Neutrophil Oxygen Research Articles

Glycolysis, monocarboxylate transport, and purinergic signaling are key events in Eimeria bovis-induced NETosis.

The protozoan parasite Eimeria bovis is the causative agent of bovine coccidiosis, an enteric disease of global importance that significantly affects cattle productivity. Previous studies showed that bovine NETosis—an important early host innate effector mechanism of polymorphonuclear neutrophil (PMN)—is elicited by E. bovis stages. So far, the metabolic requirements of E. bovis-triggered NET formation are unknown. We here studied early glycolytic and mitochondrial responses of PMN as well as the role of pH, distinct metabolic pathways, P2 receptor-mediated purinergic signaling, and monocarboxylate transporters 1 and 2 (MCT1, MCT2) in E. bovis sporozoite-induced NET formation. Seahorse-based experiments revealed a rapid induction of both neutrophil oxygen consumption rate (OCR) and early glycolytic responses, thereby reflecting immediate PMN activation and metabolic changes upon confrontation with sporozoites. The impact of these metabolic changes on NET formation was studied via chemical inhibition experiments targeting glycolysis and energy generation by the use of 2-fluor-2-deoxy-D-glucose (FDG), 6-diazo-5-oxo-L-norleucin (DON), sodium dichloroacetate (DCA), oxythiamine (OT), sodium oxamate (OXA), and oligomycin A (OmA) to block glycolysis, glutaminolysis, pyruvate dehydrogenase kinase, pyruvate dehydrogenase, lactate dehydrogenase, and mitochondrial ATP-synthase, respectively. Overall, sporozoite-induced NET formation was significantly diminished via PMN pretreatments with OmA and OXA, thereby indicating a key role of ATP- and lactate-mediated metabolic pathways. Consequently, we additionally studied the effects of extracellular pH, MCT1, MCT2, and purinergic receptor inhibitors (AR-C141900, AR-C155858, theobromine, and NF449, respectively). Pretreatment with the latter inhibitors led to blockage of sporozoite-triggered DNA release from exposed bovine PMN. This report provides first evidence on the pivotal role of carbohydrate-related metabolic pathways and purinergic receptors being involved in E. bovis sporozoite-induced NETosis.

Reducing neutrophil exposure to oxygen allows their basal state maintenance.

Neutrophils are the most abundant circulating white blood cells and are the central players of the innate immune response. During their lifecycle, neutrophils mainly evolve under low oxygen conditions (0.1–4% O2), to which they are well adapted. Neutrophils are atypical cells since they are highly glycolytic and susceptible to oxygen exposure, which induces their activation and death through mechanisms that remain currently elusive. Nevertheless, nearly all studies conducted on neutrophils are carried out under atmospheric oxygen (21%), corresponding to hyperoxia. Here, we investigated the impact of hyperoxia during neutrophil purification and culture on neutrophil viability, activation and cytosolic protein content. We demonstrate that neutrophil hyper‐activation (CD62L shedding) is induced during culture under hyperoxic conditions (24 h), compared with neutrophils cultured under anoxic conditions. Spontaneous neutrophil extracellular trap (NET) formation is observed when neutrophils face hyperoxia during purification or culture. In addition, we show that maintaining neutrophils in autologous plasma is the preferred strategy to maintain their basal state. Our results show that manipulating neutrophils under hyperoxic conditions leads to the loss of 57 cytosolic proteins during purification, while it does not lead to an immediate impact on neutrophil activation (CD11bhigh, CD54high, CD62Lneg) or viability (DAPI+). We identified two clusters of proteins belonging to cholesterol metabolism and to the complement and coagulation cascade pathways, which are highly susceptible to neutrophil oxygen exposure during neutrophil purification. In conclusion, protecting neutrophil from oxygen during their purification and culture is recommended to avoid activation and to prevent the alteration of cytosolic protein composition.

