Neutrophil Respiratory Burst Activity Research Articles

Aminopeptidase N (CD13) Regulates Tumor Necrosis Factor-α-induced Apoptosis in Human Neutrophils

Neutrophil apoptosis plays a central role in the resolution of granulocytic inflammation. We have shown previously that tumor necrosis factor-alpha (TNFalpha) enhances the rate of neutrophil apoptosis at early time points via a mechanism involving both TNF receptor (TNFR) I and TNFRII. Here we reveal a marked but consistent variation in the magnitude of the pro-apoptotic effect of TNFalpha in neutrophils isolated from healthy donors, and we show that inhibition of cell surface aminopeptidase N (APN) using actinonin, bestatin, or inhibitory peptides significantly enhanced the efficacy of TNFalpha-induced killing. Notably, an inverse correlation is shown to exist between neutrophil APN activity and the sensitivity of donor cells to TNFalpha-induced apoptosis. Inhibition of cell surface APN appears to interfere with the shedding of TNFRI, and as a consequence results in augmented TNFalpha-induced apoptosis, cell polarization, and TNFalpha-primed, formyl-methionyl-leucyl-phenylalanine-stimulated respiratory burst. Of note, actinonin and bestatin had no effect on TNFRII expression under resting or TNFalpha-stimulated conditions and did not alter CXCRI or CXCRII expression. These data suggest significant variation in the activity of APN/CD13 on the cell surface of neutrophils in normal individuals and reveal a novel mechanism whereby APN/CD13 regulates TNFalpha-induced apoptosis via inhibition of TNFRI shedding. This has therapeutic relevance for driving neutrophil apoptosis in vivo.

Liu-Shen-Wan, a traditional Chinese medicine, improves survival in sepsis induced by cecal ligation and puncture via reducing TNF-α levels, MDA content and enhancing macrophage phagocytosis

Sepsis in humans is a difficult condition to treat and is often associated with a high mortality rate. Here, we investigated putative protective effects of Liu-Shen-Wan (LSW), a well-known Chinese formula used in treating infectious diseases, against polymicrobial sepsis induced by cecal ligation and puncture (CLP). The oral administration of LSW, at the first dose of 60 mg/kg and then 30 mg/kg every 12 h, significantly improved the survival of CLP mice during a 4-day observation period. The effects of LSW on the inflammatory response (circulating tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1) levels and malondialdehyde (MDA) content—an index of lipid peroxidation), infectious degree (peritoneal bacteria counts), and innate immunity function (leukocyte counts, macrophage phagocytosis and neutrophil respiratory burst) were further examined in rats. We demonstrated that treatment of LSW significantly decreased elevated levels of circulating TNF-α at 4 h and further reduced plasma MDA levels at 24 h after CLP, at first doses of 15 and 30 mg/kg and then 7.5 and 15 mg/kg every 12 h. Moreover, LSW markedly enhanced clearance of intraperitoneal bacteria associated with the increasing count of peritoneal leukocytes and enhancing phagocytic activity of macrophages partly impaired at 24 h after CLP. In contrast, LSW lightly reduced IL-1 levels at 4 h and failed to improve deactivated respiratory burst activity of neutrophils at 24 h after CLP. Thus, LSW exerts protective effects against sepsis induced by CLP, mainly by reducing plasma TNF-α and MDA levels and enhancing peritoneal macrophage phagocytosis, suggesting that it is a potential agent in the prevention and treatment of sepsis.

