Plasma and wound fluids from trauma patients suppress neutrophil extracellular respiratory burst
Hyo In KimJin Bong ParkBarbora KonečnáWei HuangIngred RiçaDavid GalloLeo E. OtterbeinKiyoshi ItagakiCarl J. Hauser
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Trauma increases susceptibility to secondary bacterial infections. The events suppressing antimicrobial immunity are unclear. Polymorphonuclear neutrophils (PMNs) migrate toward bacteria using chemotaxis, trap them in extracellular neutrophil extracellular traps, and kill them using respiratory burst (RB). We hypothesized that plasma and wound fluids from trauma patients alter PMN function.Volunteer PMNs were incubated in plasma or wound fluids from trauma patients (days 0 and 1, days 2 and 3), and their functions were compared with PMNs incubated in volunteer plasma. Chemotaxis was assessed in transwells. Luminometry assessed total and intracellular RB responses to receptor-dependent and independent stimulants. Neutrophil extracellular trap formation was assessed using elastase assays. The role of tissue necrosis in creating functionally suppressive systemic PMN environments was assessed using a novel pig model where PMNs were incubated in uninjured pig plasma or plasma from pigs undergoing intraperitoneal instillation of liver slurry.Both plasma and wound fluids from trauma patients markedly suppress total PMN RB. Intracellular RB is unchanged, implicating suppression of extracellular RB. Wound fluids are more suppressive than plasma. Biofluids suppressed RB maximally early after injury and their effects decayed with time. Chemotaxis and neutrophil extracellular trap formation were suppressed by biofluids similarly. Lastly, plasma from pigs undergoing abdominal liver slurry instillation suppressed PMN RB, paralleling suppression by human trauma biofluids.Trauma plasma and wound fluids suppress RB and other key PMNs antimicrobial functions. Circulating suppressive signals can be derived from injured or necrotic tissue at wound sites, suggesting a key mechanism by which tissue injuries can put the host at risk for infection.Keywords:
Neutrophil Extracellular Traps
Respiratory burst
Abstract The aim of the study was to determine the effect of EM Bokashi® on the phagocytic activity of monocytes and granulocytes, oxidative burst, SWC3, and CD11b + CD18+ expression on monocytes and granulocytes, and the serum concentration of cytokine and lysozyme in pig. 60 Sixty female piglets were divided into two groups: I – control and II – experimental. For the experimental group, a probiotic in the form of the preparation EM Bokashi® was added to the basal feed. Flow cytometry was used to determine selected non-specific immune response parameters, intracellular production of hydrogen peroxide by peripheral granulocytes and monocytes, and surface particles in peripheral blood. The EM Bokashi® preparation used in the study was found to increase phagocytic activity mainly in monocytes, with an increased percentage of phagocytic cells in the experimental group. The highest serum lysozyme concentration in the piglets in the experimental group (2.89 mg/dl), was noted on day 42 of the study. In the group of pigs receiving EM Bokashi®, the percentage of phagocytic cells with SWC3 (monocyte/granulocyte) expression was statistically significantly higher than in the control. The increase in the number of cells with SWC3 (monocyte/granulocyte) expression in the peripheral circulation in combination with the greater capacity of the cells for phagocytosis and respiratory burst confirms that the non-specific immune response was modulated in the pigs supplemented with EM Bokashi®.
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Human neutrophils in whole blood and after isolation were examined for their capacity to produce superoxide anions when stimulated by zymosan. The isolation procedure caused neutrophils to lose some of this capacity. The neutrophils of smokers produced less particle-stimulated superoxide than those of non-smokers when the whole blood method was used but not when using isolated cells. Zinc inhibited this aspect of the respiratory burst of isolated neutrophils but enhanced it when the whole blood method was used. We emphasize the importance of methodology when interpreting results.