Adenosine-regulated mechanisms in the pathogenesis of ventilation disorders in patients with pulmonary tuberculosis

Uncovering involvement of the purinergic system in the pathogenesis of ventilation disorders (VD) may provide additional information about the pathophysiological mechanisms leading to the development of VD in pulmonary tuberculosis (PT). The aim was to identify a relationship between the parameters of adenosine metabolism, inflammatory response and altered ventilation metabolism in PT patients. Materials and methods. Obstructive and mixed PT patients were assigned to subgroups with/without VD for assessing adenosine deaminase activity (ADA-1, 2) in serum, mononuclear cells, neutrophils; ecto-5’-nucleotidase (ecto-5’-NT); CD26 (dipeptidyl peptidase-4, DPP-4), phagocyte oxidative burst measured by NO generation. Results. PT patients showed decreased ADA-1 and CD26 (DPP-4), but increased ADA-2. Elevated intracellular adenosine concentration was found in mononuclear cells in patients lacking VD, whereas patients with mixed and obstructive VD — had it in neutrophils. Mononuclear cells of patients with PT lacking VD as well as with obstructive VD type had decreased NO3– concentration. Neutrophil hyperactivity was recorded in all groups of PT patients. Patients with PT lacking VD as well as with mixed VD type showed that the parameters of external respiration were associated with activity of extra-/intracellular ADA, whereas obstructive VD was caused by excessive formation of serum adenosine. Changes in respiratory function in PT were associated with decreased level of serum NO radicals, impaired nitrogen-dependent bactericidal phagocyte activity, and overproduced neutrophil oxygen radicals. Conclusion. Purinergic regulation is involved in regulating inflammatory and compensatory processes in PT patients as well as impaired ventilation efficiency. The most severe respiratory disorders observed in PT patients with mixed VD type are associated with the most prominent changes in nucleotidase activity, particularly ecto-ADA-2 and DPP-4/CD26.

Investigation of the Regulatory Effect of 2-Hexadecenal on Neutrophils by the Chemiluminescence Method

The effect of 2-hexadecenal on the formation of reactive oxygen and chlorine species (ROCS) in neutrophils stimulated to phagocytosis has been determined by the chemiluminescence method. It has been established that at low concentrations this aldehyde demonstrates a priming effect on the cells, enhancing ROCS production, and at higher concentrations — significantly suppresses this process. Comparison of the results of chemiluminescence and fluorescence analysis of the cell characteristics suggests that 2-hexadecenal is a signaling molecule, which exhibits the properties of the neutrophil function regulator by modifying intracellular signaling processes associated with changes in ROCS production, cytoskeleton reorganization, increase in the level of unbound calcium ions in the cytoplasm, reduction of mitochondrial membrane potential. It also induces apoptosis.

Synthesis of 3,5-Di-tert-butyl-1,2-dihydroxybenzene Derivatives and Their Effect on Free-Radical Oxidation of Hexane and Oxygen Activation Ability of Neutrophils

C6-Substituted derivatives of 3,5-di-tert-butyl-1,2-dihydroxybenzene have been synthesized, and their effect on radiation-induced free-radical oxidation of n-hexane and production of reactive oxygen and chlorine forms in neutrophils have been studied. It has been shown the introduction of the phenylhydrazone and phenylazomethine groups significantly increases the antioxidant activity of pyrocatechol derivatives. For six compounds, the ability to prevent the development of oxidative stress due to hyperproduction of active oxygen intermediates and HOCl/OCl− in neutrophils has been revealed.

Effect of physical exercise on cortisol concentration and neutrophil oxygen metabolism in peripheral blood of horses

Abstract The aim of the study was to evaluate the effect of regular physical exercise on cortisol concentration and oxygen-dependent bactericidal activity of neutrophils in peripheral blood in recreational and competitive sport horses (racehorses - Ra, trotters - T, jumping horses - J, driving horses - D). The study was conducted on 55 clinically healthy horses. Blood samples were collected from the external jugular vein three times: before exercise, immediately after exercise, and after 30-min rest. Blood samples were subjected to haematological examination, cortisol concentration was determined by radioimmunoassay (RIA) and neutrophil oxygen metabolism of neutrophils was determined using a chemiluminescence method. The analysis showed that cortisol concentration was significantly higher (P<0.05) post-exercise only in the blood of sport horses. This finding, along with a significant correlation (P<0.00005) with the heart and respiratory rates, suggests that the magnitude of cortisol secretion is associated with the intensity and duration of exercise. The relatively small post-exercise increase in cortisol concentration during different exercises of horses resulted in a transient increase of chemiluminescence activity of neutrophils, mainly in D (P<0.00001) and J horses (P<0.01). In the studied groups analysis of the correlation between cortisol concentration and other parameters showed significant correlation only in the case of the CL total in T (P<0.02) and D horses (P<0.004). It is therefore apparent that in a limited concentration, this hormone may stimulate the activity of these cells, although the effect of other neurohormonal factors cannot be excluded. This result confirms that regular and moderate training loads have a beneficial effect on the immunological status of horses.