Delayed Neutrophil Apoptosis Attenuated by Melatonin in Human Acute Pancreatitis

This study evaluated the expression of neutrophil apoptosis and the effects of melatonin at different concentrations on delayed neutrophil apoptosis in different severities of acute pancreatitis in patients. The study population was comprised of 10 patients with severe acute pancreatitis (SAP) and 10 with mild acute pancreatitis (MAP). A total of 10 mL of blood was drawn 24 hours after the onset of the clinical disease for isolation and incubation of the human neutrophils with 4 different concentrations of melatonin. Neutrophil apoptosis activity, CD18 expression, and respiratory burst activity were assessed with flow cytometry 12 hours after incubation. Another group of neutrophils from a healthy control group was used (n = 6) for comparison. Neutrophil apoptosis in patients with SAP is delayed compared with that of patients with MAP. Neutrophils from patients with SAP or MAP are functionally activated. Melatonin at concentrations of 10(-8), 10(-7), or 10(-6) M reverses the delayed process and enhances apoptosis activity in neutrophils in patients with MAP. Melatonin at concentrations of 10(-7) and 10(-6) M reverses the delayed process and increases apoptosis activity in neutrophils in patients with SAP. Neutrophils from patients with SAP and MAP showed significantly increased CD18 expression and respiratory burst activity. Melatonin at concentrations of 10(-7) or 10(-6) M reverses CD18 expression and respiratory burst activity in neutrophils in patients with SAP. This study highlights the importance of neutrophil apoptosis in patients with SAP and raises the possibility of a therapeutic strategy. Study data show that melatonin promotes neutrophil apoptosis in human acute pancreatitis.

Exercise-Induced Muscle Damage, Plasma Cytokines, and Markers of Neutrophil Activation

Unaccustomed eccentric exercise often results in muscle damage and neutrophil activation. We examined changes in plasma cytokines stress hormones, creatine kinase activity and myoglobin concentration, neutrophil surface receptor expression, degranulation, and the capacity of neutrophils to generate reactive oxygen species in response to in vitro stimulation after downhill running. Ten well-trained male runners ran downhill on a treadmill at a gradient of -10% for 45 min at 60% VO2max. Blood was sampled immediately before (PRE) and after (POST), 1 h (1 h POST), and 24 h (24 h POST) after exercise. At POST, there were significant increases (P < 0.01) in neutrophil count (32%), plasma interleukin (IL)-6 concentration (460%), myoglobin (Mb) concentration (1100%), and creatine kinase (CK) activity (40%). At 1 h POST, there were further increases above preexercise values for neutrophil count (85%), plasma Mb levels (1800%), and CK activity (56%), and plasma IL-6 concentration remained above preexercise values (410%) (P < 0.01). At 24 h POST, neutrophil counts and plasma IL-6 levels had returned to baseline, whereas plasma Mb concentration (100%) and CK activity (420%) were elevated above preexercise values (P < 0.01). There were no significant changes in neutrophil receptor expression, degranulation and respiratory burst activity, and plasma IL-8 and granulocyte-colony stimulating factor concentrations at any time after exercise. Neutrophil count correlated with plasma Mb concentration at POST (r = 0.64, P < 0.05), and with plasma CK activity at POST (r = 0.83, P < 0.01) and 1 h POST (r = 0.78, P < 0.01). Neutrophil activation remains unchanged after downhill running in well-trained runners, despite increases in plasma markers of muscle damage.

Comparison of respiratory burst activity of inflammatory neutrophils in ayu ( Plecoglossus altivelis) and carp ( Cyprinus carpio)

Neutrophils of ayu ( Plecoglossus altivelis) were previously shown to have unusually high respiratory burst activity (RBA). To understand this unique character of ayu neutrophils, the RBAs of resting and inflammatory neutrophils of ayu and carp ( Cyprinus carpio) were compared. Inflammation was induced in the peritoneal cavity by injecting killed-bacteria. The RBA of peritoneal-exudate (inflammatory) neutrophils was measured after stimulation with phorbol myristate acetate (PMA). Resting neutrophils were obtained from kidney and blood of non-injected fish. In carp, the RBA of inflammatory neutrophils was much higher than that of resting neutrophils. On the other hand, in ayu no significant difference was observed. The RBA of neutrophils was already high in the kidney stock. The process of inflammation did not further enhance RBA. In addition to PMA, other stimulants (zymosan, opsonized-zymosan, and zymosan-treated serum) were used to measure RBA. Even with these stimulants, the RBA of inflammatory neutrophils was always higher than that of kidney neutrophils in carp. On the other hand in ayu, the RBA of kidney neutrophils was already high in the kidney stock, and no significant difference was observed between peritoneal and kidney neutrophils in ayu. These results indicate ayu neutrophils have spontaneously activated characteristics with the respect to the ROS generation in the kidney hematopoietic-stock.

