Influence of acetaminophen and trichloroethylene on liver cytochrome P450-dependent monooxygenase system

The aim of the study was to evaluate the effect of acetaminophen (APAP) and/or trichloroethylene (TRI) on the liver cytochrome P450-dependent monooxygenase system, CYP2E1 and CYP1A2 (two important P450 isoforms), and liver glutathione (GSH) content in rats. Rats were given three different doses of APAP (250, 500 and 1000 mg/kg b...) and then the above-mentioned parameters were measured for 48 h. The lowest APAP dose produced small changes in the cytochrome P450 content of liver. At 500 mg/kg APAP increased the cytochrome P450 content to 230% of the control. The inductive effect was seen at 1000 mg/kg dose but at 24 h and later. NADPH-cytochrome P450 reductase activity was the highest after the lowest dose of APAP, while after the highest dose it was equal to the control value. TRI increased both the cytochrome P450 content and the NADPH-cytochrome P450 reductase activity. When TRI was combined with APAP, both these parameters increased in the first hours of observation, but they returned to the control values at 24 h. When APAP was given at 250 mg/kg, GSH levels decreased to 55% of the control at 8 h and returned to the control values at 24 h. The higher doses of APAP decreased GSH levels more than the lowest dose, but after 24 h GSH levels did not differ from those of the control. When TRI was given at 250 mg/kg, the GSH levels decreased to 68% of the control at 2 h and then they increased gradually and tended to exceed the control values at 48 h. The effect of TRI combined with APAP on the level of GSH was virtually the same as that of APAP alone given at 500 mg/kg.     

Mechanisms of acetaminophen oxidation to N-acetyl-P-benzoquinone imine by horseradish peroxidase and cytochrome P-450

Horseradish peroxidase rapidly catalyzed the H2O2-dependent polymerization of acetaminophen. Acetaminophen polymerization was decreased and formation of GSSG and minor amounts of GSH-acetaminophen conjugates were detected in reaction mixtures containing GSH. These data suggest that horseradish peroxidase catalyzed the 1-electron oxidation of acetaminophen and that GSH decreased polymerization by reducing the product, N-acetyl-p-benzosemiquinone imine, back to acetaminophen. Analyses of reaction mixtures that did not contain GSH showed N-acetyl-p-benzoquinone imine formation shortly after initiation of reactions. When GSH was added to similar reaction mixtures at various times, 3-(glutathion-S-yl)-acetaminophen was formed. The formation and disappearance of this product were very similar to N-acetyl-p-benzoquinone imine formation and were consistent with the disproportionation of 2 mol of N-acetyl-p-benzosemiquinone imine to 1 mol of N-acetyl-p-benzoquinone imine and 1 mol of acetaminophen followed by the rapid reaction of N-acetyl-p-benzoquinone imine with GSH to form 3-(glutathion-S-yl)acetaminophen. When acetaminophen was incubated with NADPH, oxygen and hepatic microsomes from phenobarbital-pretreated rats, 1.2 nmol 3-(glutathion-S-yl)acetaminophen/nmol cytochrome P-450/10 min was formed. Formation of polymers was not observed indicating that N-acetyl-p-benzoquinone imine was formed via an overall 2-electron oxidation rather than a disproportionation reaction. However, when cumene hydroperoxide was replaced by NADPH in microsomal incubations, polymerization was observed suggesting that cytochrome P-450 might also catalyze the 1-electron oxidation of acetaminophen.     

The effects of diethylcarbamazine on the ultrastructure of lung cells in vivo

The pulmonary surfactant synthesis is disturbed in experimentally induced asthma, as are the intracellular storage capacity and its physical activity. These alterations may also be present in chronic asthmatic patients, and therefore the dysfunction of the pulmonary surfactant system may play an important role in the pathophysiology of asthma. Some clinical reports have described favorable results with the use of diethylcarbamazine (DEC) in patients with bronchial asthma showing that DEC is effective in terminating acute attacks of bronchial asthma. The present study aimed to analyze the ultrastructural alterations of lung cells after treatment in vivo with diethylcarbamazine. After 12 days of treatment with DEC, when compared with control samples, type II pneumocytes showed active nuclei with abundant euchromatin and evident nucleoli, and a substantially greater number of mature secretion vesicle. On the other hand, type I pneumocytes showed no morphological alterations. After DEC treatment, lung macrophages also presented several characteristics of cellular activation such as nuclei with a prominence of euchromatin and central nucleoli as well as an abundance of early and late endossomes distributed throughout the cytoplasm. These results confirm that DEC exerts a role in the activation of important pulmonary cellular pathways, which are probably related to the clinical improvement of asthma symptoms after DEC treatment.     