Effect of adipokine priming upon neutrophil oxygen radical and extracellular trap release in response to stimulation by periodontal bacteria

Obesity is a major public health problem worldwide of increasing prevalence. It is characterised by a low grade chronic inflammatory state, which contributes to obesity-associated morbidity. A body of evidence suggests that obesity is associated with periodontitis, and is second only to smoking as a risk factor for periodontal tissue destruction, but underlying biological mechanisms for this association remain unclear. Adipose tissue functions as an endocrine organ, secreting cytokines (adipokines) that are reported to be associated with dysregulated/inflammatory immune responses to bacterial challenge. Adipokines include tumour necrosis factor alpha (TNFα), adiponectin, resistin, leptin and interleukin-6 (IL-6), all of which have been shown to prime/affect neutrophils for a secondary stimulus. Neutrophils are the major cell type involved in periodontal immunity to infectious agents. This study therefore aimed to elucidate neutrophil reactive oxygen species (ROS) and neutrophil extracellular trap (NET) responses to bacterial stimulation in an adipokine rich (obese) verses an adipokine normal (lean) environment. Blood was isolated from healthy male volunteers, with no history of diabetes and with normal body mass indices (BMI’s) (range 20-25). Peripheral blood neutrophils were isolated by Percoll TM discontinuous gradient centrifugation, primed with TNFα, adiponectin, resistin, leptin or IL-6, alongside control two control stimuli GM-CSF and IL-8, at concentrations reflective of lean and obese individuals. Neutrophils were subsequently stimulated with Fusobacterium nucleatum (ATCC 10953) for TollLike receptor (TLR)-4 activation, opsonised Staphyloccocus aureus (NCTC 6571) for FcγRII activation or phorbol 12-myristate 13-acetate (PMA) for direct activation via protein kinase C (PKC). Neutrophil ROS were measured by luminol enhanced and chemiluminsecence and NET release was by fluorometric assay of DNA and fluorescence microscopy. Adipokine concentrations representative of those found in obese individuals have been shown in this pilot study to enhance neutrophil ROS and NET generation after exposure to bacterial stimulus. The effects are however stimulus dependent and require longer studies to validate any significant results.

Long-term olive oil-based parenteral nutrition sustains innate immune function in home patients without active underlying disease

It remains unclear whether impaired host defenses contribute to the increased risk for infectious complications seen in patients on home parenteral nutrition (HPN). The aim of this study was to compare the innate immune function of patients on olive oil-based HPN with that of healthy controls. Innate immune functions and (anti-)oxidant balance were studied in 20 patients on olive oil-based HPN without an active underlying immune-mediated disease (Clinoleic(®), ≥ 6 months; >3 times/week), and 21 age- and sex-matched healthy controls. Neutrophils of patients and controls had a similar capacity to eliminate Streptococcus pneumoniae. Also, levels of activation markers (CD66b, CD11b, CD62L) in granulocytes and monocytes, phorbol ester- and zymosan-induced neutrophil oxygen radical production were not different between patients and controls. No differences in (anti-)oxidant status were found, except for higher concentrations of oxidized glutathione and lower plasma selenium and vitamin C in patients compared to controls. Compromised innate immune function does not seem to explain the increased risk for infectious complications in HPN patients using olive oil-based lipid emulsions.

The effect of storage on whole blood chemiluminescence measurement of equine neutrophils

The aim of this study was to determine the effect of duration and temperature of sample storage on whole blood chemiluminescence measurement results. Venous blood from 18 clinically healthy Polish half-bred horses aged 4 to 11 years were used in the study. Luminol dependent chemiluminescence (CL) was used to measure neutrophil oxygen metabolism in whole blood. Blood samples were examined for spontaneous CL and stimulated by a surface receptor stimulus as well as extra-receptor stimulus. The assay was performed in two parallel experimental sets with samples stored at 4 and 22 °C, respectively. Whole blood CL was estimated at 2, 6, 24, 48, 72, 96 and 120 h after collection. The study demonstrated that temperature and duration of sample storage are factors that determine the quality of CL measurements of whole blood in horses. The study concluded that samples should be stored at 4 °C and the assay should be performed as early as possible. It was also shown that the viability period of horse blood for CL assays is relatively long. Material stored at room temperature for 24 h and even up to 48 h at 4 °C did not show any significant decrease in spontaneous or stimulated chemiluminescence.

