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1
Kuijpers, TW, AT Tool, CE van der Schoot, LA Ginsel, JJ Onderwater, D. Roos, and AJ Verhoeven. "Membrane surface antigen expression on neutrophils: a reappraisal of the use of surface markers for neutrophil activation." Blood 78, no. 4 (August 15, 1991): 1105–11. http://dx.doi.org/10.1182/blood.v78.4.1105.1105.
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Abstract Neutrophil research relies largely on studies with highly purified cells. Yet the isolation procedures induce changes in surface expression of several proteins. We used a large panel of monoclonal antibodies (MoAbs) to characterize in detail the phenotypic changes during isolation and stimulation of human neutrophils. Centrifugation on density gradients appears to be the crucial step that causes an increase in expression of antigens not detectable on neutrophils in whole blood samples (cytochrome b558 recognized by MoAb 7D5; and CD10) or expressed at significantly lower levels (CD11a, CD11b, CD11c, CD13, CD16, CD45, and CD67). Other antigens were unaffected by the density gradient centrifugation step (CD32, CD54, CD58, Leu-8, HLA class I). Upregulation of antigens was also determined by stimulation of purified neutrophils. Upregulation of CD63 was an excellent marker for release from azurophil granules. We subsequently related the surface antigen expression to functional activities of purified neutrophils. From these experiments, we concluded that 7D5-as “early activation” marker--does not necessarily discriminate between primed or resting neutrophils with respect to NADPH oxidase activity.
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Kuijpers, TW, AT Tool, CE van der Schoot, LA Ginsel, JJ Onderwater, D. Roos, and AJ Verhoeven. "Membrane surface antigen expression on neutrophils: a reappraisal of the use of surface markers for neutrophil activation." Blood 78, no. 4 (August 15, 1991): 1105–11. http://dx.doi.org/10.1182/blood.v78.4.1105.bloodjournal7841105.
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Neutrophil research relies largely on studies with highly purified cells. Yet the isolation procedures induce changes in surface expression of several proteins. We used a large panel of monoclonal antibodies (MoAbs) to characterize in detail the phenotypic changes during isolation and stimulation of human neutrophils. Centrifugation on density gradients appears to be the crucial step that causes an increase in expression of antigens not detectable on neutrophils in whole blood samples (cytochrome b558 recognized by MoAb 7D5; and CD10) or expressed at significantly lower levels (CD11a, CD11b, CD11c, CD13, CD16, CD45, and CD67). Other antigens were unaffected by the density gradient centrifugation step (CD32, CD54, CD58, Leu-8, HLA class I). Upregulation of antigens was also determined by stimulation of purified neutrophils. Upregulation of CD63 was an excellent marker for release from azurophil granules. We subsequently related the surface antigen expression to functional activities of purified neutrophils. From these experiments, we concluded that 7D5-as “early activation” marker--does not necessarily discriminate between primed or resting neutrophils with respect to NADPH oxidase activity.
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Glasser, L., and RL Fiederlein. "Functional differentiation of normal human neutrophils." Blood 69, no. 3 (March 1, 1987): 937–44. http://dx.doi.org/10.1182/blood.v69.3.937.bloodjournal693937.
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In the past differentiation of human neutrophils has been defined by morphology, cytochemistry, or surface markers. In our experiments we have sequenced the various events that occur during the functional differentiation of the normal human neutrophil and have also examined some of the functional properties in relationship to surface markers and biochemical events. Granulocytes were obtained from the bone marrow and blood of hematologically normal individuals. Cells were separated into different stages of maturation by their physical properties using counterflow centrifugal elutriation and density gradient separation. Three cell fractions were obtained that were enriched for either immature myeloid cells, band neutrophils, or segmented neutrophils. Since the enriched fractions were not entirely pure, methodologies for functional assays were chosen that allowed cytologic evaluation of the functional capacity of each cell type. The criteria used to classify the stages of differentiation included both morphology by light microscopy and DNA labeling with tritiated thymidine. Various neutrophilic properties were studied: Fc receptors, complement receptors (CR1, CR3), phagocytosis of both live and dead opsonized Staphylococcus aureus, microbial killing of S aureus, NBT dye reduction after cellular stimulation with endotoxin, and chemotaxis. Our results indicate that the functional properties of the neutrophil appear in a distinct order. The sequence for the functional differentiation of the human neutrophil appears to be the following: Fc receptors----immune phagocytosis----complement receptors----oxygen-independent microbial killing----oxygen-dependent microbial killing----chemotaxis.
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Luthfi, Muhammad, and Tuti Kusumaningsih. "Salivary neutrophils isolation of severe early childhood caries patients with flow cytometry analysis using magnetic beads and CD177 marker." Dental Journal (Majalah Kedokteran Gigi) 49, no. 1 (December 5, 2016): 32. http://dx.doi.org/10.20473/j.djmkg.v49.i1.p32-36.
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Background: Neutrophils are the first line of defense, not only serving as he killer of microbes through phagocytosis process, in which reactive oxygen species (ROS) and anti-microbial peptides were released, but also regulating activation of immune response. CD177 is a tidylinositol glycosylphosphate glycoprotein with a molecular weight of 58- 64-kDa exclusively found on neutrophils, neutrophilic metamyelocytes, and mielosit. CD177 expression, a protein on the cell surface with an average size ranging from 45% to 65%, is only found on subpopulations of neutrophils. Purpose: This study aims to analyze the effects of salivary neutrophil isolation using magnetic beads and CD177 marker on S-ECC patients. Method: The study is an observational analytic research with cross sectional approach using flow cytometry analysis on the S-ECC patients and the caries-free children who were asked to use mouthwash, NaCl 1.5%. For the isolation of neutrophils, magnetic beads labeled with FITC funds and CD177+ marker were used. Result: There were 77.66% of salivary neutrophils expressing CD177+ markers, successfully isolated in the S-ECC patients, while in the caries-free children there were 63.67% of salivary neutrophils. Conclusion: In the S-ECC patients, there were 77.66% of salivary neutrophils expressing CD177markers, successfully isolated, while in the caries-free children there were 63.67% of salivary neutrophils.
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Glasser, L., and RL Fiederlein. "Functional differentiation of normal human neutrophils." Blood 69, no. 3 (March 1, 1987): 937–44. http://dx.doi.org/10.1182/blood.v69.3.937.937.
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Abstract In the past differentiation of human neutrophils has been defined by morphology, cytochemistry, or surface markers. In our experiments we have sequenced the various events that occur during the functional differentiation of the normal human neutrophil and have also examined some of the functional properties in relationship to surface markers and biochemical events. Granulocytes were obtained from the bone marrow and blood of hematologically normal individuals. Cells were separated into different stages of maturation by their physical properties using counterflow centrifugal elutriation and density gradient separation. Three cell fractions were obtained that were enriched for either immature myeloid cells, band neutrophils, or segmented neutrophils. Since the enriched fractions were not entirely pure, methodologies for functional assays were chosen that allowed cytologic evaluation of the functional capacity of each cell type. The criteria used to classify the stages of differentiation included both morphology by light microscopy and DNA labeling with tritiated thymidine. Various neutrophilic properties were studied: Fc receptors, complement receptors (CR1, CR3), phagocytosis of both live and dead opsonized Staphylococcus aureus, microbial killing of S aureus, NBT dye reduction after cellular stimulation with endotoxin, and chemotaxis. Our results indicate that the functional properties of the neutrophil appear in a distinct order. The sequence for the functional differentiation of the human neutrophil appears to be the following: Fc receptors----immune phagocytosis----complement receptors----oxygen-independent microbial killing----oxygen-dependent microbial killing----chemotaxis.
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Giese, Morgan A., Laurel E. Hind, and Anna Huttenlocher. "Neutrophil plasticity in the tumor microenvironment." Blood 133, no. 20 (May 16, 2019): 2159–67. http://dx.doi.org/10.1182/blood-2018-11-844548.
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Abstract Neutrophils act as the body’s first line of defense against infection and respond to diverse inflammatory cues, including cancer. Neutrophils display plasticity, with the ability to adapt their function in different inflammatory contexts. In the tumor microenvironment, neutrophils have varied functions and have been classified using different terms, including N1/N2 neutrophils, tumor-associated neutrophils, and polymorphonuclear neutrophil myeloid–derived suppressor cells (PMN-MDSCs). These populations of neutrophils are primarily defined by their functional phenotype, because few specific cell surface markers have been identified. In this review, we will discuss neutrophil polarization and plasticity and the function of proinflammatory/anti-inflammatory and protumor/antitumor neutrophils in the tumor microenvironment. We will also discuss how neutrophils with the ability to suppress T-cell activation, referred to by some as PMN-MDSCs, fit into this paradigm.
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Grebenchikov, O. A., I. S. Kasatkina, K. K. Kadantseva, M. A. Meshkov, and A. A. Bayeva. "The Effect of Lithium Chloride on Neutrophil Activation on Exposure to Serum of Patients with Septic Shock." General Reanimatology 16, no. 5 (November 6, 2020): 45–55. http://dx.doi.org/10.15360/1813-9779-2020-5-45-55.
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The aim of the study: to examine the anti-inflammatory effect of lithium chloride by exposing the human neutrophils to serum of patients with septic shock in vitro.Material and methods. The study was carried out on neutrophils extracted from the blood of 6 healthy donors, which were activated with serum from patients with septic shock. The neutrophil activity was evaluated with fluorescent antibodies to the CD11b and CD66b markers of degranulation. The level of human neutrophil apoptosis and necrosis was assessed 22 hours after extraction; quantitative assessment was made using annexin V and propidium iodide with flow cytofluorimetry. Intact and activated neutrophils were treated with 0.3, 3.0 and 9.0 mmol lithium chloride solution.Results. The level of CD11b expression on the surface of intact neutrophils (healthy donors) was 3434.50 [3311.0-3799.0] arbitrary fluorescence units (AFU). Incubation of neutrophils with serum of patients with septic shock increased CD11b expression 2.5 times to 8589.0 [7279.0-11258.0] AFU (P=0.005) vs intact leukocytes, and increased CD66b expression 2.7 times up to 27 600.0 [22 999.0-28 989.0] AFU ((P=0.005) vs intact neutrophils. Lithium chloride in concentrations of 0.3, 3.0 and 9.0 mmol in a dose-dependent manner reduced the level of expression of CD11b and CD66b molecules on the surface of activated neutrophils. Septic serum reduced spontaneous neutrophil apoptosis, and 3.0 mmol and higher lithium chloride solution induced spontaneous neutrophil apoptosis.Conclusion. Lithium chloride reduces the activation of neutrophils preactivated by serum of patients with septic shock, reduces expression of CD11b and CD66b molecules on the neutrophil surface, inhibiting the process of their activation (degranulation). Lithium chloride in concentration of 3.0 mmol and higher is able to induce spontaneous apoptosis of neutrophils activated by serum of patients with septic shock.
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Mattsson, Eva, Terese Persson, Pia Andersson, Jan Rollof, and Arne Egesten. "Peptidoglycan Induces Mobilization of the Surface Marker for Activation Marker CD66b in Human Neutrophils but Not in Eosinophils." Clinical Diagnostic Laboratory Immunology 10, no. 3 (May 2003): 485–88. http://dx.doi.org/10.1128/cdli.10.3.485-488.2003.
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ABSTRACT Peptidoglycan from Staphylococcus aureus mobilized CD66b in human neutrophils but did not upregulate surface activation markers in eosinophils. In addition, Toll-like receptor 2, implicated in the recognition of peptidoglycan, was detected on the surface of resting neutrophils but not on eosinophils. These findings suggest roles for neutrophils but not eosinophils in innate recognition of peptidoglycan.
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Choi, Kyoung-Seong, Justin Garyu, Jinho Park, and J. Stephen Dumler. "Diminished Adhesion of Anaplasma phagocytophilum-Infected Neutrophils to Endothelial Cells Is Associated with Reduced Expression of Leukocyte Surface Selectin." Infection and Immunity 71, no. 8 (August 2003): 4586–94. http://dx.doi.org/10.1128/iai.71.8.4586-4594.2003.
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ABSTRACT Anaplasma phagocytophilum propagates within neutrophils and causes a disease marked by inflammatory tissue injury or complicated by opportunistic infections. We hypothesized that infection with A. phagocytophilum modifies the binding of neutrophils to endothelial cells and the expression of neutrophil adhesion molecules and studied these changes in vitro. Infected dimethyl sulfoxide-differentiated HL-60 cells and neutrophils showed reduced binding to cultured brain and systemic endothelial cells and lost expression of P-selectin glycoprotein ligand 1 (PSGL-1, CD162) and L-selectin (CD62L) (to 33 and 5% of control values, respectively), at a time when the levels of β2 integrin and immunoglobulin superfamily adhesion molecules and activation markers Mac-1 and intercellular adhesion molecule 1 increased (5 to 10 times that of the control). The loss of CD162 and CD62L expression was inhibited by EDTA, which suggests that neutrophil activation and sheddase cleavage occurred. The loss of selectin expression and the retained viability of the neutrophils persisted for at least 18 h with A. phagocytophilum infection, whereas Escherichia coli and Staphylococcus aureus rapidly killed neutrophils. The adhesion defect might increase the numbers of infected cells and their persistence in the blood prior to tick bites. However, decreased CD162 expression and poor endothelial cell binding may partly explain impaired host defenses, while simultaneous neutrophil activation may aggravate inflammation. These observations may help us to understand the modified biological responses, host inflammation, and immune response that occur with A. phagocytophilum infections.
