Neutrophil Oxidant Production Research Articles

Effect of oral trifusal treatment on nitric oxide production and endothelial nitric oxide synthase protein expression in neutrophils from healthy donors

Effect of oral trifusal treatment on nitric oxide production and endothelial nitric oxide synthase protein expression in neutrophils from healthy donors

Ruptured abdominal aortic aneurysm, a “two-hit” ischemia/reperfusion injury: Evidence from an analysis of oxidative products

Purpose: Ruptured abdominal aortic aneurysm (RAAA) remains a lethal condition despite improvements in perioperative care. The consequences of RAAA are hypothesized to result from a combination of two ischemia/reperfusion events: hemorrhagic shock and lower torso ischemia. Ischemia/reperfusion results in tissue injury by diverse mechanisms, which include oxygen free radical–mediated injury produced from activated neutrophils, xanthine oxidase, and mitochondria. Oxygen-free radicals attack membrane lipids, resulting in membrane and subsequently cellular dysfunction that contributes to postoperative organ injury/failure. The purpose of this investigation was to quantify the oxidative injury that occurs as a result of the ischemia/reperfusion events in RAAAs and elective AAAs. Methods: Blood samples were taken from 22 patients for elective AAA repair and from 14 patients for RAAA repair during the perioperative period. Plasma F 2 -isoprostanes were extracted, purified, and measured with an enzyme immunoassay. Aldehydes and acyloins were purified and quantified. Neutrophil oxidative burst was measured in response to a receptor independent stimulus (phorbol 12-myristate 13-acetate) with luminol-based chemiluminescence. Results: Plasma from patients with RAAAs showed significantly elevated F 2 -isoprostane levels on arrival at hospital and were significantly elevated as compared with the levels of patients for elective repair throughout the perioperative period (two-way analysis of variance, P < .0001). Multiple regression showed a significant relationship between the phagocyte oxidative activity and F 2 -isoprostane levels ( P < .013). Total acyloin levels were significantly higher in patients with RAAAs as compared with the levels in elective cases. Conclusion: The F 2 -isoprostane levels, specific markers of lipid peroxidation, showed that patients with RAAAs had two phases of oxidative injury: before arrival at hospital and after surgery. The significant relationship between the postoperative increases in F 2 -isoprostane levels and the neutrophil oxidant production implicates neutrophils in the oxidative injury that occurs after RAAA. New therapeutic interventions that attenuate neutrophil-mediated oxidant injury during reperfusion may decrease organ failure and ultimately mortality in patients with RAAAs. (J Vasc Surg 1999;30:219-28.)

Threshold and graded response behavior in human neutrophils: effect of varying G-protein or ligand concentrations.

Observing the qualitative characteristics of response behavior as key variables in the signal transduction cascade are changed can provide insight into the fundamental roles of these interactions in producing cellular responses. Using flow cytometric assays and pertussis toxin (PT) treatment of human neutrophils, we have shown that actin polymerization stimulated with the chemoattractants N-formyl-Met-Leu-Phe, leukotriene B4, and interleukin-8 exhibits threshold behavior in terms of G-protein number. Partial PT treatment resulted in both responding and nonresponding populations of cells upon stimulation. As PT treatment was increased, the responding population of cells continued to respond maximally, while the number of cells responding decreased. We also showed that N-formyl peptide-stimulated oxidant production exhibits threshold behavior in terms of G-protein number, and the threshold for oxidant production is significantly greater than that for actin polymerization. The threshold behavior observed with PT treatment contrasted with the graded response behavior seen when cells were stimulated with different doses of ligand. For actin polymerization, only one population of cells was observed at submaximal ligand concentrations, and as ligand concentration was decreased the whole population responded submaximally. For oxidant production, as ligand concentration was decreased there were two populations of cells, but the responding cells responded submaximally. A mathematical model incorporating receptor/ligand binding and G-protein activation was developed to account for these differences in response behavior. Our results predict that an early signal transduction event in addition to, and not initiated by G-protein activation, is necessary to account for actin polymerization and oxidant production in neutrophils.

