markedly increased in neutrophils exposed to L-NMMA at both 0.5 [4,904% of time 0 (t0) baseline] and 5 mM (6,908% of t0 baseline; P < 0.05) and was not different from that seen with control neutrophils at 24 h (2,538% of t0 baseline; P = 0.136; n = 6 experiments/group). At 24 h, L-NMMA-treated neutrophil DNA fragmentation (96% of control) measured by TUNEL assay was not different from that in control cells (P > 0.05).
GSNO exposure at 0.5 mM GSNO with hyperoxic conditions (GSNO-O2) did not increase cell death compared with GSNO in room air (80 ± 6 vs. 81 ± 4%). GSNO-O2 (1,364% of control value at 24 h, range 524-2,430%) also did not enhance apoptosis to a greater extent than GSNO in room air conditions (1,010% of control value, range 390-2,840%; not significant).
Coincubation of GSNO-exposed neutrophils with MnSOD decreased DNA fragmentation. By ELISA, addition of MnSOD decreased 24-h DNA fragmentation (median 115% of control value, range 55-172%) compared with GSNO alone (250% of control value, range 189-293%; P < 0.05 by ANOVA on ranks and Student-Newman-Keuls test).
Effects of GSNO on Neutrophil Oxidative Function
Neutrophil oxidative function was affected only at high GSNO concentrations. Five millimolar GSNO decreased superoxide generation (75 ± 6 nmol/106 neutrophils vs. 233 ± 6 nmol/106 control neutrophils; P < 0.05 by ANOVA and Student-Newman-Keuls test; n = 18 experiments). O2 consumption in response to PMA was also diminished by 5 mM GSNO (5 ± 1.3 nmol · ml ![]() |
DISCUSSION |
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GSNO is formed from the reaction of NO and GSH in the presence of O2 to form a relatively stable compound (46). Several authors (15, 43) have postulated that the S-nitrosylation reaction represents a very convenient endogenous method to store, transport, and release NO. S-nitrosothiol adducts may not only serve as physiologically active forms of NO in human plasma (44), but their formation may provide a physiological scavenging mechanism to minimize toxicity (52). Packaging of NO in this form could potentially serve both to preserve the bioactivity of NO and to limit its potential for O2-dependent toxicity.
We found a time- and dose-dependent effect of GSNO exposure on DNA fragmentation and cell death. These findings are consistent both with our previous study (11) with NO gas exposure and with recent preliminary findings (53) that the NO donor GEA-3162 and the combined NO-superoxide anion donor 3-morpholinosydnonimine induced a cGMP-independent increase in neutrophil apoptosis. Our results are also similar to findings with NO gas (11). Evaluation of GSNO as an NO donor is important because of its endogenous existence in the lung (13) and its potential for systemic delivery. GSNO also induced apoptosis in the absence of hyperoxia compared with the limited effects of NO gas alone in our previous study (11).
The TUNEL method for determining DNA fragmentation has previously been considered to correlate well with histological findings of apoptosis. However, some recent studies (18, 48) have suggested that TUNEL may not always be specific for apoptosis, particularly in autopsy material or with prolonged fixation time. Our samples were rapidly fixed after exposure and would be less likely to show such artifacts. TUNEL results were supported by elevated DNA fragmentation ELISA levels and by photomicrographs supporting the development of apoptosis. It is likely that DNA fragmentation seen in the current study does not solely represent apoptotic cell death but also primary or secondary necrosis.
We did not evaluate the effects of GSNO and hyperoxia on other cell types. Recent studies have found that NO donors in the presence of hyperoxia produced surfactant dysfunction and increased neutrophil chemotactic activity in vivo (37) and cytotoxicity to alveolar epithelial cells and lung vascular endothelial cells in vitro (35). However, other studies (23, 31) of ex vivo isolated lung models and in vivo models found that inhaled NO exposure actually decreased lung injury, suggesting that NO may not injure the lung in a more complex environment and at more clinically relevant gas concentrations. Further studies are ongoing in our laboratory to determine effects of both GSNO and inhaled NO at clinically relevant concentrations on alveolar epithelium to help address this disparity of results.