Imatinib Does Not Inhibit Neutrophil Oxidative Burst Function in Patients with Chronic Myelogenous Leukemia (CML).

Imatinib mesylate is a competitive Bcr-Abl tyrosine kinase inhibitor which has been used in treatment of chronic myelogenous leukemia (CML). In addition to the inhibition of the Bcr-Abl protein tyrosine kinases, it also shows activity against other protein kinases such as platelet-derived growth factor receptor (PDGF-R), c-Kit and protein kinase C (PKC) in higher doses (Manley et al., 2002). Myelosuppression has been reported up to 60% of patients with CML under imatinib therapy (Deininger et al., 2003). Microbicidial activity of the neutrophils is mainly generated by the enzyme NADPH oxidase which produces superoxide anion. Several protein kinases including protein kinase C (PKC) that phosphorylate components of the NADPH oxidase and regulate oxidase activity have been identified. Little has been known about the effect of imatinib on neutrophil function, so far. Therefore, we aimed to evaluate the neutrophil respiratory burst activity using Phorbol Myristate acetate (PMA), a PKC activator, in vitro, in patients with CML achieving imatinib. Heparinized peripheral blood samples were collected from 14 patients with CML under imatinib therapy (400 mg daily) for 9.4 ± 4 months and 13 voluntary healthy controls. Oxidative burst was evaluated using a standard method defined previously by flow cytometry (Robinson et al., 1993) Oxidative burst stimulation index was calculated as the ratio of mean value of fluorescence intensity of DCFH-DA labelled neutrophils before and following PMA stimulation. It has been found that the stimulation index of neutrophils in patient achieving imatinib was 14.5±5 which was comparable to healthy control group 14±6.6. Furthermore, no serious infection disease was clinically detected during 8 month follow up period. So this study suggest Imatinib which has ability to inhibit bcr-abl fusion protein selectively and also can inhibit some others kinases, however has no inhibitory effect on the neutrophil function in patients with CML.

Changes in neutrophil surface receptor expression, degranulation, and respiratory burst activity after moderate- and high-intensity exercise.

Intense exercise stimulates the systemic release of a variety of factors that alter neutrophil surface receptor expression and functional activity. These alterations may influence resistance to infection after intense exercise. The aim of this study was to examine the influence of exercise intensity on neutrophil receptor expression, degranulation (measured by plasma and intracellular myeloperoxidase concentrations), and respiratory burst activity. Ten well-trained male runners ran on a treadmill for 60 min at 60% [moderate-intensity exercise (MI)] and 85% maximal oxygen consumption [high-intensity exercise (HI)]. Blood was drawn immediately before and after exercise and at 1 h postexercise. Immediately after HI, the expression of the neutrophil receptor CD16 was significantly below preexercise values (P < 0.01), whereas MI significantly reduced CD35 expression below preexercise values (P < 0.05). One hour after exercise at both intensities, there was a significant decline in CD11b expression (P < 0.05) and a further decrease in CD16 expression compared with preexercise values (P < 0.01). CD16 expression was lower 1 h after HI than 1 h after MI (P < 0.01). Immediately after HI, intracellular myeloperoxidase concentration was less than preexercise values (P < 0.01), whereas plasma myeloperoxidase concentration was greater (P < 0.01), indicating that HI stimulated neutrophil degranulation. Plasma myeloperoxidase concentration was higher immediately after HI than after MI (P < 0.01). Neutrophil respiratory burst activity increased after HI (P < 0.01). In summary, both MI and HI reduced neutrophil surface receptor expression. Although CD16 expression was reduced to a greater extent after HI, this reduction did not impair neutrophil degranulation and respiratory burst activity.