A rapid assay of endotoxin in whole blood using autologous neutrophil dependent chemiluminescence

A rapid (30 min) whole blood assay for the detection of lipopolysaccharide (LPS) is described. This chemiluminescent (CL) assay utilizes the CR 1 and CR 3 receptor-induced oxidant production of polymorphonuclear leucocytes as a detection platform. The differential priming of neutrophils in whole blood by LPS-antibody complexes allows the specificity of the assay to be achieved. Oxidant released in response to complement opsonized zymosan results in luminol oxidation and subsequent light emission. This is dependent on heat labile putative complement proteins in the plasma. The assay consists of a control which measures baseline whole blood neutrophil oxidant production. The test assay contains murine monoclonal IgM antibody against the Lipid A epitope of LPS and measures the enhanced chemiluminescent response of the neutrophils in the presence of LPS-antibody complexes. Maximal sensitivity of the CL assay is dependent upon optimal antigen–antibody equivalence and duration of pre-incubation with the whole blood sample. The quantification of LPS is possible by inclusion of a positive control containing a maximally reactive LPS dose (800 pg/ml Escherichia coli 055:B5 LPS at an antibody concentration of 0.8 μg/assay). The CL assay is insensitive to variations in patient neutrophil concentration over a minimum range of 0.5 to 20×10 9 cells/l. The CL assay is widely reactive with the LPS of many strains of gram negative bacteria but not with the cell wall products of gram positive bacteria or Candida and Aspergillus. In comparison to acid extraction chromogenic LAL, the CL assay demonstrates superior recovery precision and accuracy in in vitro studies. This was reproducible over a wide range of LPS concentrations (0.017–1.6 EU/ml or 20–2000 pg/ml). This assay may be a clinically useful tool for the diagnosis of infection or endotoxin in patients.

Pyrrolidine dithiocarbamate attenuates endotoxin-induced acute lung injury.

Lung injury in the acute respiratory distress syndrome (ARDS) is in part due to polymorphonuclear leukocyte (PMN)-mediated oxidative tissue damage. By means of nuclear factor-kappaB (NF-kappaB) activation, oxidants may also induce several genes implicated in the inflammatory response. The dithiocarbamates are antioxidants with potent inhibitory effects on NF-kappaB. We postulated that the pyrrolidine derivative pyrrolidine dithiocarbamate (PDTC) would attenuate lung injury following intratracheal challenge with endotoxin (lipopolysaccharide; LPS) through its effect as an antioxidant and inhibitor of gene activation. Rats were given PDTC (1 mmole/kg) by intraperitoneal injection, followed by intratracheal administration of LPS. The transpulmonary flux of [125I] albumin (permeability index; PI) was used as a measure of lung injury. Northern blot analysis of total lung RNA was performed to assess induction of tumor necrosis factor-alpha (TNF-alpha) and intercellular adhesion molecule-1 (ICAM-1) messenger RNA (mRNA) as markers of NF-kappaB activation. The effect of in vivo treatment with PDTC on LPS-induced NF-kappaB DNA binding activity in macrophage nuclear extracts was evaluated with the electrophoretic mobility shift assay (EMSA). PDTC administration attenuated LPS-induced increases in lung permeability (PI = 0.16 +/- 0.02 for LPS versus 0.06 +/- 0.01 for LPS + PDTC; P < 0.05). TNF-alpha levels and PMN counts in bronchoalveolar lavage fluid (BALF) were unaffected, as were whole-lung TNF-alpha and ICAM-1 mRNA expression. PDTC had no effect on NF-kappaB activation as evaluated with EMSA. PDTC reduced lung lipid peroxidation as assessed by levels of malondialdehyde, without reducing neutrophil oxidant production. We conclude that PDTC attenuates LPS-induced acute lung injury. This effect occurs independently of any effect on NF-kappaB. PDTC reduces oxidant-mediated cellular injury, as demonstrated by a reduction in the accumulation of malondialdehyde. Administration of PDTC may represent a novel approach to limiting neutrophil-mediated oxidant injury.

Activation of neutrophil function via CD66: differential effects upon beta 2 integrin mediated adhesion.

To further define the role of CD66 glycoproteins in the regulation of neutrophil function, we analysed the effects of a CD66 monoclonal antibody, Kat4c, which recognizes an epitope present on AB domains of CD66a, CD66b and CD66c. Intact Kat4c and F(ab')2 fragments were found to augment fMLP-induced oxidation of 1,2,3-dihydrorhodamine (oxidant species production) and beta 2 integrin-mediated adhesion to fibrinogen but did not promote beta 2 integrin-mediated binding of albumin coated latex beads. Since the latter assay is a sensitive indicator of neutrophil CD11b/CD18 functional activation, these results imply CD66 may exert differential effects upon beta 2 integrin activity. Neutrophil oxidant species production and spreading on fibrinogen substrates were further potentiated by cross-linking of Kat4c F(ab')2, in keeping with the suggestion that ligation of CD66 regulates neutrophil function. However, although intact Kat4c promoted beta 2 integrin-dependent homotypic neutrophil adhesion, F(ab')2 fragments were without effect, implying a role for Fc receptors in this effect which has previously been attributed to CD66. Together these data define more clearly the role of CD66 in regulation of neutrophil function and further suggest that augmented beta 2 integrin-mediated adhesion following CD66 ligation occurs independently of affinity regulation.

