Measure Of Neutrophil Activation Research Articles

Systemic Inflammation Induces Release of Cell-Free DNA from Hematopoietic and Parenchymal Cells in Mice and Human

Cell-free DNA (cfDNA) and DNA binding proteins are released in the circulation upon systemic inflammation, the extent of which is proportional to the severity of the systemic inflammation. There is an ongoing debate on the cellular source of cfDNA during systemic inflammatory conditions. During infection, circulating histones and DNA in the form of nucleosomes have been argued to be released by activated neutrophils in the process of NETosis. However, cfDNA has also been attributed to the cell death of other cell types, such as lymphocytes and endothelial cells. The objective of the current study is to investigate the compartmental source of extracellular DNA released into the circulation during a systemic inflammatory response.We determined the plasma levels of nucleosomes as a measure of cfDNA and DNA binding proteins, and of elastase-α1-antitrypsin (EA) complexes as a measure of neutrophil activation in different systemic inflammatory responses. First, we induced a low-grade endotoxemia in eight healthy human subjects by injection of lipopolysaccharide (LPS). In these subjects, nucleosome levels increased to a maximum at 3 hours after LPS injection (mean 529 ± SE 112 AU/ml), after which they returned towards baseline levels. Interestingly, the release of EA showed similar kinetics as that of nucleosomes, reaching a maximal level of 499 ± 58 ng/ml at 3 hours. Furthermore, plasma EA levels positively correlated with nucleosome levels during LPS challenge (r=0,79; p<0.0001), suggesting that during a mild, transient inflammatory response, neutrophils are the predominant source of nucleosomes within the plasma. We next measured nucleosomes and neutrophil activation in twenty patients with severe systemic inflammation caused by sepsis. Surprisingly, sequential measurements in sepsis patients did not show a correlation between plasma EA levels and nucleosome levels (r=0,155; p=0,13), suggestive of a non-neutrophil source of cfDNA. Thus, there might be other potential cellular sources of cfDNA than neutrophils in a severe systemic inflammation.To further investigate the difference of cfDNA release between a mild and a severe systemic inflammation, we used chimeric mice transplanted with bone marrow of the opposite sex. After hematopoietic recovery, mice were challenged with LPS. We were able to follow up on the release of cfDNA from different compartmental sources, that is either the hematopoietic or the non-hematopoietic compartment, by using a specific mouse SRY qPCR as a measure for male DNA, and IL3 qPCR as a measure for total DNA. CfDNA from the hematopoietic compartment was present within the plasma of the mice at 6 and 20 hours after LPS challenge, whereas cfDNA from a parenchymal source could be detected at 20 hours but less at 6 hours after LPS challenge. This suggests that the hematopoietic compartment acts as a source of cfDNA both early and late after LPS challenge, whereas the parenchymal compartment only releases DNA at later stages of severe systemic inflammation.Collectively, our results show that in severe systemic inflammation, circulating cfDNA originates from both hematopoietic cells (e.g. neutrophils and lymphocytes) and parenchymal cells (e.g. endothelial cells). Understanding the kinetics of extracellular DNA may improve knowledge on duration and severity of sepsis and may provide a point of interference during systemic inflammation. DisclosuresNo relevant conflicts of interest to declare.

Anti-inflammatory effects of selected drugs on activated neonatal and adult neutrophils

Aim. In view of the central role of granulocytic neutrophils in the context of inflammatory reactions, the present study focuses on anti-inflammatory effects of drugs on activated neutrophils in neonates and adults. Methods. Sixteen blood samples of neonates and adults were investigated in a prospective study. Loss of deformability, morphological changes, and increases in neutrophil elastase were determined as measures of neutrophil activation due to incubation with the pro-inflammatory cytokine interleukin-8. For inhibition experiments, the blood samples were also incubated with the phosphodiesterase inhibitors milrinone and piclamilast, the protease inhibitor urinastatin, ketamine, protein C concentrate, and the nitric oxide donor FK 409. Changes in deformability were investigated with a cell transit analyzer, morphological changes by microscopic observation, and the extent of neutrophil elastase release with an enzyme immunoassay. Results. The drugs milrinone, piclamilast, urinastatin, ketamine, protein C concentrate and FK 409 showed deactivating effects on activated neutrophils in recommended clinical doses. They improved deformability as well as reduced pseudopod formation and the release of neutrophil elastase. The effects on neutrophils did not differ between neonates and adults despite their functional differences. Conclusion. We conclude that these drugs may reduce the inflammatory response and improve microcirculation in neonates and adults during inflammation.