Changes in Immunological Parameters and Disease Resistance in Juvenile Coho Salmon (Oncorhynchus kisutch) in Response to Dehydroabietic Acid Exposure under Varying Thermal Conditions

Abstract This study explored the effects of a sublethal 96-h dehydroabietic acid (DHAA) exposure on aspects of the immune system of juvenile coho salmon under varying temperature conditions. Coho were exposed to DHAA concentrations below the determined LC50 value of 0.94 mg/L (95% confidence limits of 0.81 to 1.24 mg/L) for 96 h at either their acclimation temperature (8 or 18°C), or during an acute warm-shock (8 to 18°C) or cold-shock (18 to 8°C). Acclimation temperature alone significantly affected hematocrit (Hct), neutrophil respiratory burst activity (RBA) and leucocyte proportions. With temperature-shock, leucocrit (Lct), RBA and leucocyte proportions were altered. All parameters were affected by DHAA exposure, but not always in a dose-dependent manner. Across groups, DHAA caused Hct, lysozyme, thrombocyte, neutrophil and monocyte proportions to increase, and Lct, RBA and lymphocyte proportions to decrease. DHAA-temperature interactions resulted in the exacerbation of DHAA-induced effects. Exposure temperature had the most significant effect on the susceptibility of coho to Aeromonas salmonicida; fish were more susceptible at cold temperatures and when subjected to a temperature-shock compared to their respective controls. DHAA exposure modulated the response of temperature-shocked fish to this pathogen.

Flow cytometric determination of neutrophil respiratory burst activity in workers exposed to formaldehyde.

The aim of the study was to investigate neutrophil respiratory burst activity (NRBA) in workers who were occupationally exposed to formaldehyde. NRBA, spontaneous and stimulated with E. coli, N-formyl-methionyl-leucyl-phenylalanine (fMLP) and phorbol 12-myristate 13-acetate (PMA), was studied by means of quantitative flow cytometric determination in 29 workers who were occupationally exposed to formaldehyde; 21 healthy subjects, not exposed to formaldehyde, served as controls. All subjects underwent clinical assessment, including a review of a summary of their medical history and a physical examination. Routine haematological tests were performed. A statistically significant predominance of subjective symptoms and objective clinical findings of chronic upper respiratory tract inflammation, as well as decreased resistance to infections, was observed in the 29 workers exposed to formaldehyde, compared with the controls (chi2 = 9.28, P = 0.02). No statistically significant difference in the spontaneous and stimulated NRBA between the exposed workers and the control group was observed. The spontaneous NRBA (percentage oxidizing cells) was significantly lower in the group of exposed workers with upper respiratory tract findings and frequent and long-lasting infectious inflammatory relapses (median and range 0.45 (0.02-2.03), mean values 0.65 +/- 0.74) than in the healthy controls (median and range 1.35 (0.07-8.69), mean values 2.42 +/- 2.47; P < 0.05), and in the group of exposed workers with rare and short, acute, inflammation of the upper respiratory tract or without any inflammations (median and range 1.00 (0.02-8.67), mean values 1.67 +/- 2.08; P < 0.05). A significant negative correlation between the duration of occupational exposure to formaldehyde and erythrocyte count and haematocrit was found. The observed decrease of spontaneous NRBA in workers with a history and clinical findings of frequent and long-lasting relapses of chronic inflammation of the upper respiratory tract could be due to formaldehyde exposure and individual susceptibility. The results obtained suggest that functional changes in polymorphonuclear neutrophil granulocytes could serve as an early indicator of an impact of formaldehyde on NRBA. The applied method might be used for identifying groups at increased toxicological risk.