The 1- and 2-electron oxidation of acetaminophen catalyzed by prostaglandin H synthase

Purified and microsomal preparations of prostaglandin H synthase catalyzed the arachidonic acid-dependent polymerization of acetaminophen and, in the presence of GSH, catalyzed the formation of 3-(glutathion-S-yl)acetaminophen. The formation of these products was inhibited by indomethacin and by purging reaction mixtures with argon. When H2O2 replaced arachidonic acid, neither indomethacin nor argon purging inhibited product formation. These results suggest that the peroxidase activity of prostaglandin H synthase catalyzed the oxidation of acetaminophen. Addition of GSH to reaction mixtures decreased acetaminophen polymerization; however, 3-(glutathion-S-yl)acetaminophen formation was maximal with 40 microM GSH, and higher concentrations of GSH did not substantially alter its formation. In the presence of GSH, either ascorbic acid or NADPH decreased polymerization by greater than 97% while 3-(glutathion-S-yl)acetaminophen formation was still observed. These data suggest that polymers and conjugates were formed by two different pathways. Since polymerization of acetaminophen involves radical termination of N-acetyl-p-benzosemiquinone imine whereas 3-(glutathion-S-yl)acetaminophen is formed by conjugation of N-acetyl-p-benzoquinone imine with GSH, the data suggest that prostaglandin H synthase catalyzed both the overall 1- and 2-electron oxidation of acetaminophen.     

Role of the nitric oxide pathway in ischemia-reperfusion injury in isolated perfused guinea pig lungs

We examined the role of the nitric oxide (NO) pathway on ischemia-reperfusion injury via the use of isolated perfused guinea pig lungs. We administered both L-Arginine and N-nitro-L-arginine methyl ester (L-NAME) to the lungs in or after 3 h of ischemia. We observed pulmonary artery pressures as well as tissue and perfusate malondialdehyde (MDA) and glutathione (GSH) levels. We observed that L-NAME significantly increased both tissue and perfusate GSH levels and pulmonary artery pressures, but it decreased both tissue and perfusate MDA levels. On the other hand, L-arginine significantly decreased pulmonary artery pressure and both tissue and perfusate glutathione levels, but it increased both tissue and perfusate MDA levels. Electron microscopic evaluation supported our findings by indicating the preservation of lamellar bodies of type II pneumocytes. We concluded that L-NAME administration during reperfusion improves lung recovery from ischemic injury.     

Survival and cell death in cells constitutively unable to synthesize glutathione

We examined the role of GSH in survival and cell death using GCS-2 cells that are deficient in glutamate cysteine ligase (gamma-glutamyl cysteine synthetase, gammaGCS), an enzyme essential for GSH synthesis. Cells maintained in 2.5 mM GSH have GSH levels that are approximately 2% of wild type and grow indefinitely; however, they express both pro- and anti-apoptotic Bcl-2 family members and have detectable levels of cytoplasmic cytochrome C. Withdrawal of GSH from the medium results in a fall in intracellular GSH to undetectable levels, decreased mitochondrial dehydrogenase activity, decreased anti-apoptotic factor RNAs, increased pro-apoptotic factor RNAs, additional cytochrome C release, and a fall in ATP levels; however, cells continue to grow for another 24h. At 48 h, these trends continue with the exception that mitochondrial membrane potential and ATP levels rise; DNA fragmentation begins at 48 h. Thus, severe reduction of GSH to 2% of wild type produces a metastable state compatible with survival, but complete absence of GSH triggers apoptosis.     

Proinflammatory response of alveolar epithelial cells is enhanced by alveolar macrophage-produced TNF-alpha during pulmonary ischemia-reperfusion injury