Increased Circulating Nucleosomes and Neutrophil Activation As a Measure of the Formation of Neutrophil Extracellular Traps (NETs) During Sickle Cell Painful Crisis

AbstractAbstract 821 Introduction:Sickle cell disease (SCD) is characterized by recurrent acute vaso-occlusive painful crisis frequently leading to SCD related complications such as acute chest syndrome, stroke, multi-organ failure and even sudden death. The complex pathophysiology of the vaso-occlusive painful crisis is mediated by activation of endothelial cells, adhesion of sickled erythrocytes and neutrophils, oxidative stress, coagulation activation and increased release of inflammatory mediators, resulting in ischemic organ damage. Recently, neutrophils have been demonstrated to form neutrophil extracellular traps (NETs) upon activation. Nucleosomes and histones exposed together with neutrophil proteases, such as elastase on these NETs have been shown to kill efficiently bacteria. NET formation has been shown to propagate coagulation in sepsis and in deep venous thrombosis. In addition, nucleosomes and histones exposed on NETs have been shown to be strongly cytotoxic to endothelial cells. Beside the exposure on NETs, nucleosomes can be actively released into the circulation from dead cells. Circulating nucleosomes detected in sepsis have been reported to correlate with severity of inflammation, organ dysfunction and mortality. However, no studies are available yet on the dynamics of nucleosomes and NETs in sickle cell patients suffering from painful crisis. The aim of this case-control study was to assess plasma levels of circulating nucleosomes and human neutrophil elastase–α1-antitrypsin (EA) complexes as measure of systemic neutrophil activation, in sickle cell patients during steady state and painful crisis. Methods:Plasma levels of nucleosomes and EA as a measure of neutrophil activation were measured in 74 patients in asymptomatic state (49 HbSS/HbSβ0-thalassemia, and 25 HbSC/HbSβ+-thalassemia), 70 painful crises (53 HbSS/HbSβ°-thalassemia and 17 HbSC/HbSβ+-thalassemia) in 49 patients and in 24 HbAA healthy controls using Enzyme-Linked Immunosorbent Assay (ELISA). Results:Plasma levels of nucleosomes in both HbSS/HbSβ°-thalassemia and HbSC/HbSβ+-thalassemia patients were significantly higher during painful crisis (median; interquartile range, 20.2; 8.9 – 129.0 U/ml, P < 0.0001 and 11.7; 5.1 – 67.7 U/ml, P = 0.045 respectively) as compared to patients in steady state (6.0; 3.0 – 9.8 U/ml and 7.1; 4.6 – 9.6 U/ml respectively). Nucleosomes levels in healthy controls were just above the detection limit of the assay (5.0; 5.0 – 6.5) U/ml).Plasma levels of EA in HbSS/HbSβ°-thalassemia patients were significantly increased during painful crisis as compared to steady state (75.1; 56.5 – 102.4 vs. 45.7; 34.7 – 59.7 ng/ml, P < 0.0001). Also in HbSC/HbSβ+-thalassemia patients, EA levels were higher during painful crisis than in steady state, though the difference did not reach statistical significance (62.0; 48.0 – 96.7 vs. 50.2; 33.3 – 67.7, P = 0.051). Plasma levels of EA in healthy controls (39.9; 31.5 – 62.2 ng/ml) were comparable with those in steady state patients. In a paired analysis of 36 patients, included both during steady state and painful crisis, significant increments were observed during painful crisis in levels of both nucleosomes (from 5.0; 3.0 – 10.8 to 20.2; 6.8 – 94.3 U/ml, P < 0.0001) and EA (from 47.9; 36.0 – 67.6 to 70.6; 55.9 – 101.4 ng/ml, P < 0.0001), as compared to steady state. During painful crisis, EA levels were strongly correlated with levels of nucleosomes in both HbSS/HbSβ°-thalassemia (Spearman’s rank (Sr)=0.55, P<0.0001) and HbSC/HbSβ+-thalassemia patients (Sr=0.90, P=<0.0001). In steady state the correlation was significant only in HbSC/HbSβ+-thalassemia patients (Sr=0.63, P=0.001) Four patients who developed an acute chest syndrome during painful crisis were among the patients with the highest nucleosome (359, 130, 128 and 100 U/ml) and EA levels (121, 87, 92 and 64 ng/ml respectively). Conclusion:Sickle cell painful crisis is associated with increased levels of nucleosome and stronger neutrophil activation. This might point to a crucial role of NET formation in the pathogenesis of painful crisis. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