In our study, GSNO demonstrated fragmentation effects only at concentrations significantly above previously measured serum and airway GSNO concentrations in vivo (13), although intracellular GSNO levels have not been reported. Endogenous S-nitrosothiols would therefore be unlikely to produce the effects on cell death that we observed with higher concentrations. Exogeneous GSNO at these higher concentrations could potentially diminish pulmonary inflammation secondarily induced by neutrophils, dependent on the timing of administration. Moilaen et al. (34) found that NO donors, including GSNO, inhibited polymorphonuclear leukocyte function, whereas Lefer et al. (26) showed a beneficial antineutrophil effect from GSNO in a canine model of acute myocardial ischemia and reperfusion.
The effects of GSNO on DNA fragmentation compared with those of SNAP
could result from an enhanced ability to transport NO intracellularly.
The mechanism of nitrosothiol transport and metabolism, however,
remains uncertain. Recent evidence suggests that -glutamyl transpeptidase (GGT), a dipeptide transporter enzyme
involved in GSH metabolism and transport (33), may play a role in the cytostatic effect of GSNO on Salmonella
typhimurium (10) and infers that intracellular
transport of GSNO occurs with subsequent S-nitrosylation of cytoplasmic
proteins. GGT hydrolyzes the
-glutamyl moiety of GSNO to form
glutamate and
S-nitrosocysteinylglycine (20), which
is much more susceptible to release of free NO. Although GGT is
predominantly membrane bound and localized on the external surface of
epithelial cells (33), GGT activity is also found in leukocytes,
primarily in immmature myeloid cells but also in mature granulocytes
(47). GGT is released into a surfactant-associated pool within the lung
alveolus (22), which could potentially enable NO to be generated in the
alveolar space. SNAP effects may require higher concentrations because
a previous study (35) found that SNAP at a concentration of 2 mM
(compared with 1 mM in our study) in hyperoxic conditions induced
apoptosis in an alveolar epithelial cell line. We also did not measure
NO production in our donor experiments, and it is possible that
released NO from our lower SNAP dose was inadequate.
The mechanism of this NO effect on apoptosis remains uncertain. NO
inactivation of the gene transcription factor nuclear factor-B may
induce apoptosis in some cell types (49, 50). A preliminary study (12)
in our laboratory showed that GSNO inhibits nuclear factor-
B
activation in human neutrophils. In the current study, MnSOD inhibited
DNA fragmentation with NO, suggesting that oxidants could be
participating in cell death. A speculative mechanism could involve
production of the toxic metabolite peroxynitrite from the reaction of
NO and superoxide anion (2) because peroxynitrite can induce apoptosis
in other cell types (27). However, we found that superoxide generation
was actually decreased in GSNO-exposed neutrophils, making this
possibility much less likely.
NO synthase inhibition did not slow the intrinsic progression of neutrophil DNA fragmentation in our study, in contrast to the effects of NO synthase inhibition in murine macrophages. Albina et al. (1) found that NO synthase inhibition prevented interferon- and/or endotoxin-mediated apoptosis in murine peritoneal macrophages, implying that endogenous NO induced apoptosis. Differences among cell types in intrinsic apoptosis are likely related to quantitative and qualitative differences in molecular signaling mechanisms. Neutrophils express the Fas/Apo-1 antigen but lack the antiapoptotic gene bcl-2 that is found in lymphocytes and monocytes (21). The absence of bcl-2 could allow the inexorable progression toward apoptosis seen in neutrophils but not in other cellular blood elements.
In summary, GSNO enhances DNA fragmentation in human neutrophils by a mechanism that may be, in part, mediated by interactions of NO with superoxide. Further studies are necessary to determine whether this in vitro pharmacological effect significantly affects neutrophil clearance in models of lung injury.
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ACKNOWLEDGEMENTS |
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We thank Dean Jones for guidance in performing neutrophil O2 consumption experiments, Frank Harris for technical assistance, and Reese Clark for thoughtful review of the manuscript.
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FOOTNOTES |
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This study was funded by a grant from the American Lung Association and by a Goddard Research Scholarship from the Department of Pediatrics, Emory University, Atlanta GA (both to J. D. Fortenberry).
Address for reprint requests and other correspondence: J. D. Fortenberry, Egleston Pediatric Subspecialists, Egleston Children's Hospital, 1405 Clifton Road NE, Atlanta, GA 30322 (E-mail: jfortenb{at}mail.egleston.org).
Received 18 August 1997; accepted in final form 16 November 1998.
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