Pulmonary ischemia-reperfusion (IR) injury entails acute activation of alveolar macrophages followed by neutrophil sequestration. Although proinflammatory cytokines and chemokines such as TNF-alpha and monocyte chemoattractant protein-1 (MCP-1) from macrophages are known to modulate acute IR injury, the contribution of alveolar epithelial cells to IR injury and their intercellular interactions with other cell types such as alveolar macrophages and neutrophils remain unclear. In this study, we tested the hypothesis that following IR, alveolar macrophage-produced TNF-alpha further induces alveolar epithelial cells to produce key chemokines that could then contribute to subsequent lung injury through the recruitment of neutrophils. Cultured RAW264.7 macrophages and MLE-12 alveolar epithelial cells were subjected to acute hypoxia-reoxygenation (H/R) as an in vitro model of pulmonary IR. H/R (3 h/1 h) significantly induced KC, MCP-1, macrophage inflammatory protein-2 (MIP-2), RANTES, and IL-6 (but not TNF-alpha) by MLE-12 cells, whereas H/R induced TNF-alpha, MCP-1, RANTES, MIP-1alpha, and MIP-2 (but not KC) by RAW264.7 cells. These results were confirmed using primary murine alveolar macrophages and primary alveolar type II cells. Importantly, using macrophage and epithelial coculture methods, the specific production of TNF-alpha by H/R-exposed RAW264.7 cells significantly induced proinflammatory cytokine/chemokine expression (KC, MCP-1, MIP-2, RANTES, and IL-6) by MLE-12 cells. Collectively, these results demonstrate that alveolar type II cells, in conjunction with alveolar macrophage-produced TNF-alpha, contribute to the initiation of acute pulmonary IR injury via a proinflammatory cascade. The release of key chemokines, such as KC and MIP-2, by activated type II cells may thus significantly contribute to neutrophil sequestration during IR injury.     

Paradoxical anti-inflammatory actions of TNF-alpha: inhibition of IL-12 and IL-23 via TNF receptor 1 in macrophages and dendritic cells

IL-12 and TNF-alpha are central proinflammatory cytokines produced by macrophages and dendritic cells. Disregulation of TNF-alpha is associated with sepsis and autoimmune diseases such as rheumatoid arthritis. However, new evidence suggests an anti-inflammatory role for TNF-alpha. TNF-alpha-treated murine macrophages produced less IL-12p70 and IL-23, after stimulation with IFN-gamma and LPS. Frequency of IL-12p40-producing macrophages correspondingly decreased as measured by intracellular cytokine staining. IL-12p40 production was also inhibited in dendritic cells. TNFR1 was established as the main receptor involved in IL-12p40 regulation, because IL-12p40 levels were not affected by TNF-alpha in TNFR1(-/-)-derived macrophages. Macrophages activated during Listeria monocytogenes infection were more susceptible to inhibition by TNF-alpha than cells from naive animals, which suggests a regulatory role for TNF-alpha in later stages of infection. This nonapoptotic anti-inflammatory regulation of IL-12 and IL-23 is an important addition to the multitude of TNF-alpha-induced responses determined by cell-specific receptor signaling.     

The effect of asbestos and stone-wool fibres on some chemokines and redox system of pulmonary alveolar macrophages and pneumocytes type II

The in vitro effect of stone-wool has been studied in primary cultures of pulmonary alveolar macrophages (AM) and type II pneumocytes (T2) by morphological, biochemical and immunological methods. UICC crocidolite was applied as a positive control. Although stone-wool brought about frustrated phagocytosis, it did not induce serious membrane damage, whereas crocidolite gave rise to very severe membrane alterations. Stone-wool significantly reduced the activity of Cu,Zn/superoxide dismutase (SOD) in alveolar macrophages and significantly decreased the activity of gamma-glutamyl transpeptidase (GGT) in pneumocytes type II. Crocidolite, on the other hand, decreased the activity of all enzymes (glutathione peroxidase - GSH-Px, glutathione reductase - GSH-Rd) of glutathione metabolism in alveolar macrophages. It decreased the activity of all enzymes in pneumocytes type II except for Cu,Zn/SOD. After exposure to stone-wool, the production of inflammatory proteins, macrophage chemoattractant protein-1 (MCP-1) and macrophage inhibitory protein-1alpha (MIP-1alpha) increased in both cultured cells but did not reach the level induced by crocidolite. Although this study provided a useful insight in the toxicity of the stone-wool, we can not draw the conclusions how the intact pulmonary tissue may respond on the exposure to these fibres, exclusively based on the in vitro tests.     