The Search for Diagnostic Markers in Sepsis

The Search for Diagnostic Markers in Sepsis

SB‐656933, a novel CXCR2 selective antagonist, inhibits ex vivo neutrophil activation and ozone‐induced airway inflammation in humans

Receptor antagonists that block the binding of chemokines such as CXCL8 (IL-8) are effective in animals models of neutrophil-mediated inflammation. It has been hypothesized that selective inhibition of neutrophil trafficking and activation may be a useful adjunct for the treatment of inflammatory airway diseases such as chronic obstructive pulmonary disease or cystic fibrosis. A CXCR1/2 receptor antagonist has shown activity in an ozone challenge model in humans. SB-656933, a selective CXCR2 antagonist, is safe and well-tolerated at single doses and is shown to inhibit agonist (CXCL1)-mediated expression of the CD11b on peripheral blood neutrophils as well as ozone-induced airway neutrophilia in healthy subjects. To determine the safety and tolerability of a novel selective CXCR2 antagonist and assess its pharmacodynamic effects using measures of neutrophil activation and function, including CD11b expression in whole blood and ozone-induced airway inflammation in healthy subjects. Flow cytometric determination of ex vivo CXCL1-induced CD11b expression on peripheral blood neutrophils was performed following single dose oral administration of SB-656933 (dose range 2-1100 mg). A subsequent randomized study (placebo, 50 mg and 150 mg) was performed to explore the dose-response for ozone-induced airway inflammation, as measured by sputum biomarkers. Oral administration of SB-656933 resulted in significant inhibition of CXCL1-induced CD11b expression on peripheral blood neutrophils at single doses greater than or equal to 50 mg. Maximum inhibition (70%) relative to placebo was observed following administration of SB-656933 400 mg (95% CI 60%, 77%). This was sustained up to a dose of 1100 mg. Single doses of SB-656933 reduced ozone-induced airway inflammation in a dose-dependent manner. Relative to placebo, there were 55% (95% CI 20%, 75%) and 74% (95% CI 55%, 85%) fewer neutrophils in the sputum of subjects after a single dose of 50 mg or 150 mg, respectively. There was a corresponding reduction in myeloperoxidase concentrations in the sputum supernatant of 32.8% (95% CI 9.2, 50.3) and 50.5% (95% CI 33.3, 63.3). SB-656933 was safe and well-tolerated at all doses. SB-656933 is a CXCR2 antagonist that demonstrates dose-dependent effects on neutrophil activation and recruitment within a well-tolerated dose range. These data suggest that SB-656933 may be an effective agent in neutrophil-predominant diseases.

P27 Neutrophil dysfunction: the missing link between ammonia, infection and hepatic encephalopathy?

IntroductionNeutrophil phagocytic dysfunction is associated with increased risk of infection and mortality in patients with cirrhosis. The p38-mitogen-activated protein kinase (MAPK) signalling pathway is a critical step in neutrophil activation...