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Elsner, J., R. Hochstetter, K. Spiekermann, and A. Kapp. "Surface and mRNA expression of the CD52 antigen by human eosinophils but not by neutrophils." Blood 88, no. 12 (December 15, 1996): 4684–93. http://dx.doi.org/10.1182/blood.v88.12.4684.bloodjournal88124684.
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Eosinophilic and neutrophilic granulocytes represent major effector cells in the inflammatory response. Whereas neutrophils are predominantly involved in bacterial infections, eosinophils are of essential importance in the allergic inflammation. Surface markers have been used to distinguish neutrophils (CD16+) from eosinophils (CD16-) and might indicate different functional properties of these cells. In this study, expression and functional activity of CD52 on human eosinophils and neutrophils was investigated in nonatopic healthy donors and from patients with hypereosinophilia. Flow cytometric analysis using different anti-CD52 monoclonal antibodies (MoAbs) (mouse IgG3, humanized IgG1, and rat IgM) showed significant and homogeneous expression of CD52 on human eosinophils, but not on neutrophils. In addition, reverse transcription-polymerase chain reaction and Northern blot analysis showed that CD52 mRNA was constitutively expressed in eosinophils but not in neutrophils. Furthermore, expression of CD52 could be diminished in a dose-dependent manner by preincubation of eosinophils with phosphatidylinositol-specific phospholipase C, suggesting that CD52 on eosinophils is anchored to the membrane through a glycosylphosphatidylinositol (GPI) molecule. Whereas the phorbolester phorbol myristate acetate was able to downregulate the expression of CD52 on eosinophils in a dose-dependent manner, different eosinophil activating cytokines and chemotaxins had no effect. Cross-linking of CD52 by mouse anti-CD52 MoAb (IgG3) and humanized anti-CD52 MoAb (IgG1) with goat antimouse antibody and mouse antihuman antibody, respectively, dose-dependently resulted in an inhibition of reactive oxygen species production of eosinophils after stimulation with C5a, platelet-activating factor, and granulocyte-macrophage colony-stimulating factor. In summary, this study shows that the GPI-anchored antigen CD52 is not only a useful marker to distinguish eosinophils from neutrophils. The data point out a novel role of the CD52 antigen on human eosinophils that might be of clinical relevance, because cross-linking of this molecule will stop the destructive power of human eosinophils in the inflammatory tissue.
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Sochalska, Maja, Magdalena B. Stańczyk, Maria Użarowska, Natalia Zubrzycka, Susanne Kirschnek, Aleksander M. Grabiec, Tomasz Kantyka, and Jan Potempa. "Application of the In Vitro HoxB8 Model System to Characterize the Contributions of Neutrophil–LPS Interaction to Periodontal Disease." Pathogens 9, no. 7 (July 1, 2020): 530. http://dx.doi.org/10.3390/pathogens9070530.
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(1) Background: Studying neutrophils in vitro is difficult since these cells are terminally differentiated and are easily activated during isolation. At the same time, most of the available model cell lines are associated with certain limitations, such as functional deficiency or a lack of expression of surface markers characteristic of neutrophils. P. gingivalis is a periodontopathogen that causes dysbiosis in subgingival bacterial biofilm. This triggers the accumulation of functional neutrophils in the periodontium. However, until now, the specific effects of P. gingivalis-derived lipopolysaccharide on neutrophil functions have not been analyzed. (2) Methods: The impact of two variants of commercially available P. gingivalis endotoxin on neutrophil functions was tested using the HoxB8 in vitro system that is well suited to analyze neutrophil response to different stimuli in a controlled manner. (3) Results: The Standard P. gingivalis lipopolysaccharide (LPS), known to activate cells through Toll-like receptor 2 (TLR2)- and Toll-like receptor 4 (TLR4)-dependent pathways, prolonged neutrophil survival and exhibited pro-inflammatory effects. In contrast, Ultrapure LPS, binding exclusively to TLR4, neither protected neutrophils from apoptosis, nor induced an inflammatory response. (4) Conclusion: Two variants of P. gingivalis-derived LPS elicited effects on neutrophils and, based on the obtained results, we concluded that the engagement of both TLR2 and TLR4 is required for the manipulation of survival and the stimulation of immune responses of HoxB8 neutrophils.
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Dittrich, A. Susanne, Iris Kühbandner, Stefanie Gehrig, Verena Rickert-Zacharias, Matthew Twigg, Sabine Wege, Clifford C. Taggart, Felix Herth, Carsten Schultz, and Marcus A. Mall. "Elastase activity on sputum neutrophils correlates with severity of lung disease in cystic fibrosis." European Respiratory Journal 51, no. 3 (March 2018): 1701910. http://dx.doi.org/10.1183/13993003.01910-2017.
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Neutrophil elastase (NE) is a key risk factor for severity of cystic fibrosis (CF) lung disease. Recent studies identified increased NE activity on the surface of airway neutrophils from CF-like mice and patients with CF. However, the role of surface-bound NE in CF lung disease remains unknown. We determined the relationship between surface-bound NE activity and severity of lung disease in CF.Surface-bound NE activity was measured on sputum neutrophils from 35 CF patients and eight healthy controls using novel lipidated Förster resonance energy transfer reporters and correlated with free NE activity, neutrophil counts, interleukin-8, myeloperoxidase and antiproteases in sputum supernatant, and with lung function parameters.Surface-bound NE activity was increased in CF compared to healthy controls (p<0.01) and correlated with free NE activity (p<0.05) and other inflammation markers (p<0.001). Surface-bound and free NE activity correlated with forced expiratory volume in 1 s % predicted (p<0.01 and p<0.05), but only surface-bound NE activity correlated with plethysmographic functional residual capacity % pred (p<0.01) in patients with CF.We demonstrate that surface-bound NE activity on airway neutrophils correlates with severity of lung disease in patients with CF. Our results suggest that surface-bound NE activity may play an important role in the pathogenesis and serve as novel biomarker in CF lung disease.
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Thom, Stephen R., Ming Yang, Veena M. Bhopale, Shaohui Huang, and Tatyana N. Milovanova. "Microparticles initiate decompression-induced neutrophil activation and subsequent vascular injuries." Journal of Applied Physiology 110, no. 2 (February 2011): 340–51. http://dx.doi.org/10.1152/japplphysiol.00811.2010.
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Progressive elevations in circulating annexin V-coated microparticles (MPs) derived from leukocytes, erythrocytes, platelets, and endothelial cells are found in mice subjected to increasing decompression stresses. Individual MPs exhibit surface markers from multiple cells. MPs expressing platelet surface markers, in particular, interact with circulating neutrophils, causing them to degranulate and leading to further MP production. MPs can be lysed by incubation with polyethylene glycol (PEG) telomere B surfactant, and the number of circulating MPs is reduced by infusion of mice with PEG or antibody to annexin V. Myeloperoxidase deposition and neutrophil sequestration in tissues occur in response to decompression, and the pattern differs among brain, omentum, psoas, and leg skeletal muscle. Both MP abatement strategies reduce decompression-induced intravascular neutrophil activation, neutrophil sequestration, and tissue injury documented as elevations of vascular permeability and activated caspase-3. We conclude that MPs generated by decompression stresses precipitate neutrophil activation and vascular damage.
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Grebenchikov, O. A., A. K. Shabanov, A. A. Kosov, Yu V. Skripkin, A. G. Yavorovsky, and V. V. Likhvantsev. "Synthetic leu-enkefalin analogue prevents activation of neutrophils induced by a bacterial component." Almanac of Clinical Medicine 47, no. 3 (July 31, 2019): 228–35. http://dx.doi.org/10.18786/2072-0505-2019-47-026.
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Background: Neutrophil activation is a mandatory stage and a sensitive marker of systemic inflammatory response. The development of this condition is associated with subsequent multiple organ failure which is the main indication for the patients stay in the intensive care unit. The search for drugs that could prevent the development of systemic inflammatory response and reduce mortality remains an urgent task of anesthesiology/resuscitation.Aim: To study the anti-inflammatory effect of dalargin, a synthetic analogue of lei-enkephalin, on human neutrophils in vitro.Materials and methods: The study was performed on blood neutrophils isolated from 5 healthy donors. A proportion of neutrophils were activated by 10 mkM formil-Met-Leu-Pro (fMLP) and 100 ng/mL lipopolysaccharide (LPS) with subsequent assessment of their activity by fluorescent antibodies to the degranulation markers CD11b and CD66b. Thereafter intact and activated neutrophils were treated with dalargin solution at concentrations of 50 and 100 mcg/mL.Results: Dalargin at 100 mcg/mL reduced the expression of CD11b molecules on the surface of intact neutrophils by 5.5-fold (p=0.008). On the contrary, LPS at a dose of 100 ng/mL increased the expression of the same molecules by 46% (p=0.08). The addition of dalargin at 50 mcg/mL to LPS-activated neutrophils reduced the expression of CD11b molecules (p=0.016). The addition of dalargin at 50 mcg/mL to fMLP-activated neutrophils significantly (p=0.008) reduced the expression of CD11b molecules and reversed their expression virtually to the level of the control. The addition of dalargin at 100 mcg/mL to neutrophils activated by fMLP at 10 mkM reduced the expression of CD11b on their surface to a level below the control by 23% (p=0.08).Conclusion: Dalargin at the studied concentrations has an anti-inflammatory effect on both intact and pre-activated bacterial components of neutrophils, thus inhibiting the process of activation and degranulation in a dose-dependent manner.
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Bzowska, Małgorzata, Magda Hamczyk, Anna Skalniak, and Krzysztof Guzik. "Rapid Decrease of CD16 (FcγRIII) Expression on Heat-Shocked Neutrophils and Their Recognition by Macrophages." Journal of Biomedicine and Biotechnology 2011 (2011): 1–14. http://dx.doi.org/10.1155/2011/284759.
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Accumulation of neutrophils in the site of inflammation is a typical mechanism of innate immunity. The accumulated neutrophils are exposed to stressogenic factors usually associated with inflammation. Here, we studied response of human peripheral blood neutrophils subjected to short, febrile-range heat stress. We show that 90 min heat stress slowed down the spontaneous apoptosis of neutrophils. In the absence of typical markers of apoptosis the heat-shocked neutrophils induced antiinflammatory effect in human monocyte-derived macrophages (hMDMs), yet without being engulfed. Importantly, the expression of FcγRIII (CD16) was sharply reduced. Surprisingly, concentration of the soluble CD16 did not change in heat-shocked neutrophil supernates indicating that the reduction of the cell surface CD16 was achieved mainly by inhibition of fresh CD16 delivery. Inhibitors of 90 kDa heat shock protein (HSP90), a molecular chaperone found in membrane platforms together with CD16 and CD11b, significantly increased the observed effects caused by heat shock. The presented data suggest a novel systemic aspect of increased temperature which relies on immediate modification by heat of a neutrophil molecular pattern. This effect precedes cell death and may be beneficial in the initial phase of inflammation providing a nonphlogistic signal to macrophages before it comes from apoptotic cells.
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Tranah, Thomas H., Godhev K. Manakkat Vijay, Jennifer M. Ryan, R. Daniel Abeles, Paul K. Middleton, and Debbie L. Shawcross. "Dysfunctional neutrophil effector organelle mobilization and microbicidal protein release in alcohol-related cirrhosis." American Journal of Physiology-Gastrointestinal and Liver Physiology 313, no. 3 (September 1, 2017): G203—G211. http://dx.doi.org/10.1152/ajpgi.00112.2016.