Ultrasound contrast agents can influence the respiratory burst activity of human neutrophil granulocytes

Activated leucocytes can bind and, subsequently, phagocytose microbubbles that are used as ultrasound (US) contrast agents. The purpose of this study was to investigate whether or not microbubbles can influence the inflammatory response of human neutrophil granulocytes. Granulocytes isolated from healthy volunteers were activated with various stimuli, for example, the bacterial peptide N-formyl-methyonyl-leucyl-phenylalanine (fMLP), the calcium ionophore A23187, the protein kinase C activator phorbol myristate acetate (PMA) and the cytokine tumor necrosis factor alpha (TNF-α), and incubated with albumin or phospholipid microbubbles. Neutrophil respiratory burst activity and elastase release were quantified. Albumin (Optison®) and phospholipid (SonoVue®) contrast agents induced an extensive oxidative response of human granulocytes to all the stimuli used, and these effects could be significantly impaired by preincubation of the cells with cytochalasin B. Left heart contrast agents used for contrast-enhanced US assessment can activate human neutrophil granulocytes, inducing an extensive respiratory burst to secondary stimuli. The potential clinical relevance of this effect needs to be elucidated. (E-mail: helmut_kuecherer@med.uni-heidelberg.de)

Sulfur-related air pollutants induce the generation of platelet-activating factor, 5-lipoxygenase- and cyclooxygenase-products in canine alveolar macrophages via activation of phospholipases A 2

Recent studies have shown that long-term in vivo exposure of dogs to neutral sulfur(IV)/sulfite aerosols induces mild inflammatory reactions, whereas the combination of neutral sulfite with acidic sulfur(VI)/sulfate aerosols evokes less pronounced effects. To understand underlying mechanisms, we studied in vitro the role of lipid mediators in the responses of alveolar macrophages (AMs) to sulfur-related compounds under neutral (pH 7) or moderate acidic (pH 6) conditions. Canine AMs incubated with sulfite at pH 7 released threefold higher amounts of platelet-activating factor than control ( P<0.005). Generation of arachidonic acid, leukotriene B 4, 5-hydroxy-eicosatetraenoic acid, prostaglandin E 2, thromboxane B 2 and 12-hydroxyheptadecatrienoic acid increased twofold ( P<0.0005). However, these metabolites remained unchanged following incubation of AMs with sulfite at pH 6 or with sulfate at pH 7 or pH 6. Mediator release by sulfite-treated AMs at pH 7 stimulated respiratory burst activity of neutrophils. Inhibition of MAPK pathway by PD 98059, of cytosolic (cPLA 2) and secretory phospholipases A 2 by AACOCF 3 and thioetheramide-PC, respectively, reduced sulfite-induced eicosanoid formation in AMs. Sulfite activated cPLA 2 activity twofold at pH 7. This mechanism of sulfite-stimulated responses in phospholipid metabolism predicts that chronic exposure to sulfur(IV)/sulfite is associated with a considerable health risk.

Flow cytometric analysis of the neutrophil respiratory burst of ayu, Plecoglossus altivelis: comparison with other fresh water fish

Neutrophils of vertebrates undergo respiratory burst activity (RBA) as a defense mechanism against bacterial infections. We report here that ayu ( Plecoglossus altivelis) have unusually high RBAs even when they are in a healthy condition. Kidney and blood leukocytes were obtained from ayu, rainbow trout ( Oncorhynchus mykiss), carp ( Cyprinus carpio), eel ( Anguilla japonica), and pond smelt ( Hypomesus nipponensis). Neutrophil RBA was measured by flow cytometry using dihydrorhodamine after stimulation with phorbol myristate acetate. The amount of RBA of neutrophils from both blood and kidney was significantly higher in ayu than in the other species (e.g. the fluorescence intensity of ayu blood neutrophils was about 3–7 times higher than that from trout and carp, and that of ayu kidney neutrophils was 2–19 times higher than that of rainbow trout, carp, eel, and pond smelt). This unique character of ayu neutrophils was invariable even at different ages, locations, and sex-maturation stages.

Akt phosphorylates p47phox and mediates respiratory burst activity in human neutrophils.