Effects of methylene blue in reducing cholestatic oxidative stress and hepatic damage after bile-duct ligation in rats

The aim of this study was to evaluate the effects of methylene blue against cholestatic oxidative stress and liver damage after ligation of the common bile duct in male Wistar rats. Eight animals were included in each of the following five groups: untreated control, methylene blue control, sham-operated, bile-duct ligation, and bile-duct ligation plus methylene blue. Methylene blue was administered intraperitoneally for 14 days at a daily dose of 2mg/kg per day. All rats were sacrificed 2 weeks following the experimental treatment and the livers of all groups were examined biochemically and histopathologically. The severity of cholestasis and hepatic injury were determined by changes in the plasma, including enzymatic activities: aspartate aminotransferase, alanine aminotransferase, gamma glutamine transferase, and also bilirubin levels. Malondialdehyde, nitric oxide and superoxide dismutase were measured to indicate the oxidative status in the liver tissue. Myeloperoxidase activity and levels of tissue hydroxyproline were determined as measures of neutrophil activation and collagen accumulation, respectively. Liver damage was significantly prevented in the bile-duct ligated rats treated with methylene blue compared with the control bile-duct ligated rats without methylene blue. Treatment with methylene blue markedly reduced activities of serum transaminase, gamma glutamine transferase and bilirubin levels as compared to bile-duct ligated rats without methylene blue. Positive immunolabelling for alpha-smooth muscle actin (alpha-SMA) was increased, especially in vascular smooth muscle cells, fibrotic septa and also around the proliferated bile ducts, after bile-duct ligation. Only weak alpha-SMA immunolabelling was seen in livers of rats treated with methylene blue. These results indicate that methylene blue can attenuate hepatic damage in extrahepatic cholestasis by reducing oxidative stress and inflammatory processes.

Activation of Neutrophils in Cutaneous T-Cell Lymphoma

Cutaneous T-cell lymphoma (CTCL) is a spectrum of disease of unknown etiology defined by infiltrates of activated and malignant T cells in the skin. In working with blood from CTCL patients, we noticed frequent activation of neutrophils; therefore, we tested the hypothesis that neutrophils are activated in CTCL subjects compared with normal healthy controls. Using peripheral blood of 44 subjects with CTCL and 15 normal controls, we examined three measures of neutrophil activation. These are the presence of neutrophils of reduced buoyant density, the presence of primed neutrophils in a stimulated chemiluminescence assay, and changes in surface markers by flow cytometry. In addition, we tested plasma interleukin-8 (IL-8) and leukotriene B4 (LTB4) levels using ELISA. A significantly larger fraction of hypodense neutrophils was observed in CTCL subjects compared with normals (10.6 +/- 1.7% versus 1.5 +/- 0.4%). Stimulated chemiluminescence was also significantly increased in CTCL, and analysis of neutrophil surface markers using flow cytometry showed significantly increased CD11b and CD66b and decreased CD62L, consistent with neutrophil activation. These changes were present even in early stages of CTCL. We further found that plasma IL-8 and LTB4 levels are elevated in CTCL, which could form a feedback loop contributing to disease pathophysiology. CTCL is associated with systemic neutrophil activation, even in early disease, and a feedback loop between neutrophils and T cells mediated by IL-8 and LTB4 is a potential contribution to the pathophysiology of CTCL.

Short communication: Reliability testing and applied use of a measure of neutrophil activation in stress research

AbstractThe purpose of this research was to test the reliability of a measure of neutrophil activation and to apply the measure in stress research. Two studies were carried out with university undergraduates. The first was a test‐retest reliability study of the nitro‐blue tetrazolium (NBT) test, a measure of neutrophil activity. The second was an applied study of its use in stress research. In the first study, finger‐stick blood samples were taken at three points in time (baseline, +1 hour, and at 1 month) from 30 participants. Physiological arousal was monitored (heart rate and blood pressure) before and after samples were taken. Neutrophil activity is determined by their capacity to reduce NBT and is presented as a percentage of NBT positive cells. Highly significant correlations were found between all time points (p = 0.0001) and there were no significant differences between the means. The NBT test was found to be reliable and was used in an examination of the association between neutrophil activity and perceived stress (PSS). Health related issues were also explored. A highly significant positive association was found between NBT and PSS (r = 0.840, p = 0.01). No associations are found between PSS and health. The results confirm and extend findings that stressors, even naturalistic stressors, can activate neutrophils. Copyright © 2004 John Wiley &amp; Sons, Ltd.