Influence of variable domain glycosylation on anti-neutrophil cytoplasmic autoantibodies and anti-glomerular basement membrane autoantibodies

BackgroundThe pathophysiological significance of variable region glycosylation of autoantibodies is still unclear. In the current study, the influence of the variable region N-linked oligosaccharides on the reactivity of three autoantibody specificities was investigated with Sambucus nigra agglutinin (SNA), which mainly binds to oligosaccharides with terminal α2, 6-linked sialic acid on the variable region of IgG.MethodsTwenty-seven patients with serum positive anti-neutrophil cytoplasmic autoantibodies (ANCA) against myeploperoxidase (MPO) or proteinase 3 (PR3), or autoantibodies against glomerular basement membrane (GBM) were included. Total IgG was isolated and separated into non-SNA-binding and SNA-binding fractions with SNA affinity chromatography. Antigen-specific IgG was purified by immunoaffinity chromatography.ResultsAt the same concentration of IgG, the antigen binding level of non-SNA-binding IgG was significantly lower than that of SNA-binding IgG for MPO-ANCA (absorbance value at 405 nm, 0.572 ± 0.590 vs. 0.962 ± 0.670, P < 0.001) and for PR3-ANCA (0.362 ± 0.530 vs. 0.560 ± 0.531, P = 0.003). The antigen binding level of non-SNA-binding IgG was significantly higher than that of SNA-binding IgG for anti-GBM antibodies (1.301 ± 0.594 vs. 1.172 ± 0.583, P = 0.044). The level of variable region glycosylation of total IgG was significantly lower than that of affinity-purified MPO-ANCA (1.021 ± 0.201 vs. 1.434 ± 0.134, P = 0.004). The level of variable region glycosylation of total IgG was significantly higher than that of affinity-purified anti-GBM antibodies (1.034 ± 0.340 vs. 0.734 ± 0.333, P = 0.007). The SNA-binding fraction of MPO-ANCA-containing IgG and PR3-ANCA-containing IgG induced higher levels of neutrophil oxygen radical production than the corresponding non-SNA-binding fractions (P < 0.001 and P = 0.043, respectively). The level of variable region glycosylation of affinity-purified MPO-ANCA was higher in active AAV than the same patients in remission (P = 0.001).ConclusionCharacteristics of variable region glycosylation of ANCA and anti-GBM antibodies were different from that of total IgG, which might influence the antigen-binding ability of these antibodies. Variable region glycosylation of ANCA might influence the effect of ANCA-induced neutrophils respiratory burst.

Adiponectin Protects Against Hyperoxic Lung Injury and Vascular Leak

Adiponectin (Ad), an adipokine exclusively secreted by the adipose tissue, has emerged as a paracrine metabolic regulator as well as a protectant against oxidative stress. Pharmacological approaches of protecting against clinical hyperoxic lung injury during oxygen therapy/treatment are limited. We have previously reported that Ad inhibits the NADPH oxidase-catalyzed formation of superoxide from molecular oxygen in human neutrophils. Based on this premise, we conducted studies to determine whether (i) exogenous Ad would protect against the hyperoxia-induced barrier dysfunction in the lung endothelial cells (ECs) in vitro, and (ii) endogenously synthesized Ad would protect against hyperoxic lung injury in wild-type (WT) and Ad-overexpressing transgenic (AdTg) mice in vivo. The results demonstrated that exogenous Ad protected against the hyperoxia-induced oxidative stress, loss of glutathione (GSH), cytoskeletal reorganization, barrier dysfunction, and leak in the lung ECs in vitro. Furthermore, the hyperoxia-induced lung injury, vascular leak, and lipid peroxidation were significantly attenuated in AdTg mice in vivo. Also, AdTg mice exhibited elevated levels of total thiols and GSH in the lungs as compared with WT mice. For the first time, our studies demonstrated that Ad protected against the hyperoxia-induced lung damage apparently through attenuation of oxidative stress and modulation of thiol-redox status.