Respiratory burst activity and phosphorylation of an NADPH oxidase component, p47(phox), during neutrophil stimulation are mediated by phosphatidylinositol 3-kinase (PI-3K) activation. Products of PI-3K activate several kinases, including the serine/threonine kinase Akt. The present study examined the ability of Akt to regulate neutrophil respiratory burst activity and to interact with and phosphorylate p47(phox). Inhibition of Akt activity in human neutrophils by an inhibitory peptide significantly attenuated fMLP-stimulated, but not PMA-stimulated, superoxide release. Akt inhibitory peptide also inhibited hydrogen peroxide generation stimulated by bacterial phagocytosis. A direct interaction between p47(phox) and Akt was shown by the ability of GST-p47(phox) to precipitate recombinant Akt and to precipitate Akt from neutrophil lysates. Active recombinant Akt phosphorylated recombinant p47(phox) in vitro, as shown by (32)P incorporation, by a mobility shift change detected by two-dimensional gel electrophoresis, and by immunoblotting with phospho-Akt substrate Ab. Mutation analysis indicated that 2 aa residues, Ser(304) and Ser(328), were phosphorylated by Akt. Inhibition of Akt activity also inhibited fMLP-stimulated neutrophil chemotaxis. We propose that Akt mediates PI-3K-dependent p47(phox) phosphorylation, which contributes to respiratory burst activity in human neutrophils.

Respiratory burst activity in bronchopulmonary dysplasia and changes with dexamethasone

The first objective of this study was to evaluate longitudinal changes in respiratory burst activity in circulating neutrophils and monocytes in infants of less than 30 weeks of gestation with respiratory distress syndrome (RDS), and to examine differences in neonates who subsequently developed bronchopulmonary dysplasia (BPD) compared with those neonates who did not. The second objective was to investigate the effects of dexamethasone on respiratory burst activity in neutrophils and monocytes. We measured burst activity on neutrophils and monocytes in fresh heparinized blood in response to E. coli, N-formyl-met-leu-phe (fMLP), and phorbol 12-myristate 13-acetate stimulation on days 3, 7, 14, and 21 of life, before and 2-3 days after initiating a 6-day course of dexamethasone treatment. Infants with RDS participating in the study were followed until discharge, and were classified as non-BPD and either 1) BPD d28, reflecting their oxygen requirement at day of life 28, or 2) BPD 36 weeks, reflecting oxygen dependence at 36 weeks' corrected gestational age. The diagnosis of BPD was supported by radiological changes of BPD. The percentage of activated neutrophils producing a respiratory burst increased in all premature infants with increasing postnatal days during the first 28 days of life, when the physiological stimulus E. coli was used as an activator (P < 0.02). There was no significant difference in respiratory burst activity measured either as percent activation or as mean fluorescence intensity between non-BPD and BPD infants after adjusting for the difference in weight and gestational age between the two groups. The treatment of premature infants with dexamethasone was associated with decreased activation of neutrophils (P < 0.005) when E. coli was used as a stimulus. In conclusion, a significant increase in neutrophil respiratory burst activity occurs during the first month of life in very low birth weight infants. Greater pulmonary damage in BPD cannot be attributed to reduced burst activity in either neutrophils or monocytes. Dexamethasone treatment was associated with decreased neutrophil respiratory burst activity.

Inhibition of actin polymerization by peroxynitrite modulates neutrophil functional responses