Uptake of 18fluorodeoxyglucose in the cystic fibrosis lung: a measure of lung inflammation?

Positron emission tomography is a three-dimensional imaging technique that measures physiological effects, including metabolism. 18Fluorodeoxyglucose has been extensively used as a tracer of cellular energy metabolism in the brain and in tumour detection. As neutrophils utilise glucose as an energy source during their respiratory burst, it was hypothesised that 18fluorodeoxyglucose uptake, by these cells, could be interpreted as a measure of neutrophil activation in cystic fibrosis (CF). Ten adult CF patients were given a bolus intravenous injection of 18fluorodeoxyglucose, followed by a 90-min dynamic mid-lung acquisition scan. Right-lung 18fluorodeoxyglucose uptake was assessed using a Patlak plot and values were converted to glucose utilisation. Three clinically inactive pulmonary sarcoidosis patients served as controls. From the 10 CF patients with baseline sputum neutrophils of 14 x 10(6) cells x mL(-1) who were investigated, seven were found to have sputum at a normal or slightly depressed glucose utilisation rate (mean 1.33 micromol x g(-1) x h(-1)) compared with a mean of 2.82 micromol x g(-1) x h(-1) for the sarcoidosis patients. In eight patients, receiving inhaled tobramycin therapy, no change in lung glucose utilisation or sputum neutrophil counts were found. Despite high-sputum neutrophil levels, lung glucose utilisation was not elevated in patients with cystic fibrosis.

Putting your heart into walking further

Abstract Background Leg exercise training improves maximum walking distance (MWD) in claudicants, but it also causes neutrophil activation and may, as a consequence, elicit damage to the coronary circulation. It was hypothesized that symptomatic improvements induced by exercise programmes result from systemic cardiovascular effects rather than localized changes in the lower limb. The aim of this study was to develop an upper body training programme that would enhance cardiovascular fitness while avoiding potentially damaging inflammatory responses. Methods Sixty-three stable claudicants were recruited; all underwent arm and leg ergometry exercise assessments. Peripheral blood was obtained before and 1 h after exercise. The intensity (median channel of fluorescence; MCF) of neutrophil CD11b expression (as a measure of neutrophil activation) was determined using whole blood flow cytometry. Patients were randomized to an arm (n = 24) or leg (n = 24) training programme structured to achieve identical heart rate changes. Training involved two supervised sessions per week for 6 weeks. Controls undertook no exercise (n = 15). The MWD was determined at the beginning and end of training. Results Arm exercise had no effect on neutrophil expression of CD11b, whereas expression was significantly increased by leg exercise (Table). Arm and leg training increased the maximum work rate from 75 to 100 W (P = 0·004; Wilcoxon test) and there was a concomitant decrease in the submaximal heart rate (arm group 150 to 131 beats min−1; leg group 155 to 136 beats min−1; P = 0·002). These cardiovascular changes were accompanied by an increased MWD (Table). There were no changes in the control group. Conclusion Upper body training can increase walking distance in claudicants without neutrophil activation and maximizing cardiovascular fitness appears to be the key to improving symptoms in these patients.

Superoxide generation by the human polymorphonuclear leukocyte in response to latex beads.

In this study, superoxide formation was not immediately detected when polymorphonuclear leukocytes were treated with linoleyl alcohol-coated, carboxy-modified latex beads. However, all other measures of neutrophil activation were present. Superoxide was not detected until 30 min after the initial exposure to beads. However, an O2(-)-producing, NADPH-dependent oxidase is active 15 min after exposure. When polymorphonuclear leukocytes are pretreated with cytochalasin B, superoxide production is detected immediately after exposure to the beads. Superoxide secretion after treatment with linoleyl alcohol-coated latex beads is compared with the response to other latex beads. The results imply that neutrophils form a phagolysosome around linoleyl alcohol-coated latex beads that is tightly sealed and does not allow superoxide to escape into the medium where it could be detected by the reduction of ferricytochrome c.