Advanced glycation end products enhance reactive oxygen and nitrogen species generation in neutrophils in vitro

Increased oxidative stress (OS) in diabetes mellitus is one of the major factors leading to diabetic pathology. However, the mediators and mechanism that provoke OS in diabetes is not fully understood, and it is possible that accumulation of advanced glycation end products (AGEs) formed secondary to hyperglycemic conditions may incite circulating polymorphonuclear neutrophils (PMN) to generate reactive oxygen species (ROS). In this report, we aim to investigate the effect of AGE on reactive oxygen and nitrogen species generation and subsequent OS in PMN. AGE-HSA exert dose- and time-dependent enhancement of ROS and reactive nitrogen intermediates (RNI) generation by PMN. Increased ROS and RNI generation were found to be mediated through the upregulation of NADPH oxidase and inducible nitric oxide synthase (iNOS), respectively, as evident from the fact that AGE-treated neutrophils failed to generate ROS and RNI in presence of diphenyleneiodonium, a flavoprotein inhibitor for both enzymes. Further increased generation of ROS and RNI ceased when the cells were incubated with anti-RAGE antibody suggesting the involvement of AGE-RAGE interaction. Also increased malondialdehyde (MDA) and protein carbonyl formation in AGE-exposed PMN suggest induction of OS by AGE. This study provides evidence that AGEs may play a key role in the induction of oxidative stress through the augmentation of PMN-mediated ROS and RNI generation and this may be in part responsible for development of AGE-induced diabetic pathology.

Impact of Intestinal Lavage on the Development of Pneumonia in Patients with Acute Poisonings by Psychopharmacological Agents

Objective: to evaluate the impact of intestinal lavage (IL) on the incidence of pneumonia in patients with poisonings by psychopharmacological agents (PPA). Subjects and methods. The incidence of pneumonia as a complication of acute oral poisonings by PPA was studied in 393 patients with moderate and severe poisonings. Of them, 112 patients underwent IL as a detoxification method; the other patients received standard blood detoxification methods in a package of remedial measures. The time course of changes in the blood levels of gram-negative intestinal bacterial lipopolysaccharide (LPS) and immune status were studied before and after IL. Results. Prior to treatment, 65% of the patients with PPA poisoning were found to have an 8—10-fold excess in the reference values for blood LPS. At the same time, there were suppressed immunogram readings. After IL, there was a 1.5-fold decrease in blood LPS levels, which remained during 3 days, and activation of immune processes. It was shown that the patients undergoing IL developed pneumonia as a complication of PPA poisoning 1.6 times less frequently and died of pneumonia 16 times less often. Conclusion. The better results of IL treatment are obviously associated with enteral detoxification, reduced enterogenic toxemia, and improved hemostatic parameters, which was accompanied by normalized neutrophil oxygen metabolism and lower exposure of lung tissue to reactive oxygen species. Key words: acute poisonings, endotoxins, enterohumoral barrier, pneumonia, intestinal lavage.

R2105 Gram-positive and Gram-negative bacteria induce neutrophil oxygen burst augmented by platelets; role of platelet-derived TXA2 in cellular host response

R2105 Gram-positive and Gram-negative bacteria induce neutrophil oxygen burst augmented by platelets; role of platelet-derived TXA2 in cellular host response

Inappropriate neutrophil activation in venous disease.

Neutrophil oxygen radical production was studied in 18 limbs with class 2 or 3 venous disease and compared with that of nine normal limbs. Neutrophils were isolated from arm and leg venous samples. Free radical production was determined using chemiluminescence after stimulation with the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (FMLP) or the ester phorbol myristate acetate (PMA). The ratio of leg to arm luminescence was greater after FMLP stimulation in patients with venous disease (median 1.52 (95 per cent confidence interval (c.i.) 1.27-2.60)) than in controls (median 0.97 (95 per cent c.i. 0.70-1.12); P < 0.01). These changes were not observed with PMA (venous disease 1.16 (95 per cent c.i. 1.05-1.40); controls 0.95 (95 per cent c.i. 0.78-1.24)). There were fewer FMLP receptors on activated leg neutrophils (median 20.19 (95 per cent c.i. 3.58-51.42) fluorescence units) than arm neutrophils (median 36.03 (95 per cent c.i. 13.00-65.28) fluorescence units; P < 0.05), indicating an amplification of signal transduction. Intracellular calcium imaging demonstrated a larger release of calcium after stimulation of leg neutrophils (median 25.0 per cent (95 per cent c.i. 15.7-43.9 per cent)) compared with neutrophils from the arm (median 8.0 per cent (95 per cent c.i. 5.6-16.1 per cent); P = 0.04), demonstrating calcium-dependent activation. Neutrophils in patients with chronic venous disease inappropriately produce more oxygen free radical as a result of amplification of a calcium-dependent signal pathway.