In vivo effect of diallyl sulfide and cimetidine on phenacetin metabolism and bioavailability in rat

Numerous cytochrome P450 inhibitors have been described as effective modulators of cytochrome P450 isoforms activity in vitro. Their inhibitory efficiency may be considerably modified after in vivo application. The aim of this study was to examine the effect of oral administration of diallyl sulfide--a cytochrome P450 2E1 inhibitor and cimetidine--a cytochrome P450 2C6 and 2C11 inhibitor on rat serum concentration of phenacetin and its metabolite acetaminophen. Both inhibitors increased area under the curve (AUC(0-4 h)) for phenacetin by 50%. Only cimetidine reduced AUC(0-4 h) for acetaminophen indicating inhibition of O-deethylation activity. Quinidine--a cytochrome P450 2D subfamily and P-glycoprotein inhibitor did not change significantly phenacetin bioavailability. These results suggest that diallyl sulfide inhibits the deacetylation pathway catalysed by arylamine N-acetyl transferase. Beside cytochrome P450 1A2 other cytochrome P450 isoforms (2A6 and/or 2C11) are involved in phenacetin O-deethylation in rat.     

Regulation of cyclooxygenase-2 by hypoxia and peroxisome proliferators in the corneal epithelium

Hypoxic injury provokes inflammation of many tissues including the ocular surface. In rabbit corneal epithelial cells, both peroxisome proliferator-activated receptor (PPAR)-inducible cytochrome P450 4B1 and cyclooxygenase-2 (COX-2) mRNAs were increased by hypoxia. PPAR alpha and beta but not gamma mRNAs were detected in these cells. The PPAR activator, WY-14,643 increased COX-2 expression. Similarly, non-steroidal anti-inflammatory drugs with the ability to activate PPARs induced COX-2 independently of prostaglandin synthesis inhibition. COX-2 protein overexpression by hypoxia and PPAR activation was not associated with a parallel increase in prostaglandin E(2) accumulation. However, the enzyme regained full catalytic activity when: 1) hypoxic cells were re-exposed to normoxic conditions in the presence of heme and arachidonic acid, and 2) WY-14,643-treated cells were depleted of intracellular GSH. Consistent with previous observations showing that the corneal production of cytochrome P450-derived inflammatory eicosanoids is elevated by hypoxia and inflammation, the current data suggest that hypoxic injury is a model of inflammation in which molecules other than COX-derived arachidonic acid metabolites play a major proinflammatory role. This study also suggests that increased cellular GSH may be the mechanism responsible for the characteristic dissociation of PPAR-induced COX-2 expression and activity. Moreover, we provide new insights into the commonly observed lack of efficacy of classical non-steroidal anti-inflammatory drugs in the treatment of hypoxia-related ocular surface inflammation.     

Tumor necrosis factor induces resistance of macrophages to Legionella pneumophila infection

Legionella pneumophila is an ubiquitous opportunistic intracellular pathogen that replicates readily in thioglycollate-elicited peritoneal macrophages from genetically susceptible A/J mice. Treatment of macrophage cultures in vitro with tumor necrosis factor-alpha (TNF-alpha) induced resistance of the macrophages to infection by Legionella as compared with control macrophages treated with medium alone. Addition of small amounts of monoclonal antibody to TNF-alpha restored susceptibility of the macrophages. Furthermore, antibody to the proinflammatory cytokine interleukin-1 (IL-1) alpha/beta increased resistance, but recombinant IL-1 had little effect. Such decreased susceptibility to Legionella growth in anti-IL-1 antibody-treated cultures corresponded with enhanced levels of TNF-alpha in the supernatants of the treated cells. An antibody to another proinflammatory cytokine with known immunoregulatory properties (i.e., IL-6) had little or no effect on the ability of the macrophages to be infected by Legionella and, furthermore, treatment with recombinant IL-6, similar to recombinant IL-1, did not modify the ability of the cells to be infected in vitro. These results indicate that TNF-alpha is important in controlling L. pneumophila replication, and IL-1 can regulate TNF-alpha levels, affecting susceptibility of macrophages to infection with an intracellular opportunistic pathogen like Legionella.     

Differential regulation of cytokine release and leukocyte migration by lipopolysaccharide-stimulated primary human lung alveolar type II epithelial cells and macrophages