Peroxynitrite, a potent oxidant generated in inflammatory tissues, can nitrate tyrosine residues on a variety of proteins. Based on previous studies suggesting that actin might be a potential target for peroxynitrite-mediated nitration in neutrophils, we investigated the effects of peroxynitrite on actin function. We show here that peroxynitrite and the peroxynitrite generator (SIN-1) modified actin in a concentration-dependent manner, resulting in an inhibition of globular-actin polymerization and filamentous-actin depolymerization in vitro. The effects of peroxynitrite were inhibited by the pyrrolopyrimidine antioxidant PNU-101033E, which has been shown previously to specifically block peroxynitrite-mediated tyrosine nitration. Furthermore, spectrophotometric and immunoblot analysis of peroxynitrite-treated actin demonstrated a concentration-dependent increase in nitrotyrosine, which was also blocked by PNU-101033E. Activation of neutrophils in the presence of a nitric oxide donor (S-nitroso-N-acetylpenicillamine) resulted in nitration of exogenously added actin. Nitrated actin was also found in peroxynitrite-treated neutrophils, suggesting that actin may be an important intracellular target during inflammation. To investigate this issue, we analyzed the effect of peroxynitrite treatment on a number of actin-dependent neutrophil processes. Indeed, neutrophil actin polymerization, migration, phagocytosis, and respiratory burst activity were all inhibited by SIN-1 treatment in a concentration-dependent manner. Therefore, the ability of peroxynitrite to inhibit actin dynamics has a significant effect on actin-dependent, cellular processes in phagocytic cells and may modulate their host defense function.

Peripheral blood leucocyte functional responses to acute eccentric exercise in humans are influenced by systemic stress, but not by exercise-induced muscle damage

The effects of comparable lower-limb eccentric exercise that induces high (bench-stepping; STEP) and low (repeated eccentric muscle action; ECC) systemic stress on neutrophil and monocyte phagocytic and respiratory burst activity, and activation antigen (CD11b, CD66b, CD64) expression, were compared in recreationally active subjects (20–37 years old). Leucocyte responses were determined before and 4, 24, 48 and 72h after exercise using whole-blood flow cytometry. Serum creatine kinase (CK) activity and perceived muscle soreness [delayed-onset muscle soreness (DOMS)] were assessed at the same time points up to 96 h; as a control, measurements were taken during 5 days of rest. DOMS in quadriceps and contralateral triceps surae peaked 24–72h after STEP (P&lt;0.05) and 48–72h after ECC (P&lt;0.05), whereas serum CK activity (mean±S.E.M.) was only higher than baseline after ECC (15123±3488 at 96h compared with 115±29 units·l-1 pre-exercise; P&lt;0.01). The total leucocyte count increased from (5.4±0.4)×109·l-1 and (5.7±0.5)×109·l-1 at baseline to (7.6±0.5)×109·l-1 and (7.0±0.5)×109·l-1 at 4h after STEP and ECC respectively; this was largely attributable to changes in the neutrophil count (P&lt;0.05). The proportion of neutrophils undergoing phagocytosis and respiratory burst was unchanged 4h after ECC and STEP, which, given the increase in neutrophil count after exercise, would suggest an overall improvement in systemic neutrophil microbicidal potential. The intensity of neutrophil (P = 0.01) and monocyte (P&lt;0.05) phagocytosis and neutrophil respiratory burst responses (P&lt;0.05) was only increased 24h after STEP, whereas no changes in these measures were observed after ECC. Activation antigen expression was unchanged in all groups. These findings suggest that systemic stress evoked during an acute bout of eccentric exercise has a greater influence on subsequent leucocyte functional responses than the degree of muscle damage induced.

Xenon preserves neutrophil and monocyte function in human whole blood.

Most volatile anesthetics are known to inhibit the oxidative and phagocytic function of neutrophils. In the present study, we investigated the effect of xenon on phagocytosis and respiratory burst activity of neutrophils and monocytes in human whole blood. Heparinized whole blood from 22 healthy volunteers was incubated for 60 min in the presence of 65% xenon. Sixty-five percent nitrous oxide was used as a positive control to prove the reliability of our in vitro system. Phagocytosis of fluorescein isothiocyanate labelled, opsonized Escherichia coli (E. coli) by neutrophils and monocytes was measured using flow cytometry. After induction with either N-formyl-methionyl-leucyl-phenylalanine (FMLP), phorbol-12-myristate-13-acetate or opsonized E. coli, respiratory burst activity was assessed by measuring the oxidation of dihydrorhodamine 123 to rhodamine 123 with a flow cytometer. Exposure of human whole blood to xenon increased the percentage of neutrophils showing phagocytosis (94 +/- 3% vs 92 +/- 4%; P < 0.01), and the amount of ingested bacteria (P < 0.01). Respiratory burst activity in neutrophils and monocytes was not affected by xenon. Nitrous oxide significantly reduced the percentage of neutrophils showing respiratory burst after FMLP stimulation. Furthermore, E. coli-induced stimulation resulted in a decreased number of reacting neutrophils (84 +/- 15% vs 95 +/- 5%; P < 0.05) and monocytes (70 +/- 22% vs 83 +/- 11%; P < 0.05) as well as a reduced production of hydrogen peroxide in both cell lines compared to control. In contrast to nitrous oxide, xenon preserves neutrophil and monocyte antibacterial capacity in vitro.