PMA-enhanced neutrophil [ 18F]FDG uptake is independent of integrin occupancy but requires PI3K activity

Objective While respiratory burst enhances neutrophil glucose utilization, many neutrophil functions are critically influenced by extracellular matrix interaction and phosphoinositide-3-OH kinase (PI3K) signaling. We thus evaluated the role of RGD integrin occupancy and PI3K inhibition on respiratory burst and [ 18F]fluorodeoxyglucose ([ 18F]FDG) uptake of stimulated neutrophils. Methods Human neutrophils were stimulated by 100 ng/ml phorbol–myristate–acetate (PMA), and respiratory burst was measured by cumulative luminescence with lucigenin. [ 18F]FDG uptake and total hexokinase activity was measured 20 min after PMA stimulation in the presence or absence of soluble RGD peptides (200 μg/ml) and/or the PI3K inhibitor wortmannin (200 nM). Results Phorbol–myristate–acetate induced a 71.7±0.9 fold increase in neutrophil oxygen intermediate generation. [ 18F]FDG uptake was increased to 194.6±3.7% and hexokinase activity to 145.0±2.0% of basal levels (both P<.0005). RGD peptides attenuated respiratory burst activation to 35.6±0.2% ( P<.005) but did not inhibit stimulated [ 18F]FDG uptake or hexokinase activity. In contrast, without affecting respiratory burst activation, wortmannin inhibited PMA-stimulated [ 18F]FDG uptake to 66.9±1.6% and hexokinase activity to 81.0±4.2% (both P<.0005), demonstrating its dependence on PI3K activity. Neither RGD nor wortmannin reversed the other's inhibitory effect on stimulated [ 18F]FDG uptake and hexokinase activity or respiratory burst, which suggests the involvement of distinct signaling pathways. Conclusion Neutrophil [ 18F]FDG uptake is enhanced by PMA through a mechanism that requires PI3K activity but is independent of integrin receptor occupancy or respiratory burst activation.

Effects of platinum and palladium ions on the production and reactivity of neutrophil-derived reactive oxygen species

This study was designed to investigate the effects of platinum, as hydrogen hexachloroplatinate (Pt; 0.0025–25 μM), on the production of reactive oxygen species (ROS) by human neutrophils in vitro. ROS were measured by lucigenin-enhanced chemiluminescence (LECL). Addition of Pt to neutrophils was accompanied by a lag phase of about 1 min, followed by a linear dose-related increase in LECL, which peaked at around 4 min and achieved statistical significance at concentrations of 0.025 μM Pt and higher. Interestingly, Pt-mediated enhancement of LECL was not associated with meaningful alterations in neutrophil oxygen consumption, assembly of NADPH oxidase, or cytosolic Ca 2+ and was completely attenuated by superoxide dismutase and inhibitors of NADPH oxidase, but not by catalase or scavengers of hydroxyl radical, and was undetectable with cells from individuals with chronic granulomatous disease. Exposure of α1-proteinase inhibitor to Pt-treated neutrophils resulted in inactivation of elastase-inhibitory capacity, underscoring the potential toxicity of neutrophil/Pt interactions. The pro-oxidative actions of Pt were mimicked by palladium (Pd), but not by cisplatin or rhodium. These observations demonstrate that Pt and Pd potentiate the reactivity, as opposed to the generation of neutrophil-derived oxidants, an activity that may contribute to airway inflammation in occupationally and possibly environmentally exposed individuals.

Methylglyoxal: a stimulus to neutrophil oxygen radical production in chronic renal failure?