Bacterial colonization is a secondary feature of many lung disorders associated with elevated cytokine levels and increased leukocyte recruitment. We hypothesized that, alongside macrophages, the epithelium would be an important source of these mediators. We investigated the effect of LPS (0, 10, 100, and 1000 ng/ml LPS, up to 24 h) on primary human lung macrophages and alveolar type II epithelial cells (ATII; isolated from resected lung tissue). Although macrophages produced higher levels of the cytokines TNF-alpha and IL-1beta (p < 0.0001), ATII cells produced higher levels of chemokines MCP-1, IL-8, and growth-related oncogene alpha (p < 0.001), in a time- and concentration-dependent manner. Macrophage (but not ATII cell) responses to LPS required activation of ERK1/2 and p38 MAPK signaling cascades; phosphorylated ERK1/2 was constitutively up-regulated in ATII cells. Blocking Abs to TNF-alpha and IL-1beta during LPS exposure showed that ATII cell (not macrophage) MCP-1 release depended on the autocrine effects of IL-1beta and TNF-alpha (p < 0.003, 24 h). ATII cell release of IL-6 depended on autocrine effects of TNF-alpha (p < 0.006, 24 h). Macrophage IL-6 release was most effectively inhibited when both TNF-alpha and IL-1beta were blocked (p < 0.03, 24 h). Conditioned media from ATII cells stimulated more leukocyte migration in vitro than conditioned media from macrophages (p < 0.0002). These results show differential activation of cytokine and chemokine release by ATII cells and macrophages following LPS exposure. Activated alveolar epithelium is an important source of chemokines that orchestrate leukocyte migration to the peripheral lung; early release of TNF-alpha and IL-1beta by stimulated macrophages may contribute to alveolar epithelial cell activation and chemokine production.     

Diethylmaleate and buthionine sulfoximine, glutathione-depleting agents, differentially inhibit expression of inducible nitric oxide synthase in endotoxemic mice.

Diethylmaleate (DEM) and buthionine sulfoximine (BSO), glutathione (GSH)-depleting agents, reduced the metabolic activity and the protein level of iNOS in both macrophages and hepatocytes activated by lipopolysaccharide (LPS). In this study, we examined the effects of DEM and BSO on iNOS expression in LPS-treated mice under the assumption that the level of GSH may alter the expression of nitric oxide synthase. Serum levels of interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) were also determined. DEM markedly decreased the levels of hepatic GSH in response to LPS. Treatment of mice with DEM significantly reduced serum nitrite/nitrate levels and hepatic iNOS protein and mRNA induction by LPS. Although BSO inhibited the level of hepatic GSH in LPS-treated mice, the agent did not alter serum nitrite/nitrate levels and hepatic iNOS expression. DEM completely inhibited an increase of serum IL-1beta level by LPS, whereas BSO failed to inhibit it. Neither DEM nor BSO significantly affected the induction of serum TNF-alpha level by LPS. These results showed that DEM and BSO differentially affect the expression of iNOS in endotoxemic mice, suggesting the possibility that suppression of iNOS expression by DEM may be associated with the inhibition of IL-1beta but not of TNF-alpha.     

In vitro cytokine release by activated murine peritoneal macrophages: role of prostaglandins in the differential regulation of tumor necrosis factor alpha, interleukin 1, and interleukin 6

Distinct subpopulations of macrophages or differently activated macrophages display various functions in immune reactions. Some of their activities depend on specific sets of factors (i.e., cytokines and eicosanoids) produced by activated macrophages. We have studied the ability of murine (CBA/ca) peritoneal macrophages to selectively release tumor necrosis factor alpha (TNF-alpha), interleukin 1 (IL-1), and IL-6. We have found that the priming of cells (Mo) with different stimulants (thioglycolate vs. LPS) induces the release of particular cytokines by reactivated macrophages. The increased release of TNF-alpha correlates with lower levels of IL-1 and IL-6. We have also found that prostaglandin E2 (PGE2) and prostacyclin (PGI2) have opposing effects on the production of two of these cytokines. The release of TNF-alpha is inhibited by prostaglandins, whereas increased levels of PGE2 and PGI2 correlate with higher levels of IL-6.     

Glutathione metabolism in resting and phagocytizing peritoneal macrophages

The steady state GSH content of cultured mouse resident peritoneal macrophages was 34 +/- 5 pmol/microgram of cell protein. Intracellular GSH content decreased concomitantly with zymosan ingestion. The half-life of GSH decreased from 1.9 h in resting cells to 0.58 h during phagocytosis as determined by inhibition of GSH synthesis with buthionine sulfoximine. The decrease in GSH half-life was directly related to the extent of particle uptake. In cytochalasin D-treated cells, attachment of zymosan to the macrophage plasma membrane in the absence of particle interiorization was sufficient to stimulate GSH turnover. Efflux was the major route of GSH loss in [35S]cystine-labeled macrophages, and was enhanced 3-fold by a zymosan challenge. GSH was lost intact since resident macrophages lack gamma-glutamyl transpeptidase (less than 1 pmol of L-gamma-glutamyl-p-nitroanilide/microgram of protein . h). Macrophages obtained from mice challenged in vivo with Corynebacterium parvum maintained higher intracellular GSH levels (50 +/- 5 pmol/microgram of cell protein) than did resident cells. The half-life of GSH in buthionine sulfoximine-treated C. parvum-elicited macrophages was 3.8 +/- 0.2 h while resting and 1.3 +/- 0.2 h during phagocytosis. C. parvum-elicited macrophages, in contrast to resident cells, contained sufficient levels of gamma-glutamyl transpeptidase activity to hydrolyze 55 pmol of L-gamma-glutamyl-p-nitroanilide/microgram of cell protein . h. These studies indicate that phagocytosis and cellular activation have profound effects on GSH metabolism in macrophages.     