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Patients with alcohol-related cirrhosis (ALD) are prone to infection. Circulating neutrophils in ALD are dysfunctional and predict development of sepsis, organ dysfunction, and survival. Neutrophil granules are important effector organelles containing a toxic array of microbicidal proteins, whose controlled release is required to kill microorganisms while minimizing inflammation and damage to host tissue. We investigated the role of these granular responses in contributing to immune disarray in ALD. Neutrophil granular content and mobilization were measured by flow cytometric quantitation of cell-surface/intracellular markers, [secretory vesicles (CD11b), secondary granules (CD66b), and primary granules (CD63; myeloperoxidase)] before and after bacterial stimulation in 29 patients with ALD cirrhosis (15 abstinent; 14 actively drinking) compared with healthy controls (HC). ImageStream Flow Cytometry characterized localization of granule subsets within the intracellular and cell-surface compartments. The plasma cytokine environment was analyzed using ELISA/cytokine bead array. Circulating neutrophils were primed in the resting state with upregulated surface expression of CD11b ( P = 0.0001) in a cytokine milieu rich in IL-8 ( P < 0.001) and lactoferrin ( P = 0.035). Neutrophils showed exaggerated mobilization to the cell surface of primary granules at baseline ( P = 0.001) and in response to N-formyl-l-methionyl-l-leucyl-l-phenylalanine ( P = 0.009) and Escherichia coli ( P = 0.0003) in ALD. There was no deficit in granule content or mobilization to the cell membrane in any granule subset observed. Paradoxically, active alcohol consumption abrogated the hyperresponsive neutrophil granular responses compared with their abstinent counterparts. Neutrophils are preprimed at baseline with augmented effector organelle mobilization in response to bacterial stimulation; neutrophil degranulation is not a mechanism leading to innate immunoparesis in ALD.NEW & NOTEWORTHY Neutrophil granule release is dysregulated in patients with alcohol-related cirrhosis (ALD) with augmented effector organelle mobilization and microbiocidal protein release. Neutrophil granules are upregulated in ALD at baseline and demonstrate augmented responses to bacterial challenge. The granular responses in ALD did not contribute to the observed functional deficit in innate immunity but rather were dysregulated and hyperresponsive, which may induce bystander damage to host tissue. Paradoxically, active alcohol consumption abrogated the excessive neutrophil granular responses to bacterial stimulus compared with their abstinent counterparts.
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Parrinello, Nunziatina, Alessandra Romano, Concetta Conticello, Maide Cavalli, Alessia La Fauci, Giuseppina Rizzo, Piera La Cava, et al. "Neutrophils Of Multiple Myeloma Are Dysfunctional and Immunosuppressive." Blood 122, no. 21 (November 15, 2013): 3138. http://dx.doi.org/10.1182/blood.v122.21.3138.3138.
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Abstract Introduction Multiple Myeloma (MM) is a plasma cell malignancy with a well documented immune dysfunction. However the role and function of neutrophils in MM and monoclonal gammopathy of undetermined significance (MGUS) has been poorly investigated. Methods on neutrophils (N) of 65 MM at diagnosis, 74 MGUS and 30 healthy subjects we evaluated, by flow cytometer, phagocytic activity and surface expression of CD64, CD16, CD62L and CD11b, markers of neutrophil activation. We tested also the immunosuppressive properties of N isolated from MGUS or MM patients, through functional assays, based on in vitro co-culture of N isolated from patients and T-lymphocytes from healthy subjects and we evaluated the expression of the immunosuppressive molecule arginase-1 (Arg-1) by RT-PCR. Results Despite no differences in the absolute number of N between MM, MGUS and healthy subjects, we found a functional impairment in MM not evident in MGUS patients. The phagocytic activity of MM-N was significantly reduced compared to healthy subjects-N (p<0.001) and MGUS-N (p<0.0001) and restored after induction chemotherapy (p=0.02). Expression of CD64 was significantly elevated in MM-N compared to MGUS-N or healthy subjects-N (p=0.01 and p= 0.007 respectively) and was inversely correlated with phagocytic activity (p=0,01). No differences were observed among MM, MGUS and healthy subjects for the other surface markers evaluated. MM-N exhibit an increased expression of ARG-1 compared to MGUS and healthy subjects (25.5 vs 6.2 vs 1 fold changes in gene expression, p=0.003), confirmed by functional assay of enzymatic activity of ARG-1, positively correlated with advanced disease. After PHA-P stimulation,T-lymphocytes isolated from healthy subjects missed the expression of activation markers such CD71, CD69, CD25, CD3ζ in the presence of MM-N for 72 hours, and in a less extensive way in the presence of MGUS-N. Conclusion Compared to controls, neutrophils obtained from MM patients have a reduced phagocytic activity, a greater expression of Arg-1 and exhibit an immunosuppressive function on T lymphocytes. Taken together, these findings indicated that neutrophils may contribute to impairment of immune function that characterizes MM patients. Disclosures: No relevant conflicts of interest to declare.
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Döring, Yvonne, Peter Libby, and Oliver Soehnlein. "Neutrophil Extracellular Traps Participate in Cardiovascular Diseases." Circulation Research 126, no. 9 (April 24, 2020): 1228–41. http://dx.doi.org/10.1161/circresaha.120.315931.
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Neutrophil extracellular traps (NETs) have recently emerged as a newly recognized contributor to venous and arterial thrombosis. These strands of DNA extruded by activated or dying neutrophils, decorated with various protein mediators, become solid-state reactors that can localize at the critical interface of blood with the intimal surface of diseased arteries and propagate and amplify the regional injury. NETs thus furnish a previously unsuspected link between inflammation, innate immunity, thrombosis, oxidative stress, and cardiovascular diseases. In response to disease-relevant stimuli, neutrophils undergo a specialized series of reactions that culminate in NET formation. DNA derived from either nuclei or mitochondria can contribute to NET formation. The DNA liberated from neutrophils forms a reticular mesh that resembles morphologically a net, rendering the acronym NETs particularly appropriate. The DNA backbone of NETs not only presents intrinsic neutrophil proteins (eg, MPO [myeloperoxidase] and various proteinases) but can gather other proteins found in blood (eg, tissue factor procoagulant). This review presents current concepts of neutrophil biology, the triggers to and mechanisms of NET formation, and the contribution of NETs to atherosclerosis and to thrombosis. We consider the use of markers of NETs in clinical studies. We aim here to integrate critically the experimental literature with the growing body of clinical information regarding NETs.
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Weiss, J., and I. Olsson. "Cellular and subcellular localization of the bactericidal/permeability- increasing protein of neutrophils." Blood 69, no. 2 (February 1, 1987): 652–59. http://dx.doi.org/10.1182/blood.v69.2.652.bloodjournal692652.
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Human and rabbit polymorphonuclear leukocytes contain a bactericidal/permeability-increasing protein (BPI), a potent cytotoxin active specifically against gram-negative bacteria. To identify the cell population(s) producing BPI, we have examined mature and immature human blood cells for BPI by immunofluorescence of intact cells and radioimmunoassay and bioassay of cell extracts. By immunofluorescence and radioimmunoassay of cells from peripheral blood, BPI was detected only in neutrophils; immunofluorescent staining was punctate, indicative of the granule localization of BPI. Nearly all (greater than 90%) BPI was recovered during the subcellular fractionation of neutrophils (N2 cavitation and discontinuous Percoll gradient) in fractions containing primary granules. Little BPI was released from intact cells during degranulation (cytochalasin B and f-Met-Leu-Phe) or could be extracted from isolated granules with salt or weak acid, which suggests that most granule-associated BPI is membrane bound. Double staining of bone marrow smears for BPI and lactoferrin revealed BPI only in neutrophil precursors including (pro)myelocytelike cells lacking lactoferrin, a marker of neutrophil secondary granules. Of several human cell lines tested, only the promyelocytelike HL-60 (and to a lesser extent, KG-1) cells contained BPI. BPI was present in a more mature subpopulation (less than 25%) of untreated HL-60 cells, recognized by surface marker analysis (rosetting with IgG-sensitized sheep RBC, the absence of proliferation-associated cell surface antigen). Induction of neutrophilic or monocytic differentiation caused, respectively, a small (approximately 50%) rise or fall in the BPI content. These findings indicate that BPI is a specific product of the neutrophil lineage and, hence, of the specialized cytotoxic apparatus of the neutrophil that plays an essential role in host defense v gram-negative bacteria.
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Kremserova, Silvie, Tomas Perecko, Karel Soucek, Anna Klinke, Stephan Baldus, Jason P. Eiserich, and Lukas Kubala. "Lung Neutrophilia in Myeloperoxidase Deficient Mice during the Course of Acute Pulmonary Inflammation." Oxidative Medicine and Cellular Longevity 2016 (2016): 1–13. http://dx.doi.org/10.1155/2016/5219056.
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Systemic inflammation accompanying diseases such as sepsis affects primarily lungs and induces their failure. This remains the most common cause of sepsis induced mortality. While neutrophils play a key role in pulmonary failure, the mechanisms remain incompletely characterized. We report that myeloperoxidase (MPO), abundant enzyme in neutrophil granules, modulates the course of acute pulmonary inflammatory responses induced by intranasal application of lipopolysaccharide. MPO deficient mice had significantly increased numbers of airway infiltrated neutrophils compared to wild-type mice during the whole course of lung inflammation. This was accompanied by higher levels of RANTES in bronchoalveolar lavage fluid from the MPO deficient mice. Other markers of lung injury and inflammation, which contribute to recruitment of neutrophils into the inflamed lungs, including total protein and other selected proinflammatory cytokines did not significantly differ in bronchoalveolar lavage fluid from the wild-type and the MPO deficient mice. Interestingly, MPO deficient neutrophils revealed a decreased rate of cell death characterized by phosphatidylserine surface expression. Collectively, the importance of MPO in regulation of pulmonary inflammation, independent of its putative microbicidal functions, can be potentially linked to MPO ability to modulate the life span of neutrophils and to affect accumulation of chemotactic factors at the inflammatory site.
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Weimann, Andreas, Andreas Lun, Sebastian Lun, Mathias Zimmermann, Adrian C. Borges, Reinhard Ziebig, Jose B. Gonzalez, et al. "Leukocyte, neutrophil, immature granulocyte counts and interleukin-6 are superior to procalcitonin, C-reactive protein and delta-He for detection of mild inflammation: data from marathon runners producing mild systemic inflammation visible immediately after the run / Leukozyten, Neutrophile, unreife Granulozyten und Interleukin-6 sind zum Nachweis geringgradiger Entzündungen Procalcitonin, C-reaktivem Protein und Delta-He überlegen: Ergebnisse aus einer Untersuchung von Marathonläufern." LaboratoriumsMedizin 34, no. 1 (February 1, 2010): 53–59. http://dx.doi.org/10.1515/jlm.2010.005.
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AbstractImmature granulocytes (IGs) and differences between reticulocyte and erythrocyte haemoglobin content (delta-He) are now available as modern parameters on routine haematology analysers for detecting inflammation. Are these markers more suitable to detect mild inflammation when compared with traditional inflammation markers such as leukocyte count, C-reactive protein (CRP), procalcitonin (PCT), interleukin-6 (IL-6) or diverse leukocyte surface molecules mainly used in research programs? To answer this question, a marathon race was used as a model of mild inflammation. Full blood counts, CRP, PCT, IL-6, expression of surface molecules on granulocytes, monocytes and lymphocytes were measured before and immediately after the race (inflammatory state) and were compared with each other. A further blood sample was taken after a 10-day rest. In the inflammatory state leukocytes, neutrophil counts and IL-6 concentration were considerably increased compared with basic conditions. Diagnostic sensitivity and specificity came up to 100%. CRP was not increased and delta-He did not drop to negative values, as it occurs in severe inflammation. Leukocyte surface molecules were able to indicate a mild inflammatory state induced by the marathon race, but these markers did not achieve the same discriminatory power when compared with IL-6 levels or neutrophil count. In conclusion, leukocytes, neutrophils and IG counts as well as IL-6 levels are the best indicators in a mild inflammation model similar to a marathon race.
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Stockfelt, Marit, Karin Christenson, Anders Andersson, Lena Björkman, Médea Padra, Bettina Brundin, Koustav Ganguly, et al. "Increased CD11b and Decreased CD62L in Blood and Airway Neutrophils from Long-Term Smokers with and without COPD." Journal of Innate Immunity 12, no. 6 (2020): 480–89. http://dx.doi.org/10.1159/000509715.
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There is incomplete mechanistic understanding of the mobilization of neutrophils in the systemic and local compartment in smokers with chronic obstructive pulmonary disease (COPD). In this pilot study, we characterized how the adhesion molecules CD11b and CD62L, surface markers indicative of priming, are altered as neutrophils extravasate, and whether surface density of CD11b and CD62L differs between long-term tobacco smokers (LTS) with and without COPD compared with healthy never-smokers (HNS). Unstimulated blood neutrophils from LTS with (<i>n</i> = 5) and without (<i>n</i> = 9) COPD displayed lower surface density of CD62L compared with HNS (<i>n</i> = 8). In addition, surface density of CD11b was higher in bronchoalveolar lavage (BAL) neutrophils from LTS without COPD compared with those with COPD and HNS. Moreover, in BAL neutrophils from all study groups, CD62L was lower compared with matched blood neutrophils. In addition, BAL neutrophils responded with a further decrease in CD62L to ex vivo TNF stimulation. Thus, neutrophils in the airway lumen display a higher state of priming than systemic neutrophils and bear the potential to be further primed by local cytokines even with no smoking or the presence of COPD, findings that may represent a universal host defense mechanism against local bacteria. Moreover, systemic neutrophils are primed in LTS regardless of COPD. Further studies in larger materials are warranted to determine whether the priming of neutrophils is protective against COPD or merely preceding it.
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Boateng, Lydia A., Rahima Zennadi, and Marilyn J. Telen. "Sickle Red Blood Cell Induced Adhesion of Neutrophils to Endothelial Cells and Biologic Correlates of Leukocyte Activation." Blood 118, no. 21 (November 18, 2011): 1055. http://dx.doi.org/10.1182/blood.v118.21.1055.1055.