Prolactin modulation of nitric oxide and TNF-α production by peripheral neutrophils in rats

It has been demonstrated that prolactin (PRL) is a potent immunomodulator that exerts stimulatory effects on physiological responses of immune cells. In the present research we have investigated whether PRL may influence nitric oxide (NO) and/or tumor necrosis factor-alpha (TNF-α) production in neutrophils obtained from inflammatory exudate of carrageenin-induced experimental pleurisy in the rat. In this acute model of inflammation the role of endogenous NO was evaluated using an inhibitor of NO-synthase, N G-nitro-L-arginine methyl ester (L-NAME). A treatment of animals with L-NAME (10 mg kg s.c.) induced a reduction of volume and cell number of pleural exudate and a decrease of nitrite production (measured by the Griees reaction) by polymorphonuclear cells after 24 h of incubation, while D-NAME, the inactive isomer, was without effect. Neutrophils from ovine prolactin (oPRL) treated rats (5 mg kg for 5 times s.c.) or from rats with a hyperprolactinaemia induced by pituitary gland graft produced higher amounts of NO both after 24 and 48 h of incubation. On the contrary, a clear reduction in the production of NO was found in neutrophils from rats treated with bromocriptine (BRC) (2 mg kg s.c.), a dopamine D 2-receptor agonist. TNF-α production (measured by MTT/cytotoxic assay) by neutrophils was markedly increased in PRL-treated or pituitary-grafted rats in comparison to controls, whereas BRC treatment reduced TNF-α production.

Stimulation of Primed Neutrophils by Soluble Immune Complexes

Soluble IgG-containing immune complexes are unable to initiate oxidant production in unprimed neutrophils. However, priming of the neutrophils with either granulocyte-macrophage colony-stimulating factor (GM-CSF) or cytochalasin B prior to exposure to these complexes results in activation of a rapid and extensive secretion of reactive oxidants. In this study, we have investigated the ability of soluble immune complexes to: (1) induce oxidant generation; (2) bind to the cell surface; and (3) induce Ca2+transients in neutrophils primed by incubation with GM-CSF of cytochalasin B. Our findings give new insight into the molecular processes involved in the “priming” phenomenon.

Modification of neutrophil oxidant production with diphenyleneiodonium and its effect on bacterial killing

Diphenyleneiodonium (DPI), an inhibitor of the NADPH oxidase, has been used to distinguish between oxidative and nonoxidative killing of Staphylococcus aureus and Escherichia coli by neutrophils. The rate of killing of S. aureus was inhibited by 77% in the presence of 10 μM DPI, compared to 81% measured under anaerobic conditions. DPI represents a convenient and accessible alternative to an anaerobic environment or using neutrophils from patients with chronic granulomatous disease, for eliminating oxidative killing. The killing of E. coli was also inhibited by DPI. The effect was more apparent at 30 min than at 10 min, suggesting that E. coli can be killed rapidly by nonoxidative mechanisms that become less efficient at later times. DPI was used at concentrations less than 10 μM to determine how this affected production of the three major neutrophil oxidants, superoxide, hydrogen peroxide, and hypochlorous acid, and to determine the effect of partial inhibition of oxidant production on the killing of S. aureus. Unexpectedly, lower concentrations of DPI (0.1–2 μM) inhibited hydrogen peroxide and hypochlorous acid production 10–30% more than they inhibited superoxide production. Correlation of hydrogen peroxide or hypochlorous acid production with the killing of S. aureus showed that up to 30% inhibition had no effect on the rate of killing, implying that agents that impair neutrophil oxidant production less than this will not compromise bacterial killing. Higher inhibition of oxidant production led to a linear decline in the rate of killing.