The effects of dexamethasone and prostaglandin F2alpha on production and release of surfactant in type II alveolar cells

Incubation of adult rat Type II alveolar pneumocytes with dexamethasone microM induced an increase of intracellular 3Hphosphatidyl choline (PC), a component of lung surfactant, from precursor 3Hcholine. Release of 3HPC into the media did not differ from control unless prostaglandin F2alpha-THAM microM was added to the dexamethasone-treated cells. This observation corresponds to the ontogenetically observed endogenous increases in cortisol during pregnancy followed by increases in prostaglandins associated with contractions. The model system will serve well to test whether various substances effect synthesis and/or release of pulmonary surfactants.     

Metabolism of 2-acetylaminofluorene by clara cells,type II cells and alveolar macrophages isolated from rabbit lung,and use of a new chamber incubation mutagenicity test system

Clara cells, alveolar type II cells and pulmonary alveolar macrophages (PAM) were isolated in high yield from rabbit lung. The purity of the cell fractions was 80–90%, 98% and above 99%, respectively. Cytochrome P-450 total content was determined in microsomes from freshly prepared cells. The Clara cells contained significantly more cytochrome P-450 than was found in whole lung microsomes. Furthermore, the cytochrome content of the Clara cells was 2 -fold higher than in the type II cells and 4 -fold higher than in the macrophages. 2-aminofluorene (AF) was the major metabolite in all preparations when intact cells were incubated with 2-acetylaminofuorene (AAF). The PAMs produced AF in the highest rates, while the Clara cells showed the largest rates of cytochrome P-450-dependent, ring hydroxylation of AAF. Mutagenic activation of AAF by isolated lung cells was assayed with a chamber-incubation method. The Clara cells were far more active than the type II cells in this respect, while the macrophages were inactive.Abbreviations AAF 2-acetylaminofluorene - AF 2-aminofluorene - DMSO dimethyl sulfoxide - NBT nitro blue tetrazolium - 7-OH-AAF 7-hydroxy-AAF - 9-OH-AAF 9-hydroxy-AAF     

Nitrite generation and antioxidant effects during neutrophil apoptosis

Neutrophil apoptosis is important for the resolution of airway inflammation in a number of lung diseases. Inflammatory mediators, endogenous reactive oxygen and nitrogen species, and intracellular and extracellular antioxidants may all influence neutrophil apoptosis. This study investigated the involvement of these factors during apoptosis of neutrophils cultured in vitro. Neutrophils undergoing spontaneous apoptosis in culture as assessed by annexin V binding generated significant amounts of nitrite. Incubation with agonistic anti-Fas monoclonal antibody or tumor necrosis factor-alpha (TNF-alpha) enhanced neutrophil apoptosis at 6 h, although it decreased nitrite accumulation. Although granulocyte-macrophage colony-stimulating factor significantly reduced neutrophil apoptosis, this was also associated with decreased nitrite accumulation. In contrast, inhibition of apoptosis at 16 h by dibutyryl cyclic adenosine monophosphate was associated with increased nitrite accumulation. Exogenous glutathione (GSH) or N-acetylcysteine significantly enhanced neutrophil apoptosis at 6 h and stimulated the production of H(2)O(2), which may mediate apoptosis through intracellular hydroxyl radical production. Intracellular GSH concentrations decreased in neutrophils undergoing apoptosis, and this was more marked in neutrophils treated with anti-Fas or TNF-alpha. These results suggest a causal association between reduced endogenous nitric oxide production, reduced intracellular GSH, and Fas- and TNF-alpha-mediated neutrophil apoptosis, whereas enhanced neutrophil survival mediated by dibutyryl cyclic adenosine monophosphate is associated with increased nitrite generation and maintenance of intracellular GSH. The interaction of endogenous reactive oxygen species with extracellular antioxidants such as GSH could also contribute to the complex processes regulating neutrophil apoptosis and hence the resolution of inflammation in the lung.