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Abstract 1055 In sickle cell disease (SCD), any event that slows the passage of red blood cells (RBCs) in the microcirculation can promote hemoglobin polymerization, red cell sickling, and vaso-occlusion. Homozygous (SS) RBCs have a sticky surface and, compared to normal (AA) RBCs, attach readily to both endothelial cells and other blood cells. Furthermore, the adhesive function of SS RBCs is up-regulated by the stress hormone epinephrine, resulting in significant vaso-occlusion and increased mononuclear leukocyte adhesion to the vascular endothelium in a mouse model. We therefore postulated that direct interaction between SS RBCs and neutrophils may also lead to activation and adhesion of neutrophils to endothelial cells. In order to explore whether SS RBCs can induce adhesion of neutrophils to endothelial cells independent of exogenous cytokine stimulation, neutrophils were isolated from healthy donors using density gradient media and then labeled with fluorescent dye. Fluorescent neutrophils were then co-incubated with epinephrine-treated SS or sham-treated (untreated) SS, AA or no added RBCs (control) at 37°C. Graduated height flow chambers were used to quantify adhesion of neutrophils to human umbilical vein endothelial cells (HUVECs) grown on glass slides coated with gelatin. We then also examined markers of hematologic status and endothelial activation to determine if any of these were correlated with the ability of SS RBCs to induce neutrophil adhesion. Enzyme linked immunosorbent assays (ELISAs) were used to quantify inflammatory markers (IL-1B, IL-6, IL-8), soluble L selectin (sL-SEL) and soluble P selectin (sP-SEL) in the plasma of the SS patient samples used in adhesion assays. We found that neither sham-treated nor epi-treated AA RBCs (n=3) significantly increased neutrophil adhesion to HUVECs, while both sham-treated and epinephrine-stimulated (epi) SS RBCs significantly increased neutrophil adhesion to endothelial cells (n=17, 42.0% adhesion for epi-SS RBCs vs 20.0% for no RBCs and 27.2% for untreated SS RBCs, with p values as shown in Figure 1). ELISA immunoassays confirmed elevated levels of sP-SEL and sL-SEL in SS patients, and levels of sL-SEL correlated with total leukocyte count (p=0.005). However, neither these biomarkers nor leukocyte count correlated with the ability of patient RBCs to stimulate normal leukocyte adhesion. Levels of IL-1B, IL-6 and IL-8 also did not correlate with the ability of patients' RBCs to stimulate neutrophil adhesion. However, we also found that HbF levels correlated inversely with sL-SEL levels (P=0.014), suggesting that HbF not only helps prevent RBC sickling but also reduces leukocyte activation. Other hematologic markers (Hb level, platelet count, reticulocyte count, LDH) also did not correlate with the ability of epi-SS RBCs to induce neutrophil adhesion. Therefore, we conclude that SS RBCs are able to stimulate neutrophil adhesion, especially under conditions that activate the adhesion receptors responsible for RBC-neutrophil interaction. In addition, we found that sL-SEL was increased in the presence of higher WBC counts, while it was inversely related to HbF levels. Taken together, these data suggest that the effect of HbF on SS RBCs reduces leukocyte activation, and this may occur in part through reduction of SS RBC-leukocyte interaction. Disclosures: No relevant conflicts of interest to declare.
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MOLLINEDO, Faustino, Motowo NAKAJIMA, Ana LLORENS, Enrique BARBOSA, Sagrario CALLEJO, Consuelo GAJATE, and Angels FABRA. "Major co-localization of the extracellular-matrix degradative enzymes heparanase and gelatinase in tertiary granules of human neutrophils." Biochemical Journal 327, no. 3 (November 1, 1997): 917–23. http://dx.doi.org/10.1042/bj3270917.
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The expression of cell-surface adhesion proteins and the release of extracellular-matrix degradative enzymes constitute crucial processes for the attachment of neutrophils to the endothelium and for the subsequent extravasation of these cells through the endothelial layer. We have analysed in resting human neutrophils the subcellular localization of heparanase, a heparan-sulphate-degrading endoglycosidase that can degrade basement-membrane components, thereby facilitating neutrophil passage into the tissue during an inflammatory reaction. By subcellular fractionation of postnuclear supernatants from resting human neutrophils on continuous sucrose gradients, we have found that heparanase activity was mainly located in gelatinase-containing tertiary granules. Using a specific antibody, the 96-kDa heparanase protein was further located in the gelatinase-rich subcellular fractions. Following immunoblotting and immunoprecipitation analysis in the distinct subcellular fractions, we also found co-localization of heparanase and Mo1 (CD11b/CD18), a leucocyte integrin involved in the attachment of neutrophils to the endothelium, in the fractions enriched in gelatinase-containing tertiary granules. Treatment of human neutrophils with tumour necrosis factor or granulocyte/macrophage colony-stimulating factor induced an increase in the CD11b/CD18 cell-surface expression, as well as the release of both gelatinase (matrix metalloproteinase-9) and heparanase, but not of other granule markers, indicating a major co-localization of gelatinase, heparanase and CD11b/CD18 in the same organelle. Furthermore, confocal laser scanning microscopy using specific antibodies against gelatinase and heparanase revealed a major co-localization of both enzymes in intracellular cytoplasmic granules. The major localization of heparanase and CD11b/CD18 in the gelatinase-containing tertiary granule supports the notion that mobilization of this organelle can regulate extravasation of human neutrophils.
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Weiss, J., and I. Olsson. "Cellular and subcellular localization of the bactericidal/permeability- increasing protein of neutrophils." Blood 69, no. 2 (February 1, 1987): 652–59. http://dx.doi.org/10.1182/blood.v69.2.652.652.
Full textAbstract:
Abstract Human and rabbit polymorphonuclear leukocytes contain a bactericidal/permeability-increasing protein (BPI), a potent cytotoxin active specifically against gram-negative bacteria. To identify the cell population(s) producing BPI, we have examined mature and immature human blood cells for BPI by immunofluorescence of intact cells and radioimmunoassay and bioassay of cell extracts. By immunofluorescence and radioimmunoassay of cells from peripheral blood, BPI was detected only in neutrophils; immunofluorescent staining was punctate, indicative of the granule localization of BPI. Nearly all (greater than 90%) BPI was recovered during the subcellular fractionation of neutrophils (N2 cavitation and discontinuous Percoll gradient) in fractions containing primary granules. Little BPI was released from intact cells during degranulation (cytochalasin B and f-Met-Leu-Phe) or could be extracted from isolated granules with salt or weak acid, which suggests that most granule-associated BPI is membrane bound. Double staining of bone marrow smears for BPI and lactoferrin revealed BPI only in neutrophil precursors including (pro)myelocytelike cells lacking lactoferrin, a marker of neutrophil secondary granules. Of several human cell lines tested, only the promyelocytelike HL-60 (and to a lesser extent, KG-1) cells contained BPI. BPI was present in a more mature subpopulation (less than 25%) of untreated HL-60 cells, recognized by surface marker analysis (rosetting with IgG-sensitized sheep RBC, the absence of proliferation-associated cell surface antigen). Induction of neutrophilic or monocytic differentiation caused, respectively, a small (approximately 50%) rise or fall in the BPI content. These findings indicate that BPI is a specific product of the neutrophil lineage and, hence, of the specialized cytotoxic apparatus of the neutrophil that plays an essential role in host defense v gram-negative bacteria.
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Lizcano, Anel, Ismael Secundino, Simon Dohrmann, Ross Corriden, Lingquan Deng, Sandra Diaz, Victor Nizet, and Ajit P. Varki. "Erythrocyte Sialoglycoproteins Engage Siglec-9 to Maintain Neutrophil Quiescence in the Bloodstream." Blood 128, no. 22 (December 2, 2016): 1022. http://dx.doi.org/10.1182/blood.v128.22.1022.1022.
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Abstract Neutrophils remain functionally quiescent in the bloodstream of healthy humans. Characterized by a short lifespan (for which estimates vary widely), they exit the bloodstream to mediate various functions such as pathogen elimination or wound healing--or undergo spontaneous apoptosis and macrophage-mediated clearance in the absence of an inflammatory stimulus. Understanding of neutrophil quiescence, activation and lifespan is relevant to many clinical situations such as infection, sepsis, reperfusion injury, granulocyte transfusions, and other neutrophil-mediated inflammatory pathologies. It has long been known that neutrophils purified from blood tend to be short-lived and show signs of activation, but this technical challenge has been assumed to be a reflection of their natural biology in vivo. We found that when purified from whole blood, neutrophils indeed rapidly express activation markers (low L-selectin; high CD11b) and progress to apoptosis. However, using a novel method to label neutrophils in situ followed by flow cytometry, we found that these activation events occurred at a much slower rate in neutrophils that remained in whole blood. This finding led us to hypothesize that during separation of neutrophils from other blood components, one may be removing an inhibitor that normally maintains neutrophil quiescence in the bloodstream. Co-incubation studies showed that erythrocytes are the primary blood component that dampens neutrophil activation, including chemotaxis, generation of reactive oxygen species, and release of neutrophil extracellular traps. The duration of neutrophil viability was also lengthened upon re-incubation with erythrocytes. We found that this maintenance of functional quiescence is mediated largely by erythrocyte surface sialoglycoproteins (particularly glycophorin A, GPA), which engage neutrophil Siglec-9, a sialic acid-binding receptor that is known to dampen innate immune cell activation via cytosolic inhibitory motifs. Modification of erythrocyte sialic acid side chains using a newly developed gentle method eliminated Siglec-9 binding, and allowed neutrophil activation as measured by reactive oxygen species production. We next sought evidence that this interaction occurs in vivoby studying freshly collected whole blood. Smears made from fresh whole blood showed a high degree of Siglec-9 clustering on neutrophils, which was evident at points of contact with GPA on erythrocytes. This clustering was markedly reduced when smears were made from buffy coat preparations, which involves initial physical separation of neutrophils from erythrocytes during centrifugation. Taken together, this data indicates that erythrocyte sialic acids have an unexpected function as carriers of "self-associated molecular patterns" (SAMPs), regulating innate immunity and maintaining neutrophil quiescence in the bloodstream, apparently by tonic engagement of inhibitory Siglec-9. Notably, this SAMP effect blunts but does not completely inhibit bacterial killing by neutrophils in whole blood, and yet presumably helps to limit unwanted neutrophil inflammatory activation in the bloodstream. Our findings are relevant to many physiological, pathological and clinical situations involving neutrophil biology, and may be useful in reevaluation of prior studies of activation, function and reinfusion-based kinetics that were performed using neutrophils isolated away from whole blood. Disclosures No relevant conflicts of interest to declare.
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Al-Ruzzeh, Sharif, Ginette Hoare, Nandor Marczin, George Asimakopoulos, Shane George, Kenneth Taylor, and Mohamed Amrani. "Off-Pump Coronary Artery Bypass Surgery Is Associated with Reduced Neutrophil Activation as Measured by the Expression of CD11b: A Prospective Randomized Study." Heart Surgery Forum 6, no. 2 (February 2, 2005): 89. http://dx.doi.org/10.1532/hsf.1205.
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<P>Background: Coronary artery bypass grafting (CABG) surgery is associated with systemic inflammation. Activation of neutrophils is a crucial step in inflammation and results in neutrophil sequestration within the tissues. One of the potential advantages of performing off-pump coronary artery bypass (OPCAB) surgery is the attenuation of the systemic inflammatory response. This prospective randomized study compares neutrophil activation in patients undergoing OPCAB versus those undergoing CABG with cardiopulmonary bypass (CPB). </P><P>Methods: Twenty patients undergoing primary isolated CABG were randomly divided prospectively into 2 groups: 1 group underwent CABG with CPB, and the other group underwent OPCAB. Central venous blood samples were obtained before skin incision and at 15 minutes, 60 minutes, 2 hours, 5 hours, and 24 hours following the initiation of CPB or application of the stabilization device. Differential white cell counts were measured with routine laboratory techniques. CD11b surface expression on neutrophils was measured by flow cytometry. Interleukin 8 levels in the plasma were measured by enzyme-linked immunosorbent assays. </P><P>Results: The 2 groups were matched with respect to preoperative and operative characteristics. White cell and neutrophil counts rose in both groups following the operation but were significantly higher in the OPCAB group at 5 hours (P < .001 and P = .002, respectively). Interleukin 8 concentrations were significantly higher in the CPB group at 5 hours following the initiation of CPB (P = .034). CD11b levels were significantly higher in the CPB group at 60 minutes (P = .002). Conclusion: This prospective randomized study demonstrates that the activation of circulating neutrophils as measured by CD11b expression is lower following OPCAB than in CPB. Although OPCAB is associated with significantly higher neutrophil counts, these neutrophils exhibit fewer activation markers. The lower postoperative neutrophil counts occurring in the CPB group may be explained by the activation and consequent sequestration of the neutrophils in the CPB circuit and tissues.</P>
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Savchenko, А. А., A. G. Borisov, D. V. Cherdancev, O. V. Pervova, I. V. Kudryavtsev, I. I. Gvozdev, and A. V. Moshev. "FEATURES OF THE PHENOTYPE AND NAD(P)-DEPENDENT DEHYDROGENASES ACTIVITY IN NEUTROPHIL BY PATIENTS WITH WIDESPREAD PURULENT PERITONITIS IN PROGNOSIS FOR SEPSIS DEVELOPMENT." Russian Journal of Infection and Immunity 8, no. 3 (November 4, 2018): 369–76. http://dx.doi.org/10.15789/2220-7619-2018-3-369-376.