Rapid neutrophil accumulation and protein oxidation in irradiated rat lungs

Exposure of lungs to high doses of ionizing radiation can initiate an injurious acute inflammatory response. We show that neutrophil content in the lungs of rats exposed to 10 Gy whole body gamma radiation increased threefold 4.5 h after irradiation and returned to normal by 24 h. Oxidized methionine in the proteins of the lungs, heart, liver, kidney, and jejunum increased significantly in 2 h. Treatment with the antioxidant dithiothreitol immediately after irradiation prevented methionine oxidation. Methionine oxidation was also observed after intrabronchial instillation of phorbol myristate acetate, a model of neutrophil oxidant-mediated pulmonary injury, as well as in isolated lungs perfused with hypochlorous acid, confirming the ability of neutrophil oxidants to cause protein oxidation in lungs. No change in glutathione or protein sulfhydryl content was detected in irradiated lungs 4.5 h after irradiation, possibly as a result of protection by the observed increase in pulmonary glutathione reductase. We therefore show that the acute pulmonary inflammatory response to radiation involves rapid neutrophil accumulation, oxidant production, and protein oxidation.

Intratracheal instillation of silica up-regulates inducible nitric oxide synthase gene expression and increases nitric oxide production in alveolar macrophages and neutrophils.

Alveolar macrophages (AM) exposed to cytokines or bacterial lipopolysaccharide (LPS) produce the free radical nitric oxide (NO.) by an inducible nitric oxide synthase (iNOS). They also release reactive oxygen free radicals following exposure to silica dust. The purpose of the present study was to determine whether NO. is produced by rat AM and/or recruited leukocytes following the intratracheal (IT) instillation of silica. Male Sprague-Dawley rats (175 to 225 g) were IT instilled with either silica dust (10 mg/100 g body wt) or LPS (0.25 mg/100 g body wt). After 24 h, bronchoalveolar lavage cells (BALC) and lavaged lung tissue were assayed for iNOS mRNA. Cell counts of BALC and iNOS-dependent (N omega-nitro-L-arginine methyl ester [L-NAME]-inhibitable) chemiluminescence generated by AM were also determined. Northern blot analysis demonstrated that the steady-state levels of BALC iNOS mRNA were significantly increased by 3-fold following IT silica and by 7-fold following IT LPS. Partially enriched fractions of either AM or leukocytes from silica-treated rats both exhibited significantly elevated iNOS mRNA in Northern analysis. iNOS-dependent chemiluminescence was significantly increased in AM by 36-fold following IT silica and by 89-fold following IT LPS. Differential counts of BALC showed that AM numbers did not change in any of the treatments; however, red blood cells increased by 30-fold following IT silica and by 23-fold following IT LPS. Total leukocytes (polymorphonuclear leukocytes plus lymphocytes) increased by 58-fold following IT silica and by 274-fold following IT LPS.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of Fc gamma receptors in the activation of neutrophils by soluble and insoluble immunoglobulin aggregates isolated from the synovial fluid of patients with rheumatoid arthritis.

Synovial fluid from patients with rheumatoid arthritis contains both soluble and insoluble immunoglobulin aggregates which activate reactive oxidant production in human neutrophils. The objectives were to determine the roles played by Fc gamma receptors in activation of neutrophils by these complexes. Pronase treatment was used to remove Fc gamma RIII from the neutrophil surface and blocking monoclonal antibodies were used to prevent the binding of complexes to Fc gamma RII and Fc gamma RIII. When Fc gamma RIII was removed from the cell surface by pronase treatment, activation by the soluble aggregates did not occur [mean (SD) inhibition 89 (16)%, n = 6] whereas activation via the insoluble aggregates was less affected [34 (16)%, n = 6]. Blocking the binding to Fc gamma RIII with antibodies decreased activation in response to the soluble aggregates [mean (SD) inhibition 71 (22)%, n = 8] but again had a lower effect on activation by the insoluble aggregates [40 (17)%, n = 9]. When binding to Fc gamma RII was blocked, activation via the soluble aggregates was substantially inhibited [mean (SD) 93 (13)%, n = 8] whereas that via the insoluble aggregates was inhibited to a much lesser extent [28 (38)%, n = 9]. When Fc gamma RII and III were simultaneously blocked, activation by the insoluble aggregates was only inhibited by 45% [(19), n = 5]. These data thus indicate that activation of human neutrophils by soluble immunoglobulin aggregates from rheumatoid synovial fluid occurs via cooperative occupancy of both Fc gamma RII and III: perturbation of binding to either of these receptor classes will abrogate activation.

Stimulation of reactive oxidant production in neutrophils by soluble and insoluble immune complexes occurs via different receptors/signal transduction systems

Stimulation of reactive oxidant production in neutrophils by soluble and insoluble immune complexes occurs via different receptors/signal transduction systems

Intestinal brush border membrane function.