Respiratory burst
Zymosan
Human blood
Isolation
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Granulocytes play a key role in the body's innate immune response to bacterial and viral infections. While methods exist to measure granulocyte function, in general these are limited in terms of the information they can provide. For example, most existing assays merely provide a percentage of how many granulocytes are activated following a single, fixed length incubation. Complicating matters, most assays focus on only one aspect of function due to limitations in detection technology. This report demonstrates a technique for simultaneous measurement of granulocyte phagocytosis of bacteria and oxidative burst. By measuring both of these functions at the same time, three unique phenotypes of activated granulocytes were identified: 1) Low Activation (minimal phagocytosis, no oxidative burst), 2) Moderate Activation (moderate phagocytosis, some oxidative burst, but no co-localization of the two functional events), and 3) High Activation (high phagocytosis, high oxidative burst, co-localization of phagocytosis and oxidative burst). A fourth population that consisted of inactivated granulocytes was also identified. Using assay incubations of 10, 20, and 40-min the effect of assay incubation duration on the redistribution of activated granulocyte phenotypes was assessed. A fourth incubation was completed on ice as a control. By using serial time incubations, the assay may be able to able to detect how a treatment spatially affects granulocyte function. All samples were measured using an image-based flow cytometer equipped with a quantitative imaging (QI) option, autosampler, and multiple lasers (488, 642, and 785 nm).
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Quercetin, a flavonoid found in fruits and vegetables, is a strong antioxidant with anti-inflammatory, antimicrobial and immune-modulating properties. The purpose of the present study was to investigate the effects of long-term quercetin supplementation on innate immune function and inflammation in human subjects. Female subjects ( n 120; aged 30–79 years) were recruited from the community and randomised to one of three groups, with supplements administered using double-blinded procedures: 500 mg quercetin/d ( n 38), 1000 mg quercetin/d ( n 40) or placebo ( n 42). Subjects ingested two soft chew supplements twice daily during the 12-week study period. Fasting blood samples were obtained pre- and post-study and were analysed for plasma quercetin, IL-6, TNF-α and leucocyte subset cell counts. Natural killer cell activity (NKCA) and lymphocyte subsets were assessed in a subset of seventy-four subjects. Granulocyte oxidative burst activity (GOBA) and phagocytosis were assessed in sixty-four subjects. Eighteen subjects had overlapping data. Quercetin supplementation at two doses compared with placebo increased plasma quercetin (interaction effect; P < 0·001) but had no significant influence on blood leucocyte subsets, plasma IL-6 or TNF-α concentration, NKCA, GOBA or phagocytosis. NKCA was inversely correlated with BMI ( r − 0·25; P = 0·035) and body fat percentage ( r − 0·38; P = 0·001), and positively correlated with self-reported physical fitness level ( r 0·24; P = 0·032). In summary, results from the present double-blinded, placebo-controlled, randomised trial indicated that quercetin supplementation at 500 and 1000 mg/d for 12 weeks significantly increased plasma quercetin levels but had no influence on measures of innate immune function or inflammation in community-dwelling adult females.
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Objective To observe the varieties of peripheral leukocyte count and the percent of neutrophile gyanulocyte with acute cerebral infarction patients. Methods One hundred and fifty one patients with acute cerebral infarction were divided by the area of infarction into three groups: large, middle, small area group divided by the neural default degree into three groups :low grade, moderate grade, heavy grade, record their leukocyte count and the percent of neutrophile granulocyte in blood circulation. Results The leukocyte count and the percent of neutrophile granulocyte with the group of large area cerebral infarction increased obviously(P0.01) . The leukocyte count and the percent of neutrophile granulocyte with the heavy group increase obviously(P0.01). Conclusion We can estimate morbid state by the leukocyte count and the percent of neutrophile granulocyte with acute cerebral infarction patients.