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The aim of the study was to examine the features of phenotype and the levels of NAD(P)-dependent dehydrogenases activity of blood neutrophils in the prognosis of abdominal sepsis development in patients with widespread purulent peritonitis (WPP). 50 patients with WPP of community and hospital origin in the pre-operative period were examined. From the 5th to the 10th day of the postoperative period 35 patients (70%) had developed abdominal sepsis, 15 patients (30%) had absence of complications. As controls 67 respect healthy people were examined. The research blood neutrophils phenotype was performed by f low cytometry using a direct immunofluorescence whole peripheral blood. The levels of surface receptor expression was assessed by the mean fluorescence intensity. The NAD(P)-dependent dehydrogenases activity in the blood neutrophils studied using bioluminescence method. It was established that the inflammatory reaction in patients with WPP is characterized by neutrophilia and changes in the phenotype of blood neutrophils. The markers of the subsequent development of sepsis in WPP are less pronounced (in comparison with the indices of uncomplicated patients), an increase in the number of neutrophils, a decrease in the HLA-DR + -cell count against the background of a high level of neutrophils expressing a high affinity receptor for IgG. The patients without subsequent complications had the number of neutrophils with CD23 receptor expression is increased. Metabolism of neutrophils in patients with WPP is characterized by a decrease in the intensity of plastic processes due to low activity of glucose-6-phosphate dehydrogenase and an imbalance in the activity of the enzymes of the mitochondrial compartment. A feature of the neutrophil metabolism in patients with WPP without subsequent development of sepsis is a high activity of anaerobic lactate dehydrogenase reaction and a decrease in the activity of NADP-dependent decarboxylating malate dehydrogenase. The patients with WPP with subsequent development of sepsis had a high level of NAD-dependent out flow of substrates from the tricarboxylic acid cycle on the amino acid exchange reaction via glutamate dehydrogenase which can affect the activity of aerobic respiration of blood neutrophils. The established differences in the phenotype and activity of enzymes in the blood neutrophils in patients with WPP depending on the subsequent development of sepsis determine the possibility of creating a method for predicting the development of complications and developing immunoactive therapy in the postoperative period of WPP.
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Pelikan, Zdenek. "Expression of Surface Markers on the Blood Cells during the Delayed Asthmatic Response to Allergen Challenge." Allergy & Rhinology 5, no. 2 (January 2014): ar.2014.5.0087. http://dx.doi.org/10.2500/ar.2014.5.0087.
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Patients with bronchial asthma develop various types of asthmatic response to bronchial challenge with allergen, such as immediate/early asthmatic response (IAR), late asthmatic response (LAR) or delayed asthmatic response (DYAR), because of different immunologic mechanisms. The DYAR, occurring between 24 and 56 hours after the bronchial allergen challenge (p < 0.01), differs from IAR and LAR in clinical as well as immunologic features. This study investigates the expression of CD molecules (markers) on the surface of particular cell populations in the peripheral blood and their changes during the DYAR. In 17 patients developing the DYAR (p < 0.01), the bronchial challenge with allergen was repeated 2–6 weeks later. The repeated DYAR (p < 0.001) was combined with recording of CD molecule expression on various types of blood cells by means of flow cytometry up to 72 hours after the challenge. The results were expressed in percent of the mean relative fluorescence intensity. The DYAR was accompanied by (a) increased expression of CD11b, CD11b/18, CD16, CD32, CD35, CD62E, CD62L, CD64, and CD66b on neutrophils; CD203C on basophils; CD25and CD62L on eosinophils; CD14, CD16, CD64, and CD86 on monocytes; CD3, CD4, CD8, CD11a, CD18, and CD69 on lymphocytes; CD16, CD56, CD57, and CD94 on natural killer (NK) cells; and CD31, CD41, CD61, CD62P, and CD63 on thrombocytes and (b) decreased expression of CD18 and CD62L on eosinophils, CD15 on neutrophils, and CD40 on lymphocytes. These results suggest involvement of cell-mediated hypersensitivity mechanism, on participation of Th1- lymphocytes, neutrophils, monocytes, NK cells, and thrombocytes in the DYAR.
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Kinhult, J., A. Egesten, M. Benson, R. Uddman, and L. O. Cardell. "Increased expression of surface activation markers on neutrophils following migration into the nasal lumen." Clinical & Experimental Allergy 33, no. 8 (August 2003): 1141–46. http://dx.doi.org/10.1046/j.1365-2222.2003.01682.x.
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Roervig, Sara, Christian Honore, Lars-Inge Larsson, Sophie Ohlsson, Corinna cavan Pedersen, Lars Christian Jacobsen, Jack B. Cowland, Peter Garred, and Niels Borregaard. "Ficolin-1 Is Present in a Highly Mobilizable Subset of Human Neutrophil Granules and Associates with the Cell Surface after Stimulation with fMLP." Blood 112, no. 11 (November 16, 2008): 1267. http://dx.doi.org/10.1182/blood.v112.11.1267.1267.
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Abstract Ficolins are soluble pattern recognition molecules that bind carbohydrate structures on the surface of microorganisms. Three ficolins have been identified in man: ficolin-1, ficolin-2, and ficolin-3. Ficolin-1 is derived from the FCN1 gene on chromosome 9 and is synthesized in monocytes and type II alveolar epithelial cells. Ficolin-1 has been shown to be present in secretory granules of human neutrophils, but which subset of the neutrophils secretory granules harbors ficolin-1 is not known. In order to determine the exact subcellular localization of ficolin-1 in neutrophils, recombinant ficolin-1 was expressed in Chinese hamster ovary cells and used for generation of polyclonal antibodies. This allowed detection of ficolin-1 in subcellular fractions of human neutrophils by ELISA and western blotting, and by immunohistochemistry. Real time PCR examination of normal human bone marrow showed FCN1 gene expression largely in myelocytes, metamyelocytes, and band cells with a profile quite similar to that of gelatinase. In accordance with this, immunohistochemistry and subcellular fractionation demonstrated that ficolin-1 is primarily localized in gelatinase granules, but also in highly exocytosable gelatinase poor granules, not previously described. Ficolin-1 is released from neutrophil granules by stimulation with fMLP or PMA, and a significant part becomes associated with the surface membrane of the cells and can be detected by flow cytometry. Our studies show that neutrophils are a major source of ficolin-1, which can be readily released to the surroundings by stimulation. Figure. Left: Distribution profile of ficolin-1 in unstimulated (control) neutrophils and neutrophils stimulated with fMLP or PMA. Right: Distribution profile of ficolin-1 and granule marker proteins in subcellular fractions of unstimulated human neutrophils. Figure. Left: Distribution profile of ficolin-1 in unstimulated (control) neutrophils and neutrophils stimulated with fMLP or PMA. Right: Distribution profile of ficolin-1 and granule marker proteins in subcellular fractions of unstimulated human neutrophils.
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Hanson, Madelyn S., Nancy J. Wandersee, Martin Hessner, Kirkwood A. Pritchard, Neil Hogg, and Cheryl A. Hillery. "Neutrophil Activation In Sickle Cell Disease: Biochemical and Functional Changes At Baseline and During Acute Vaso-Occlusive Crises." Blood 122, no. 21 (November 15, 2013): 992. http://dx.doi.org/10.1182/blood.v122.21.992.992.
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Abstract Neutrophilia is a well-recognized complication of sickle cell disease (SCD) and is a risk factor for increased disease severity and early death. Increased neutrophil activation, adhesion and oxidant production have been reported in SCD. While changes in neutrophil surface marker expression, degranulation, migration, adhesion and intracellular oxidative stress have been reported in SCD, there is little mechanistic underpinning for these observed functional changes. Moreover, how neutrophils specifically contribute to SCD pathology, particularly during acute vaso-occlusive crises, remains unknown. We hypothesize that chronic biochemical and functional changes of neutrophils in SCD contribute to disease pathology and are amplified during vaso-occlusive crisis. To define biochemical differences between neutrophils isolated from individuals with SCD and those isolated from healthy controls, we performed global gene expression analysis using the Affymetrix HG133 plus 2 GeneChip. Highly distinct transcriptional profiles were observed in neutrophils isolated from controls versus individuals with SCD at baseline and SCD in crisis. These were sufficiently distinct as to be separable by disease group in unsupervised hierarchical clustering analyses. Ontological analyses identified enriched transcriptional networks in SCD neutrophils related to elevated reactive oxygen species (ROS) production (p<6.3E-4) as well as potentiated respiratory burst (p<0.008). Using extracellular flux technology, we were able to measure oxygen consumption rates (OCR) of intact cells and observed that neutrophils from individuals with SCD have an elevated basal OCR over control cells. The elevated basal OCR could be due to increased NADPH oxidase 2 activity or increased mitochondrial respiration. While mitochondrial activity in healthy neutrophils is minimal, these organelles are essential to drive apoptosis, which is required for adequate resolution of inflammatory processes. In turn, neutrophil apoptosis is suppressed in multiple inflammatory diseases, including SCD. The transcriptional studies identified an upregulation of genes that promote mitochondrial stability (2.1- to 3.5-fold increase, p<0.02), a potential mechanism for both increased neutrophil lifespan and perhaps ROS production. Normally, neutrophils maintain ample antioxidant capacity to prevent intracellular damage from ROS generation. Vitamin E is a particularly important antioxidant in neutrophil biology as it attenuates multiple pro-inflammatory activities of these cells. We find that SCD neutrophils have markedly diminished vitamin E levels (p=0.031). Thus, loss of this important antioxidant may promote a pro-inflammatory phenotype of neutrophils in SCD. Next, we examined the potential contribution of neutrophils to the SCD pathobiology in vivo particularly during acute crisis. Berkeley sickle mice (SS mice) and controls (AA mice) were exposed to normoxia (baseline) or hypoxia (3 hrs 10% FIO2) to induce acute sickling followed by reoxygenation (2 hrs room air, H/R). Liver histopathology of SS mice displayed areas of leukocyte infiltration at baseline, with a potentiation in the number of these inflammatory sites after acute sickling induced by H/R. Importantly, examination of these sites at higher magnification reveals significant neutrophil infiltration near congested vessels, suggesting that H/R may potentiate neutrophil recruitment to injured tissues in SCD. To further assess neutrophil activation post H/R, plasma levels of myeloperoxidase (MPO) and DNA, evidence of neutrophil extracellular trap (NET) formation, were measured. SS mice have elevated plasma MPO levels compared to AA mice at baseline (p=0.007), with a trend towards a further increase in SS mice post H/R (no change in plasma MPO in AA mice post H/R). Plasma DNA levels were also elevated in SS mice compared to AA mice at baseline (p=0.01) and exposure of SS mice to H/R significantly increased plasma DNA levels (p=0.009), consistent with an acute increase in NET formation. In summary, chronic neutrophil activation in SS mice at baseline is exacerbated by acute sickling. Taken together, our data supports a role for neutrophils in SCD pathology which is amplified during vaso-occlusive crisis. Thus, targeting neutrophils may offer a novel therapeutic strategy for the prevention and treatment of vaso-occlusive complications in SCD. Disclosures: Wandersee: Bayer: Consultancy. Hillery:Bayer: Consultancy; Biogen Idec: Consultancy.
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Perez-Ladaga, Albert, Bennett Caughey, Huafeng Xie, Stuart H. Orkin, David B. Sykes, Benjamin L. Ebert, and Rafael Bejar. "Functional Defects In Neutrophils Derived From Ezh2 Null Mice." Blood 122, no. 21 (November 15, 2013): 1556. http://dx.doi.org/10.1182/blood.v122.21.1556.1556.