The gastrointestinal tract is a complex organ responsible for. for example, nutrient metabolism, absorption and transport. and hormonal production and action. Furthermore, it serves as an immune barrier against invasion of enteric bacteria and toxins. Recently, a series of reviews have been published on a wide variety of aspects of the intestine, including gu1 molecular and cellular biology, intestinal absorption and secretion, small-intestinal circulation, water and electrolyte absorption and secretion, the enteric nervous system, small-intestinal motility, and celiac disease (1-8). Enteric bacteria and toxins might serve as triggers to initiate, perpetuate, 01 exacerbate a septic state and promote the development oi multiple system organ failure by inducing cytokine secretion from resident tissue macrophages, promoting a proinflammatory endothelial cell phenotype, stimulating neutrophil protease and oxidant production, and activating multiple humoral protein cascades, such as complement and coagulation (9).

The Stress Response in Skin

In this study we tested the hypothesis that neutrophil products are present in ischemic skin flaps and that they are abolished with preconditioning of the skin. Random back flaps were created on rats, and the sequential appearance of neutrophil products and tissue oxidants was measured in the skin flaps. These flaps had predictable skin necrosis (4.7 +/- 0.8 cm) in the distal ends, while preconditioned flaps had no skin necrosis. Neutrophil products were assayed by both histomorphometrics and myeloperoxidase assays. Lipid peroxidation products were measured to assess tissue oxidant production. These data demonstrate that there is an increase in myeloperoxidase activity in skin flaps that is statistically significantly greater in the distal ends of the flaps at 24 hours (p < 0.05). The lipid peroxidation products were statistically significantly elevated at 48 hours in the distal ends (p < 0.05). Preconditioning the skin as a bipedicled skin flap for 7 days and then dividing the distal attachment abolished neutrophil products and tissue oxidant activity in the skin flaps (p < 0.05). These data suggest that neutrophil products and oxidant production are increased in ischemic skin and that preconditioning of the flap markedly attenuates this response.

Effect of cytotoxic drugs on mature neutrophil function in the presence and absence of granulocyte-macrophage colony-stimulating factor.

The effects of the cytotoxic drugs, adriamycin, cyclophosphamide, daunomycin (daunorubicin), prednisolone, actinomycin D, azacytidine and vincristine at concentrations of 1 microM on mature neutrophil function were examined. Up to 5 h incubation with adriamycin, azacytidine, cyclophosphamide, daunomycin and prednisolone had no effect on either luminol chemiluminescence or superoxide secretion. However, after 15 min or 1 h (but not 5 h) incubation vincristine enhanced fMet-Leu-Phe stimulated chemiluminescence, whilst after 5 h incubation with actinomycin D the ability of neutrophils to generate reactive oxidants in response to all stimuli tested was impaired: after 5 h incubation with adriamycin reactive oxidant production was also impaired, but only when fMet-Leu-Phe was used as stimulant. All of the drugs tested except azacytidine inhibited neutrophil oxidant production after 5 h incubation in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF). Actinomycin D and cyclophosphamide also inhibited GM-CSF stimulated protein biosynthesis. These data indicate that cytotoxic drugs may compromise the potentially beneficial effects of CSFs on mature neutrophil function during therapy.

Flow cytometric analysis of nitric oxide production in human neutrophils using dichlorofluorescein diacetate in the presence of a calmodulin inhibitor.

Dichlorofluorescein (DCFH) oxidation assay measures hydrogen peroxide (H2O2), which is a derivative of superoxide anion. We found that a calmodulin antagonist, W-13, which is known to inhibit superoxide anion generation enhanced the capacity of human neutrophils to oxidize DCFH. To investigate this discrepancy we studied the role of nitric oxide (NO) in DCFH oxidation. Pure NO was capable of oxidizing DCFH, and the product formed had spectral properties identical to oxidized DCFH produced by H2O2. The arginine analog, NG-monomethyl-L-arginine (NMMA), which inhibits NO production, in combination with W-13 completely inhibited the stimulus-induced increase in DCFH oxidation. We conclude that the oxidation of DCFH in human neutrophils can occur by either H2O2 or NO.

Pentoxifylline reduces injury to isolated lungs perfused with human neutrophils.