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A method has been described for the labeling, separation, and estimation of radioactivity in samples of DFP32-tagged neutrophilic granulocytes. Studies were performed to identify the potential errors of this procedure, and it was then applied to a group of 12 normal men. A mean of 43% of transfused, labeled neutrophilic granulocytes was found in the circulation, resulting in the calculation of a mean maximum of 0.68 x 109/kg neutrophilic granulocytes in the total peripheral vascular pool. The mean half-time of disappearance of these cells was 3.8 hr, and a mean maximum neutrophile turnover rate through peripheral blood of 3.2 x 109/kg per day was calculated. The failure of labeled cells to disappear from the blood at an exponential rate suggested that the instantaneously equilibrating blood neutrophile pools may be in slower equilibrium with a larger, extravascular neutrophile compartment. Note: (With the Technical Assistance of Yvonne Betson and Ruth Henderson) DFP 32 ; liquid scintillation counting of granulocytes; circulating and noncirculating blood neutrophile pools; extravascular neutrophile pool; neutrophilic granulocyte turnover Submitted on August 31, 1964
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Granulocytes play a key role in the body's innate immune response to bacterial and viral infections. While methods exist to measure granulocyte function, in general these are limited in terms of the information they can provide. For example, most existing assays merely provide a percentage of how many granulocytes are activated following a single, fixed length incubation. Complicating matters, most assays focus on only one aspect of function due to limitations in detection technology. This report demonstrates a technique for simultaneous measurement of granulocyte phagocytosis of bacteria and oxidative burst. By measuring both of these functions at the same time, three unique phenotypes of activated granulocytes were identified: 1) Low Activation (minimal phagocytosis, no oxidative burst), 2) Moderate Activation (moderate phagocytosis, some oxidative burst, but no co-localization of the two functional events), and 3) High Activation (high phagocytosis, high oxidative burst, co-localization of phagocytosis and oxidative burst). A fourth population that consisted of inactivated granulocytes was also identified. Using assay incubations of 10, 20, and 40-min the effect of assay incubation duration on the redistribution of activated granulocyte phenotypes was assessed. A fourth incubation was completed on ice as a control. By using serial time incubations, the assay may be able to able to detect how a treatment spatially affects granulocyte function. All samples were measured using an image-based flow cytometer equipped with a quantitative imaging (QI) option, autosampler, and multiple lasers (488, 642, and 785 nm).
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Neutrophil Extracellular Traps
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Background Activated human polymorphonuclear leukocytes (PMNs) participate in the innate immune response precipitated by tissue injury or bacterial invasion. As a recently described response to bacterial invasion, PMNs secrete lattices of DNA and antimicrobial proteins termed neutrophil extracellular traps (NETs) to effect extracellular killing of bacteria. However, PMNs isolated from infants born prematurely exhibit in vitro and in vivo functional deficits in bacterial killing. Whether these deficits result from impaired NET formation in preterm PMNs remains unknown. Hypothesis We hypothesize that preterm PMNs fail to robustly form NETs following activation in vitro. Methods We therefore stimulated human PMNs isolated from adults, term infants, and preterm infants with platelet-activating factor (PAF) (10−8M), lipopolysaccharide (LPS) (100 ng/mL), or live bacteria (Escherichia coli, 0.1-10 MOI) for 30 to 120 minutes. We visualized NET formation of live and fixed PMNs stained for extracellular DNA and myeloperoxidase expression via confocal microscopy, with and without time-lapse photography. We also obtained high-resolution images of NET formation in all three cell types via scanning electron microscopy of fixed PMNs following stimulation with PAF, LPS, or E. coli. Results We visualized limited or absent NET formation via confocal microscopy at 30 and 120 minutes in LPS- and E. coli-stimulated preterm PMNs compared with robust NET formation at 30 and 120 minutes in term and adult PMNs following the same stimuli. PAF failed to induce NET formation in preterm PMNs but did induce NET formation in term and adult PMNs at 30 minutes. Time-lapse confocal microscopy allowed visualization of NET formation in term and adult PMNs but failed to show NET formation in preterm PMN preparations. Furthermore, scanning electron microscopy confirmed robust NET formation at 30 minutes in term and adult PMNs following stimulation with PAF, LPS, and E. coli but noted minimal NET formation under those conditions in preterm PMNs. Conclusion We conclude that in vitro NET formation is impaired in preterm PMNs when compared with term and adult PMNs. Speculation We speculate that impaired NET formation may contribute to bacterial killing deficits documented in preterm infants, thus contributing to the morbidity and mortality in preterm infants associated with neonatal sepsis.
Neutrophil Extracellular Traps
Platelet-activating factor
Trap (plumbing)
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