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Abstract Introduction We investigate the role of Ezh2 in neutrophil function using murine progenitor cells differentiated into neutrophils lacking the Ezh2 gene. Ezh2 is the catalytic component of the polycomb repressive complex 2, which methylates lysine 27 of histone H3. It is frequently disrupted in myelodysplastic syndromes (MDS) leading to loss of function (Ernst et al., 2010). Mutations in EZH2 are found in 6% of MDS patients and while not strongly linked to cytopenias or blast proportion, they are independently associated with worse overall survival compared to patients with wildtype EZH2 (Bejar R. et al., 2011 and 2012). We hypothesize that Ezh2 mutations may cause qualitative defects in myeloid cells that impact their function and could contribute to the adverse prognosis observed in EZH2 mutant MDS. Methods Bone marrow from Ezh2 null (Ezh2-/-) and littermate control mice (WT) were transduced with HOXB8 fused to the estrogen receptor ligand-binding domain to produce immortalized myeloid progenitor cells. Removal of estrogen from the media allows these cells differentiate into mature neutrophils (Wang G.G., 2006). Differentiated cells were characterized for surface markers by flow cytometry and for gene expression by PCR of mRNA. Spontaneous cell death was measured by annexin/PI staining. Cell cycle patterns were determined by measuring the red emission of PI. Chemotactic function was assessed by counting cells that migrated across a transwell in presence/absence of the attractant zymosan. For phagocytosis experiments, cells were incubated with Fluoresbrite YG carboxylate beads at 37°C or 4°C. Reactive oxygen species (ROS) generation was measured by the oxidation of dihydrorhodamine 123 into fluorescent rhodamine 123. Results Estrogen withdrawal caused differentiation of both WT and Ezh2-/- lines into cells with mature neutrophil morphology after six days (Figure 1a). Both differentiated lines expressed the neutrophil surface markers CD11b and CD62L and the neutrophil-specific genes lactoferrin and Itgb2l. Ezh2 -/- cells had an increased rate of spontaneous cell death compared to WT in undifferentiated (32.81% vs. 20.33%) and mature cells (32.82% vs. 14.23%). Nevertheless, both progenitor cell lines showed similar cell cycle patterns, demonstrating that Ezh2 absence had no other effect on cell cycle progression. Ezh2 -/- neutrophils failed to migrate towards zymosan (Figure 1b). Expression of Tlr2, which binds zymosan, and other Toll-like receptors (Tlr4/5/9) were similar between the differentiated cell lines. Cells incubated with FITC-zymosan at 37°C showed no fluorescence differences between cell lines, indicating similar adherence. Experiments with neutrophils from an MDS patient with homozygous EZH2 mutations demonstrated a similar migration defect. Additional studies in MDS patient samples are ongoing and will be presented. Phagocytosis was reduced in Ezh2-/-cells. Unstimulated, the number of cells ingesting and adhering YG-beads was significantly greater with WT cells than with Ezh2-/-cells. When activated with fMLP, both lines showed increased adherence of YG-beads but the number of phagocytosing Ezh2-/- cells was reduced. The average number of beads ingested by each cell was lower for Ezh2-/- cells compared to WT (5.95 vs 2.94, p < 0.001) in resting cells, and 9.47 vs. 3.73 in fMLP-activated cells, p < 0.01. The fraction of Ezh2-/- neutrophils generating ROS when stimulated with PMA is 2.4-fold higher than for WT cells. ROS production was greatly reduced in the presence of diphenyleneiodonium (DPI), confirming the role of NADPH oxidase in the generation of ROS. Conclusion Our results indicate impaired function of neutrophils derived from Ezh2-/- mice, demonstrating increased spontaneous cell death, impaired migration, decreased phagocytosis, and overproduction of ROS. Qualitative defects observed in neutrophils deficient for EZH2 may help explain the adverse prognosis associated with these mutations in MDS patients. Disclosures: Bejar: Genoptix: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity’s Board of Directors or advisory committees.
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Kosko, James R., Patricia B. Williams, and Michael F. Pratt. "Correlation of Neutrophil Activation and Skin Flap Survival in Pharmacologically Altered Pigs." Annals of Otology, Rhinology & Laryngology 106, no. 9 (September 1997): 790–94. http://dx.doi.org/10.1177/000348949710600916.
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In inflamed tissue, neutrophils produce tissue necrosis factors such as free oxygen radicals. We investigated the role of neutrophils in random flap survival using the tissue neutrophil marker myeloperoxidase (MPO), and in whole blood using flow cytometry with the neutrophil activation marker 2′7′ dichlorofluorescein diacetate. Hypopigmented pigs were treated with the experimental 21-aminosteroid lipid antioxidant U-74389G (oxygen free radical scavenger) before dorsal random skin flaps were elevated. Extent of flap survival was measured by surface planimetry 7 days after surgery. Mean flap survival was 64.1% ± 3.4% in the 3-mg/kg—treated group, and 68.0% ± 3.4% in the 1-mg/kg—treated group — both significantly greater than the survival in vehicle-treated controls (48.6% ± 2.3%). We measured MPO in tissue extracts using an enzyme-linked immunoassay, which showed less MPO in treated animals than in controls. Flow cytometry results were nonspecific. These data suggest that U-74389G improves random skin flap viability by inhibiting neutrophil infiltration into the flap.
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Almkvist, Jenny, Jenny Fäldt, Claes Dahlgren, Hakon Leffler, and Anna Karlsson. "Lipopolysaccharide-Induced Gelatinase Granule Mobilization Primes Neutrophils for Activation by Galectin-3 and Formylmethionyl-Leu-Phe." Infection and Immunity 69, no. 2 (February 1, 2001): 832–37. http://dx.doi.org/10.1128/iai.69.2.832-837.2001.
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ABSTRACT We have earlier shown that galectin-3, a lactose-binding mammalian lectin that is secreted from activated macrophages, basophils, and mast cells, induces activation of the NADPH oxidase in exudated but not in peripheral blood neutrophils (A. Karlsson, P. Follin, H. Leffler, and C. Dahlgren, Blood 91:3430–3438, 1998). The alteration in responsiveness occurring during extravasation correlated with mobilization of the gelatinase and/or specific granules to the cell surface, indicating a role for mobilizable galectin-3 receptors. In this study we have investigated galectin-3-induced NADPH oxidase activation, measured as superoxide production, in lipopolysaccharide (LPS)-primed neutrophils. Upon galectin-3 challenge, the LPS-primed cells produced superoxide, both extracellularly and intracellularly. A primed extracellular response to formylmethionyl-Leu-Phe (fMLF) was also achieved. The exposure of complement receptors 1 and 3 as well as the formyl peptide receptor on the cell surface was markedly increased after LPS treatment, indicating that granule fusion with the plasma membrane had occurred. Further assessment of specific markers for neutrophil granules showed that the LPS treatment had mobilized the gelatinase granules but only a minor fraction of the specific granules. We thus suggest that the mechanism behind LPS priming lies at the level of granule (receptor) mobilization for galectin-3 as well as for fMLF.
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Mahdy, Amr M., Damon A. Lowes, Helen F. Galley, Jane E. Bruce, and Nigel R. Webster. "Production of Soluble Triggering Receptor Expressed on Myeloid Cells by Lipopolysaccharide-Stimulated Human Neutrophils Involves De Novo Protein Synthesis." Clinical and Vaccine Immunology 13, no. 4 (April 2006): 492–95. http://dx.doi.org/10.1128/cvi.13.4.492-495.2006.
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ABSTRACT The triggering receptor expressed on myeloid cells (TREM-1) is a recently identified receptor expressed on neutrophils and monocytes. Activation of the receptor induces neutrophils to release the enzyme myeloperoxidase and inflammatory cytokines such as interleukin-8. TREM-1 has an alternatively spliced variant that lacks the transmembrane region, resulting in the receptor being secreted in a soluble form (sTREM-1). Soluble TREM-1 has been detected in plasma during experimental and clinical sepsis and has been advocated as a diagnostic marker of infection for pneumonia and as a prognostic marker for patients with septic shock. We studied TREM-1 surface expression, using flow cytometry, and simultaneously measured sTREM-1 concentrations in culture supernatants of lipopolysaccharide (LPS)-stimulated neutrophils. TREM-1 surface expression was constitutive and was not upregulated upon LPS stimulation. However, sTREM-1 release from neutrophils was significantly upregulated by LPS stimulation (P < 0.0001), an effect that was abrogated by cycloheximide. Soluble TREM-1 is therefore secreted by human neutrophils in response to LPS challenge in a process involving de novo protein synthesis that is not accompanied by an upregulation of the TREM-1 receptor on the surfaces of the cells.
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Kuckleburg, Christopher, Sarah Tilkens, Sentot Santoso, and Peter J. Newman. "Neutrophil Proteinase (PR3) Regulates Neutrophil Transendothelial Cell Migration." Blood 116, no. 21 (November 19, 2010): 1492. http://dx.doi.org/10.1182/blood.v116.21.1492.1492.
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Abstract Abstract 1492 Neutrophil transmigration requires the localization of neutrophils to endothelial cell junctions where receptor-ligand interactions between these cells promotes leukocyte diapedesis. Neutrophils contain several different proteases which are thought to play a role in aiding in transendothelial cell migration, either by degrading extracellular matrix components or junctional proteins, or by inducing endothelial cell activation. Proteinase 3 (PR3) is a serine protease stored in azurophil granules that is released by activated neutrophils and can rebind to the neutrophil expressed cell surface protein NB1 (CD177). The neutrophil marker NB1 is expressed on a subset of neutrophils (∼50%) and has recently been demonstrated to be a heterophilic binding partner for PECAM-1, a protein highly expressed at endothelial cell junctions. Disrupting NB1-PECAM interactions has been reported to significantly inhibit neutrophil transmigration. Because of the critical role of NB1 in neutrophil transmigration we believe that the interactions between NB1 and PECAM-1 have the potential to localize PR3 to endothelial cell junctions where it may aid in leukocyte transmigration. For this study we sought to test the hypothesis that NB1-PR3 interactions contribute to neutrophil transmigration. Human umbilical vein endothelial cells (HUVEC) were cultured on transwell membranes, treated with IL-1β, TNFα or fMLP and then incubated with NB1+ or NB1- PMN. Using flow cytometry we observed that transmigration alone resulted in a significant increase in PR3 expression on NB1+ but not NB1- neutrophils. Using a pan-serine protease inhibitor (AEBSF) total neutrophil transmigration was significantly inhibited. However, using a highly specific PR3 inhibitor (Elafin) we observed a selective inhibition in NB1+ but not NB1- neutrophil transmigration on IL-1β stimulated HUVEC. Similarly, antibodies against the NB1 recognition site on PECAM (Ig domain 6) inhibited neutrophil transmigration of NB1+ but not NB1- cells. Interestingly, in the presence of different stimuli (TNFα, fMLP), neutrophil transmigration was significantly less dependent on NB1-PECAM interactions and inhibition of PR3 activity did not inhibit transmigration. This is despite the fact that PR3 expression was highly up-regulated on NB1+ neutrophils incubated with either of these stimuli or following neutrophil transmigration. In conclusion, the serine protease PR3 appears to play a significant role in the transmigration of NB1+ but not NB1- neutrophils. Likewise, the contribution of NB1 and PR3 in neutrophil transmigration is regulated in a stimulus-dependent mechanism which involves NB1 interactions with PECAM. These data therefore suggest that NB1 and PR3 may regulate recruitment of a neutrophil subset in response to specific inflammatory signals and this regulation may play a role in modulating the immune response. Disclosures: No relevant conflicts of interest to declare.
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Heiple, J. M., and L. Ossowski. "Human neutrophil plasminogen activator is localized in specific granules and is translocated to the cell surface by exocytosis." Journal of Experimental Medicine 164, no. 3 (September 1, 1986): 826–40. http://dx.doi.org/10.1084/jem.164.3.826.
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The subcellular localization of plasminogen activator (PA) in human neutrophils was studied. The cells were disrupted by nitrogen cavitation and fractionated on Percoll density gradients into three major components containing the plasma membranes, the specific granules, and the azurophilic granules. The biochemical markers we used to identify these organelles were alkaline phosphatase, vitamin B12-binding protein, and beta-glucuronidase, respectively. Using the radioactive fibrin plate method, PA activity and plasminogen-independent fibrinolytic activity were measured. In resting neutrophils, PA was associated mainly with the membranes of the specific granules. In five individual experiments the activity of this fraction varied from 79 to 100% of the total; the remaining activity was found to be associated with the plasma membrane, and no activity was present in the azurophilic granules. In neutrophils that were activated by exposure to PMA (20 ng/ml for 15 min at 37 degrees C), the total recoverable PA activity remained unchanged; however, the main peak of activity (85% of total) shifted from the specific granules to the plasma membranes. The magnitude of the reduction of the enzyme in the specific granules paralleled that of vitamin B12-binding protein. PMA-activated, intact neutrophils had approximately 12-fold more surface-bound PA activity than resting cells. Recovery of PA activity from neutrophils was critically dependent on pretreatment of the intact cells with DFP before cavitation; 100-fold more PA activity was detected in DFP-pretreated cells. At the same time, this pretreatment reduced the plasminogen-independent fibrinolytic activity by approximately sevenfold. We determined that PA present in the neutrophils is of the urokinase (UK) type and that the enzyme is produced and stored as a pro-UK, a form insensitive to DFP inhibition. The reduction in the level of proteases (measured as fibrinolytic activity) and the resistance of pro-UK to DFP are most likely the two major reasons for the greatly improved recovery of PA from the DFP-pretreated cells. These findings show that in resting neutrophils PA is stored in the specific granules, and that during activation, it translocates to the outer surface of the plasma membranes, thus equipping the cell with an ecto-proteolytic potential.