Chronic renal failure is characterized by oxidant stress, resulting in part from increased reactive oxygen species production by neutrophils. Plasma concentrations of methylglyoxal are increased in uraemia. Methylglyoxal activates p38 mitogen-activated protein kinase (MAPK) in endothelial cells. Activation of p38 MAPK in neutrophils enhances reactive oxygen species production through exocytosis of intracellular storage granules. We tested the hypothesis that methylglyoxal enhances reactive oxygen species production by activating p38 MAPK in neutrophils. Neutrophils were exposed to methylglyoxal in vitro. Activation of p38 MAPK was determined by immunoblot analysis. Exocytosis was determined by measuring plasma membrane expression of CD35 and CD66b, specific markers of secretory vesicles and specific granules, respectively. Reactive oxygen species production was determined by measuring H(2)O(2) and O(-)(2) production. Methylglyoxal activated p38 MAPK and caused dose-dependent increases in CD35 and CD66b expression, which were blocked by the methylglyoxal scavenger, aminoguanidine, or the p38 MAPK inhibitor, SB203580. Methylglyoxal caused dose-dependent increases in basal and Staphylococcus aureus-stimulated H(2)O(2) production and basal and formyl-methionyl-leucyl-phenylalanine-stimulated O(-)(2) production. Enhancement of reactive oxygen species production was blocked by aminoguanidine and SB203580. Methylglyoxal enhances reactive oxygen species production in neutrophils through a process involving p38 MAPK-dependent exocytosis of intracellular storage granules. These findings, together with the observation that methylglyoxal concentrations are increased in renal failure, suggest a possible role for methylglyoxal as a uraemic toxin that contributes to the oxidant stress associated with renal failure.

Effects of platinum and palladium ions on the production and reactivity of neutrophil-derived reactive oxygen species

This study was designed to investigate the effects of platinum, as hydrogen hexachloroplatinate (Pt; 0.0025–25 μM), on the production of reactive oxygen species (ROS) by human neutrophils in vitro. ROS were measured by lucigenin-enhanced chemiluminescence (LECL). Addition of Pt to neutrophils was accompanied by a lag phase of about 1 min, followed by a linear dose-related increase in LECL, which peaked at around 4 min and achieved statistical significance at concentrations of 0.025 μM Pt and higher. Interestingly, Pt-mediated enhancement of LECL was not associated with meaningful alterations in neutrophil oxygen consumption, assembly of NADPH oxidase, or cytosolic Ca 2+ and was completely attenuated by superoxide dismutase and inhibitors of NADPH oxidase, but not by catalase or scavengers of hydroxyl radical, and was undetectable with cells from individuals with chronic granulomatous disease. Exposure of α1-proteinase inhibitor to Pt-treated neutrophils resulted in inactivation of elastase-inhibitory capacity, underscoring the potential toxicity of neutrophil/Pt interactions. The pro-oxidative actions of Pt were mimicked by palladium (Pd), but not by cisplatin or rhodium. These observations demonstrate that Pt and Pd potentiate the reactivity, as opposed to the generation of neutrophil-derived oxidants, an activity that may contribute to airway inflammation in occupationally and possibly environmentally exposed individuals.

Influence of neopterin on generation of reactive species by myeloperoxidase in human neutrophils.

Increased neopterin concentrations in human serum indicate activation of cell-mediated immune response. Earlier we have shown that neopterin enhanced generation of singlet oxygen, hydroxyl radical and nitric oxide in human peripheral blood neutrophils by NADPH-independent pathways. To further investigate a participation of neopterin in reactive species production by neutrophils, we studied its influence on myeloperoxidase (MPO) activity. MPO was isolated from human peripheral blood neutrophils from healthy donors. Generation of reactive species by MPO/H(2)O(2) in Earl's solution (pH=7.2) at 37 degrees C was investigated by monitoring of chemiluminescence using luminol as light emitter. In the MPO/H(2)O(2) system, neopterin increased singlet oxygen in a concentration-dependent manner, but it decreased formation of other oxidizing species. Comparing several oxygen scavengers, formation of reactive species was totally blocked by sodium azide (NaN(3)), both in the presence and in the absence of neopterin. Superoxide dismutase (SOD) and d-mannitol insignificantly decreased chemiluminescence of this reaction, but diazabicyclo[2.2.2]octane (DABCO) strongly inhibited it. We conclude that the effects of neopterin on neutrophils' MPO are directed to increase singlet oxygen and to decrease other reactive species via inhibition of MPO and/or scavenging of reactive species.