Neutrophils and neutrophil-derived oxidants have been implicated in the development of acute lung injury such as that seen in the adult respiratory distress syndrome (ARDS), in bronchopulmonary dysplasia (BPD), and in animal models of lung injury, including the isolated perfused lung. Both neutrophil-derived oxidant production and retention of neutrophils in the lung are required for injury in this model. Pentoxifylline can reduce lung injury from sepsis in the guinea pig and endotoxin-induced neutrophil sequestration and lung injury in the dog. It is also known to increase neutrophil deformability, which may affect retention in the pulmonary microvasculature. We evaluated neutrophil oxidant production, retention in isolated lungs, and neutrophil-mediated acute lung injury after phorbol myristate acetate (PMA) in the presence of pentoxifylline. Pentoxifylline (2 mM) significantly reduced superoxide anion and hydrogen peroxide production in vitro from PMA-stimulated neutrophils when pentoxifylline was directly added to the incubation mixtures, but not when neutrophils were preincubated with the agent. Pentoxifylline did not reduce retention of neutrophils in isolated lungs as determined by infusion of 111In-labeled neutrophils and gamma counting. Pentoxifylline prevented increases in total lung weight, lung-to-body-weight ratio, and perfusate thromboxane concentrations when it was present in perfusate buffer, whether or not neutrophils were preincubated in pentoxifylline prior to infusion into the lung. Pentoxifylline did not reduce injury to lungs perfused with glucose and glucose oxidase. We conclude that pentoxifylline reduces neutrophil oxidant production and neutrophil-dependent lung injury.

Measurement of intracellular fluorescence of human monocytes relative to oxidative metabolism.

Human monocytes (MN) produce O2- and H2O2 when stimulated by agonists. Dichlorofluorescin diacetate (DCFH-DA) has been used as a substrate for measuring intracellular oxidant production in neutrophils. DCFH-DA is hydrolyzed by esterases to dichlorofluorescin (DCFH), which is trapped within the cell. This nonfluorescent molecule is then oxidized to fluorescent dichlorofluorescin (DCF) by action of cellular oxidants. DCFH-DA can not be appreciably oxidized to a fluorescent state without prior hydrolysis. We have examined the utility of DCFH-DA for the assessment of monocyte oxidative responses. The levels of intracellular fluorescence measured by flow cytometry were considerably less than expected from reported levels of O2--production or chemiluminescence assays. Compared with neutrophils, monocytes produced minimal increases in DCF fluorescence after stimulation with phorbol myristate acetate as measured by flow cytometry, but both cell types showed increases in fluorescence when bulk cell suspensions were measured by spectrofluorometry. To determine the intracellular location of the DCFH, bulk fluorescence measurements were made on both whole and sonicated cell preparations. When intact mononuclear cells were preloaded with DCFH-DA, then sonicated and oxidized with added excess H2O2, the increase in fluorescence was only 30% of the fluorescence of mononuclear cell sonicates to which DCFH-DA was added and oxidized in a similar manner. These results suggest that a portion of the DCFH-DA incorporated by intact cells, is not susceptible to oxidation by the added H2O2. Addition of NaOH to induce hydrolysis of any residual DCFH-DA in the sonicates of DCFH-DA-loaded intact mononuclear cells resulted in a further increase in fluorescence upon addition H2O2, suggesting that a significant portion of the DCFH-DA was not hydrolyzed despite ample uptake of this dye by these cells. In contrast, no further increase in fluorescence was observed in sonicates of DCFH-DA-loaded intact neutrophils, suggesting complete hydrolysis of all incorporated DCFH-DA to DCFH. When monocytes were allowed to phagocytose DCFH-DA-loaded Staphylococcus aureus, intracellular fluorescence was measurable by flow cytometry, indicating intracellular oxidation of the fluorochromes. We therefore propose that in monocytes the mechanism of intracellular processing of these fluorochromes differs from that in neutrophils owing to differences in intracellular localization of fluorochromes, site of oxidant production, and/or accessibility of the DCFH-DA to esterolysis.

OXIDANT ACTIVITY IN EXPIRED BREATH OF PATIENTS WITH ADULT RESPIRATORY DISTRESS SYNDROME

Hydrogen peroxide levels were measured in the breath condensate of 43 patients receiving mechanical ventilation. In 16 patients the mean breath condensate peroxide level was 1·68±0·35 μmol/l on the day they met diagnostic criteria for adult respiratory distress syndrome (ARDS). The peak breath condensate peroxide level in the 27 patients in whom ARDS did not develop was significantly lower (0·34±0·08 μmol/l). Plasma lysozyme, a measure of in-vivo neutrophil turnover, was significantly higher in ARDS than in non-ARDS patients (9·2±2·2 U/ml v 3·4±1·1 U/ml). These findings support the hypothesis that neutrophil activation and oxidant production are involved in the pathogenesis of ARDS.