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Jesaitis, A. J., G. M. Bokoch, J. O. Tolley, and R. A. Allen. "Lateral segregation of neutrophil chemotactic receptors into actin- and fodrin-rich plasma membrane microdomains depleted in guanyl nucleotide regulatory proteins." Journal of Cell Biology 107, no. 3 (September 1, 1988): 921–28. http://dx.doi.org/10.1083/jcb.107.3.921.
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Subcellular fractions were prepared from human neutrophils desensitized at 15 degrees C with stimulatory doses of the photoaffinity derivative F-Met-Leu-Phe-N epsilon-(2-(rho-azido[125I]salicylamido)ethyl-1,3'- dithio-propionyl)-Lys. The covalently labeled receptors were found in a membrane fraction of higher density than those from cells preexposed to ligand at 4 degrees C but not desensitized. The denser fraction (rho approximately equal to 1.155 g/cc) was the cellular locus of the membrane associated cytoskeletal proteins, actin, and fodrin, as detected immunologically on western blots. The light fraction (rho approximately equal to 1.135), cosedimented with neutrophil plasma membrane markers, plasma membrane guanyl nucleotide regulatory proteins, and several characteristic polypeptides identified by SDS-PAGE, including a major 72-kD species. The photoaffinity-labeled species in either case showed the same mobility on SDS-PAGE (Mr = 50,000-70,000) corresponding to previously reported values for N-formyl chemotactic receptors. These labeled receptors were sensitive to proteolysis after exposure of the intact photoaffinity-labeled cells to papain at 4 degrees C. We conclude that (a) the fractions isolated are probably derived from different lateral microdomains of the surface of human neutrophils; (b) the higher density fraction contains occupied N-formyl-chemotactic receptors previously shown to have been converted, to a high affinity, slowly dissociating form coisolating with neutrophil cytoskeleton and implicated in the termination of formyl peptide-induced neutrophil activation; and (c) the translocation of receptors to these microdomains may serve to compartmentalize receptors and perhaps regulate the interaction of the receptor/G-protein transduction pair.
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Cross, A., J. Hawkes, H. Frankland, A. Mediana, H. Wright, N. Goodson, S. Edwards, and R. Moots. "AB0115 SECUKINUMAB THERAPY DOES NOT AFFECT NEUTROPHIL HOST DEFENCE IN PSORIATIC ARTHRITIS." Annals of the Rheumatic Diseases 79, Suppl 1 (June 2020): 1357.1–1358. http://dx.doi.org/10.1136/annrheumdis-2020-eular.4477.
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Background:Biologic therapies have revolutionised therapy in inflammatory diseases such as psoriatic arthritis (PsA), driving major improvements in outcomes. Th17 cells appear to play a key role in the pathogenesis of PsA, and IL-17 can trigger the release of chemoattractants such as CXCL8 and CCL20, leading to the further infiltration of other immune cells including neutrophils. Infiltrating activated neutrophils can themselves generate a range of chemoattractants which may amplify and sustain the inflammatory response. Therapeutic targeting of IL-17 with biologics such as secukinumab offers great benefit in PsA by blocking this inflammatory cycle: however the interaction of this agent with neutrophils, key components of host defence as well as potential mediators of this disease, is not known.Objectives:This study aimed to measure key aspects of neutrophil function to determine: a) changes in the functions of circulating neutrophils in PsA patients pre-therapy, compared to age- and sex-matched healthy controls and b) if these functions changed in PsA patients 12-weeks post-secukinumab therapy.Methods:Neutrophils were isolated from venous blood of 16 PsA patients and 10 healthy controls. Key neutrophil functions were measured at baseline and 12 weeks: reactive oxygen species (ROS) production, apoptosis (+/- TNF and GM-CSF), phagocytosis, receptor expression and chemotaxis. Changes in gene expression pre- and 12-weeks post-therapy (n=5 PsA) were measured using RNAseq.Results:PsARC response was observed in 70.6% of participants on secukinumab therapy at 12 weeks. There were no significant differences in ROS production, phagocytosis or chemotaxis in PsA patients at baseline (compared to healthy controls) or during therapy. Chemotaxis towards IL-8 in PsA patients at baseline was decreased compared to that of healthy controls, but this difference did not reach statistical significance. Surface levels of activation markers CD11b/CD18 and CD63 were increased in PsA patients at 12-weeks compared to baseline, while surface levels of CD16 decreased. RNA-seq analysis indicated down-regulation of pathways mediated by IL-17A, oncostatin M, TWEAK (TNFSF12) and CCL2 during therapy, but up-regulated expression of pathways involvingde novoprotein biosynthesis.Conclusion:Therapy with secukimumab in PsA did not significantly affect neutrophil host defence functions. The changes that were seen in circulating neutrophils indicate selective up- and down-regulation of functions that may reflect potential alterations in local or systemic cytokines, and/or an increase in the circulating pool of activated neutrophils that are no longer recruited into sites of inflammation because of the down-regulation of the local IL-17/CXCL8 signalling network.Disclosure of Interests:Andrew Cross: None declared, Jennifer Hawkes: None declared, Helen Frankland: None declared, Ayren Mediana: None declared, Helen Wright Grant/research support from: Novartis supporting this study, Nicola Goodson Grant/research support from: Novartis supporting this research, Steven Edwards Grant/research support from: Novartis supporting this work, Robert Moots Grant/research support from: Novartis supporting this work, Consultant of: a variety of companies including Novartis, Speakers bureau: a variety of companies including Novartis
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Christin, Laurent, Deborah R. Wysong, Tova Meshulam, Ryan Hastey, Elizabeth R. Simons, and Richard D. Diamond. "Human Platelets Damage Aspergillus fumigatus Hyphae and May Supplement Killing by Neutrophils." Infection and Immunity 66, no. 3 (March 1, 1998): 1181–89. http://dx.doi.org/10.1128/iai.66.3.1181-1189.1998.
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ABSTRACT Neutropenia is considered a significant risk factor for invasive aspergillosis but is almost always associated with concurrent thrombocytopenia. Studies determined that platelets, like neutrophils, attached to cell walls of the invasive hyphal form of Aspergillus fumigatus. Organisms were damaged as shown by loss of cell wall integrity in scanning laser confocal microscopy and release of defined hyphal surface glycoproteins. Rapid expression appearance of surface antigen CD63 and release of markers of platelet degranulation confirmed activation during attachment to hyphae. Optimal platelet activation required opsonization of hyphae with fresh or heat-inactivated whole plasma. These effects of opsonization with whole plasma could not be duplicated by pooled human serum, immunoglobulin G, or fibrinogen, whether used separately or combined. Thus, platelets in the presence of whole plasma have the potential to play an important role in normal host defenses against invasive aspergillosis.
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Hutchison, Michael L., Ian R. Poxton, and John R. W. Govan. "Burkholderia cepacia Produces a Hemolysin That Is Capable of Inducing Apoptosis and Degranulation of Mammalian Phagocytes." Infection and Immunity 66, no. 5 (May 1, 1998): 2033–39. http://dx.doi.org/10.1128/iai.66.5.2033-2039.1998.
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ABSTRACT Burkholderia cepacia is an opportunistic pathogen that has become a major threat to individuals with cystic fibrosis (CF). In approximately 20% of patients, pulmonary colonization with B. cepacia leads to cepacia syndrome, a fatal fulminating pneumonia sometimes associated with septicemia. It has been reported that culture filtrates of clinically derived strains of B. cepacia are hemolytic. In this study, we have characterized a factor which contributes to this hemolytic activity and is secreted from B. cepacia J2315, a representative of the virulent and highly transmissible strain belonging to the recently described genomovar III grouping. Biochemical data from the described purification method for this hemolysin allows us to hypothesize that the toxin is a lipopeptide. As demonstrated for other lipopeptide toxins, the hemolysin from B. cepacia was surface active and lowered the surface tension of high-pressure liquid chromatography-grade water from 72.96 to 29.8 mN m−1. Similar to reports for other pore-forming cytotoxins, low concentrations of the hemolysin were able to induce nucleosomal degradation consistent with apoptosis in human neutrophils and the mouse-derived macrophage-type cell line J774.2. Exposure of human neutrophils to higher concentrations of toxin resulted in increased activities of the neutrophil degranulation markers cathepsin G and elastase. Based on the results obtained in this study, we suggest a role that allows B. cepacia to thwart the immune response and a model of the events that may contribute to the severe inflammatory response in the lungs of CF patients.
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Duzh, Еlena V., and Аndrei Y. Hancharou. "Functional properties of immune cells under the influence of extracts of mushrooms Ganoderma lucidum, Lentinula edodes, Boletus edulis." Journal of the Belarusian State University. Biology, no. 3 (December 31, 2020): 29–36. http://dx.doi.org/10.33581/2521-1722-2020-3-29-36.
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Тhe water extracts of Basidiomycota division mushrooms such as Boletus edulis, Ganoderma lucidum, Lentinula edodes posessed a wide spectrum of immunomodulatory activity. It was shown that Boletus edulis enhances the expression of surface markers on lymphoid cell lines Jurkat-tat and Daudi, and also increases the expression of reactive oxygen species by dendritic cells, monocytes and peripheral blood neutrophils, activates basophils. There was observed no effect of fungal extracts on NK-cells.
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Gourlay, Terence, Ioannis Samartzis, and Kenneth M. Taylor. "The effect of haemodilution on blood/biomaterial contact-mediated CD11b expression on neutrophils: ex vivo studies." Perfusion 18, no. 2 (March 2003): 87–93. http://dx.doi.org/10.1191/0267659103pf648oa.
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Modern cardiopulmonary bypass (CPB) systems are getting smaller, both in terms of the exposed surface area of biomaterials and the priming volume. In a series of studies utilizing a rat recirculation model, we demonstrated that the magnitude of the inflammatory response seen under these conditions is proportional to the surface area of exposed material, a finding that supports the use of miniature systems in terms of moderating the inflammatory response. However, the second impact of miniature perfusion systems, the reduced priming volume with concomitant reduction in haemodilution, was not investigated with reference to inflammation. The present study was designed to determine whether this change in CPB haematocrit profile has any effect on the inflammatory response. In common with previous studies by this group, we employed the expression of the integrin CD11b on neutrophils as a marker of neutrophil activation, and hence the inflammatory response, in a rat recirculation biomaterial testing model, containing di-(2-ethyl-hexyl)-phthalate plasticized polyvinyl chloride of the type commonly employed in CPB circuits. The results demonstrated that neutrophil activation is influenced by haemodilution. We studied five groups of animals, each with different mean induced haematocrit: Group 1 (41.39 /1.27%); Group 2 (30.939 /2.85%); Group 3(24.839 /1.36%); Group 4 (20.609 /3.47%); Group 5(20.489 /1.31%). Groups 1 and 5 animals were controls, neither of which underwent the period of recirculation. Rather, these controls were employed to isolate the noncontact effect of haemodilution on CD11b expression. We found that there were differences in per cent change in CD11b expression from start to end of the recirculation period between Group 1 (109.549 /49.53%), Group 2(189.19 /18.68%), Group 3 (224.289 /43.97), Group 4(368.979 /24.28%) and Group 5 (1279 /57.8%). There were intergroup statistically significant differences (p B /0.05). These results confirm that there is a relationship between haematocrit level and biomaterial contact-mediated activation of neutrophils. Furthermore, these studies confirm that haemodilution alone has no effect on neutrophil activation. One possible explanation for this outcome is that with higher levels of haemodilution, neutrophils have a greater opportunity to contact surface ‘receptor’ sites on the biomaterial, resulting in more neutrophil activation. Whatever the mechanism, these data tend to support the modern trend towards lower circuit surface area and higher haematocrit.
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Zinsly Sampaio Camargo, Thiago, Alexandre R. Marra, Nydia Strachman Bacal, Eduardo Casaroto, Lilian Moreira Pinto, Jacyr Pasternak, Elivane da Silva Victor, Oscar Fernando Pavão dos Santos, and Michael B. Edmond. "Evaluation of Two Methods for Determination of CD64 as a Diagnostic Marker of Infection in Critically Ill Adults." BioMed Research International 2016 (2016): 1–7. http://dx.doi.org/10.1155/2016/6593232.
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Objectives. Diagnostic markers of infection have had little innovation over the last few decades. CD64, a marker expressed on the surface of neutrophils, may have utility for this purpose. Methods. This study was conducted in an adult intensive care unit (ICU) in São Paulo, Brazil, with 89 patients. We evaluated CD64 in patients with documented or clinically diagnosed infection (infection group) and controls (patients without any evidence of infection) by two different methodologies: method #1, an in house assay, and method #2, the commercial kit Leuko64 (Trillium Diagnostics). Results. CD64 displayed good discriminating power with a 91.2% sensitivity (95% CI 90.7–91.6%) for detecting infection. The commercial kit (Leuko64) demonstrated higher specificity (87.3%) compared with method #1 as well as better accuracy (88.8%). Conclusions. CD64 seems to be a promising marker of infection in the intensive care setting, with Leuko64 showing a slight advantage.