The hepatic response to portal endotoxaemia in obstructive jaundice down-regulates neutrophil respiratory burst activity

Both pro- and anti-inflammatory cytokines are elevated in this model of obstructive jaundice. The global effect of the increased inflammatory response is a down-regulation of neutrophil respiratory burst activity. This may result in subsequent immunological dysfunction and anergy, as a result of a prominent anti-inflammatory response, increasing the susceptibility to septic complications. The use of the neutrophil chemiluminescence assay may be a more reliable assessment of the effects of the inflammatory balance in obstructive jaundice rather than measurement of individual cytokines.

Oral vitamin C attenuates acute ischaemia-reperfusion injury in skeletal muscle

Ischaemia-reperfusion injury (IRI) is caused by endothelial and subendothelial damage by neutrophil-derived oxidants. Vitamin C is an antioxidant which attenuates endothelial injury after IRI. Our aim was to evaluate the effect of oral vitamin C in the prevention of IRI in skeletal muscle. We used a model of cross-clamping (3 hours) and reperfusion (1 hour) of the cremaster muscle in rats. Muscle function was assessed electrophysiologically by electrical field stimulation. Infiltration by neutrophils was determined by the activity of tissue myeloperoxidase (MPO) and tissue oedema by the wet-to-dry ratio. Neutrophil respiratory burst activity was measured in control animals and groups pretreated with vitamin C. IRI significantly decreased muscle function and increased muscle neutrophil MPO activity and muscle oedema. Pretreatment with vitamin C preserved muscle function and reduced tissue oedema and neutrophil infiltration. Neutrophil respiratory burst activity was reduced in the group treated with vitamin C compared with the control group. We conclude that pretreatment with oral vitamin C protects against acute muscle IRI, possibly by attenuating neutrophil respiratory burst activity.

Induction of apoptosis and inhibition of migration of inflammatory and vascular wall cells by cerivastatin

Statins are thought to play a role in directly affecting immune and mesenchymal cells. Since cerivastatin's pleiotropic effects are poorly investigated, we were interested to find out whether this drug can modulate leukocyte and vessel wall cell functions. Leukocyte migration was tested in modified Boyden microchemotaxis chambers and oxygen radical production was measured fluorometrically. Transendothelial migration experiments were performed with human umbilical vein endothelial cells and neutrophils. Neutrophil, monocyte, and vascular smooth muscle cell caspase-3 activity and annexin-V binding were quantified by FIENA and FACS, respectively. Cerivastatin [10 pM to 100 μM] decreased leukocyte chemotaxis towards interleukin-8 or RANTES. Migration of cells was completely restored by addition of mevalonic acid. In neutrophils, cerivastatin [100 μM] reduced transendothelial migration, whereas treatment of endothelial cells failed to affect transmigration. Neutrophil respiratory burst activity was unaffected by cerivastatin. At concentrations of 10 nM or higher, cerivastatin increased the rate of apoptosis in phagocytes and smooth muscle cells. Results show that cerivastatin is able to inhibit leukocyte chemotaxis, and that cerivastatin induces neutrophil, monocyte, and smooth muscle cell apoptosis. The drug's impact on transendothelial migration is due to its effects on neutrophils. In addition to its lipid-lowering effects, pharmacological properties of cerivastatin may include modulatory actions in leukocytes and mesenchymal cells.