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Kobayashi, T., J. M. Robinson, and H. Seguchi. "Identification of intracellular sites of superoxide production in stimulated neutrophils." Journal of Cell Science 111, no. 1 (January 1, 1998): 81–91. http://dx.doi.org/10.1242/jcs.111.1.81.
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In this study, we show that superoxide production is carried out within intracellular compartments of human neutrophils and not at the plasma membrane following stimulation with phorbol myristate acetate. Oxidant production was not observed in unstimulated cells. Stimulated cells exhibited superoxide production in two distinct types of intracellular organelles. Initially, activity was detected in slender rod-shaped granules and in spherical or elliptical granules. The oxidant-producing granules fused directly with the plasma membrane or fused to form larger intracellular vesicles which then became associated with the plasma membrane. Longer periods of stimulation with PMA resulted in a decrease in the number of vesicles containing oxidant reaction product only, and an increase in structures containing both the oxidant-reaction product and ferritin particles; the latter was used herein as a marker for endocytosis. Thus a complex pattern of intracellular vesicular trafficking occurs in stimulated neutrophils. Alkaline phosphatase activity, a marker enzyme for a type of intracellular neutrophil granule was co-localized in the oxidant reaction-positive intracellular compartments. The time course of up-regulation of alkaline phosphatase activity to the cell surface parallelled the release of superoxide from stimulated cells. Results from this study demonstrate for the first time cytochemical and morphological evidence that superoxide is released from stimulated neutrophils through exocytosis of an oxidant-producing intracellular granule.
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Walter, Patrick B., Annie Higa, Vivian Ng, Marcela G. Weyhmiller, Nick R. Slater, Patricia Evans, John B. Porter, et al. "Association Of Cardiac Iron By T2* With Innate Immune Markers In Transfusion-Dependent Thalassemia Patients Undergoing Combined Chelation Therapy." Blood 122, no. 21 (November 15, 2013): 3450. http://dx.doi.org/10.1182/blood.v122.21.3450.3450.
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Abstract Introduction The thalassemias are inherited anemias sometimes characterized by severe transfusion dependence that can lead to extra-hepatic cardiac iron overload, causing cardiomyopathy. Despite improved chelation therapies, patients with transfusion-dependent thalassemia still endure cardiomyopathy and chronic inflammation. The innate immune system provides the first line of defense against infection and specificity depends on pattern recognition receptors (PRRs) specific to microbial pathogens. One class of PRR called the toll-like receptors (TLRs) interacts with CD14 on innate immune cells transducing the signal for bacterial lipopolysaccharide. Another cell surface protein that is not a PRR, but aids phagocytosis and is important in granulocytes function and chemotaxis is the adhesive polysaccharide antigen, CD15. The role that excess iron plays in determining expression level of these innate immune proteins is unknown. Thus, the goal in these studies is to investigate the relationship of cardiac iron overload and its chelation to innate immune cell expression of TLR4 and CD15 in patients with transfusion-dependent thalassemia. Patients and Methods Eighteen patients with transfusion dependent thalassemia (11 – 29 years old) (participating in the Novartis sponsored CICL670AUS24T) were enrolled in a substudy investigating innate immunology (Novartis sponsored CICL670AUS42T). Patients were investigated at baseline, then after 6 months and one year of combined chelation therapy with deferasirox and deferoxamine. Fasting blood samples were obtained after a 72 hr washout with no chelators. Fourteen healthy controls (10 - 35 yrs old) were also enrolled. Changes in LIC (ferritometer), cardiac function (MRI) and myocardial iron (MRI T2*) were monitored. Peripheral blood mononuclear cells (PBMCs) and granulocytes were isolated from blood samples using density gradients. Monocytes and granulocytes were further purified using antibody-linked magnetic microbeads. Highly enriched populations of CD14+ monocytes and CD15+ granulocytes were verified by flow cytometry. The expression level of CD15 and TLR4 was determined. Results Previously we found that transfusion-dependent thalassemia patients had 37% higher TLR4+ neutrophils than control patients and a smaller percentage of CD15+ neutrophils. We have also observed a decrease in TLR4 expression during the course of combined chelation therapy on neutrophils but not monocytes, indicating that TLR4 is differentially modulated on neutrophils compared to monocytes. Now we find that these flow cytometry parameters show significant relationships to markers of iron burden. The percentage of TLR4+ monocytes was related to liver iron concentration (r=- 0.49, p = 0.039), ferritin concentration (r=-0.47, p = 0.049), serum iron level (r=0.61, p = 0.008), and total iron binding capacity (TIBC; r=0.51, p = 0.021), while the percentage of CD15 positive neutrophils predicted myocardial iron, as measured by MRI T2* (r= 0.69, p<0.001), and left ventricular ejection fraction (LVEF; r=0.50, p = 0.022). Lastly, analysis of covariance, controlling for age and gender, revealed that the number of CD15+ neutrophils increased significantly from baseline (90.84%) to 52 weeks (95.09%) of combined chelation therapy (p = 0.007). Conclusions This study found evidence that the innate immune system may be modulating iron trafficking not only to the liver but to the heart as well. The negative correlation between LIC and TLR4 expression suggests that severe iron overload may lead to heptocellular damage causing monocyte dysfunction, the pathology of which could be due to altered TLR4 expression. This relationship may also be driving the positive correlation observed between TLR4 expression and TIBC. CD15 seems to play an important role in cardiac health as it is positively correlated to LVEF and MRI T2*. This relationship is further validated by our previous finding that transfusion-dependent thalassemia patients had a smaller percentage of CD15+ neutrophils, which we now show improved during one year of combination chelation therapy. Taken together, this suggests that chelation therapy may enhance cardiac health by increasing the percentage of CD15+ neutrophils. Disclosures: Walter: Novartis: Research Funding. Porter:Novartis: Consultancy, Honoraria, Research Funding; Shire: Consultancy, Honoraria; Celgene: Consultancy. Vichinsky:Novartis: Honoraria, Research Funding.
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Van Dyke, T. E., M. Warbington, M. Gardner, and S. Offenbacher. "Neutrophil Surface Protein Markers as Indicators of Defective Chemotaxis in LJP." Journal of Periodontology 61, no. 3 (March 1990): 180–84. http://dx.doi.org/10.1902/jop.1990.61.3.180.
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Гребенчиков, О. А., И. С. Касаткина, А. Н. Кузовлев, А. В. Лобанов, and А. В. Ершов. "Influence of lithium chloride on neutrophil activation in the development of systemic inflammatory response syndrome in patients after on-pump cardiac surgery." ZHurnal «Patologicheskaia fiziologiia i eksperimental`naia terapiia», no. 4() (December 18, 2020): 47–53. http://dx.doi.org/10.25557/0031-2991.2020.04.47-53.
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Цель исследования - изучение in vitro действия хлорида лития на активность нейтрофилов человека при действии сывороток пациентов с синдромом системного воспалительного ответа, развившемся после операций на сердце с искусственным кровообращением. Методика. Исследование проводили in vitro на нейтрофилах, выделенных из крови 6 здоровых доноров. Нейтрофилы активировали при помощи сыворотки пациентов с синдромом системного воспалительного ответа (ССВО), перенесших операции на сердце с искусственным кровообращением (ИК). Активность нейтрофилов оценивали с использованием флуоресцентных антител к маркерам дегрануляции CD11b и CD66b. Уровень апоптоза и некроза нейтрофилов оценивали через 22 ч после выделения из крови здоровых доноров; количественная оценка была проведена с использованием аннексина V и иодистого пропидия на проточном цитофлуориметре. Интактные и активированные нейтрофилы обрабатывали раствором хлорида лития в концентрациях 0,3; 3,0 и 9,0 мМ. Результаты. Инкубация нейтрофилов с сывороткой крови пациентов с ССВО после операций на сердце с ИК увеличивала экспрессию CD11b в 1,5 раза и экспрессию CD66b в 1,4 раза в сравнении с экспрессией на интактных нейтрофилах. Инкубация нейтрофилов с сывороткой крови пациентов с ССВО и раствором хлорида лития в концентрациях 3,0 и 9,0 мМ приводило к статистически значимому снижению уровня экспрессии CD11b CD66b на поверхности нейтрофилов в сравнении с активированными контрольными. Установлено, что хлорид лития в концентрациях 3,0 и 9,0 мМ возвращал уровни экспрессии CD11b и CD66b на активированных нейтрофилах к уровню экспрессии на интактных нейтрофилах. В концентрации 0,3 мМ хлорид лития, используемый при инкубации с активированными нейтрофилами, не вызывал значимого снижения экспрессии CD11b и CD66b относительно контрольных активированных нейтрофилов. Экспрессия CD11b и CD66b на активированных нейтрофилах при их инкубации с хлоридом лития в концентрации 0,3 мМ была значимо выше относительно экспрессии данных молекул на интактных нейтрофилах. Сыворотка пациентов с развившемся ССВО снижала спонтанный апоптоз нейтрофилов, а раствор хлорида лития в концентрации 3,0 или 9,0 мМ, добавленный в среду инкубации, увеличивал способность нейтрофилов к спонтанному апоптозу. Заключение. Хлорид лития оказывал противовоспалительный эффект снижал дегрануляцию и активацию нйтрофилов посредством уменьшения уровня экспрессии молекул CD11b и CD66b на поверхности нейтрофилов, которые предварительно были активированы сыворотками пациентов с ССВО. В концентрации 3,0 мМ и выше хлорид лития индуцировал спонтанный апоптоз нейтрофилов, активированных сыворотками пациентов с ССВО после операций на сердце с ИК. The aim of this work was to study the anti-inflammatory effect of lithium chloride on human neutrophils in vitro under the action of the serum of patients with systemic inflammatory response syndrome (SIRS), which developed after on-pump cardiac surgery. Methods. The study was performed on neutrophils isolated from the blood of five healthy donors, which was activated using serum from patients with SIRS. Neutrophil activity was assessed using fluorescent antibodies to CD11b and CD66b degranulation markers. The level of apoptosis and necrosis of human neutrophils was evaluated 22 hours after isolation. Quantification was performed using annexin V and propidium iodide on a flow cytometer. Intact and activated neutrophils were treated with 0.3, 3.0 аnd 9.0 mM lithium chlorides. Results. Incubation of neutrophils with the blood serum of patients with SIRS after on-pump cardiac surgery increased the expression of CD11b by 1.5 times and the expression of CD66b by 1.4 times compared to expression on intact neutrophils. Incubation of neutrophils with blood serum of patients with SIRS and 3.0 and 9.0 mM lithium chloride solutions led to a statistically significant decrease in the level of expression of CD11b CD66b on the surface of neutrophils in comparison with control activated neutrophils. It was found that 3.0 and 9.0 mM lithium chloride solutions returned the expression levels of CD11b and CD66b on activated neutrophils to the expression level on intact neutrophils. 0.3 mM of lithium chloride, used during incubation with activated neutrophils, did not cause a significant decrease in the expression of CD11b and CD66b relative to control activated neutrophils. The expression of CD11b and CD66b on activated neutrophils during their incubation with 0.3 mM of lithium chloride was significantly higher relative to the expression of these molecules on intact neutrophils. The serum of patients with advanced SIRS decreased the ability of neutrophils to spontaneous apoptosis. 3.0 or 9.0 mM lithium chloride solutions added to the incubation medium increased the ability of neutrophils to spontaneous apoptosis. Conclusion. Lithium chloride reduced the degranulation and activation of neutrophils by reducing the expression level of CD11b and CD66b molecules on the surface of neutrophils that were previously activated by the serum of patients with SIRS. This effect determines the anti-inflammatory influence of lithium chloride. Lithium chloride at 3.0 mM and higher induced spontaneous apoptosis of neutrophils activated by the serum of patients with SIRS after on-pump cardiac surgery.
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Homburg, CH, M. de Haas, AE von dem Borne, AJ Verhoeven, CP Reutelingsperger, and D. Roos. "Human neutrophils lose their surface Fc gamma RIII and acquire Annexin V binding sites during apoptosis in vitro." Blood 85, no. 2 (January 15, 1995): 532–40. http://dx.doi.org/10.1182/blood.v85.2.532.532.
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Abstract We have previously reported that neutrophilic granulocytes rapidly release part of their Fc gamma RIII from the plasma membrane upon in vitro activation, probably by proteolytic cleavage. In plasma and other body fluids, released or soluble Fc gamma RIII has been found in considerable amounts. In the present study, neutrophils were kept in maintenance culture for 18 to 24 hours. Forty percent of the neutrophils completely lost Fc gamma RIII, and the remainder of the cells showed a 60% decrease in Fc gamma RIII expression on their surface. Released Fc gamma RIII was detected in the culture supernatant. Nevertheless, more than 90% of the cells was viable as judged by hydrolysis of fluorescein diacetate. The presence of interferon gamma, granulocyte colony-stimulating factor, or granulocyte- macrophage colony-stimulating factor, but not interleukin-3 (IL-3), IL- 6, or IL-8, in the culture medium increased the number of cells that still expressed Fc gamma RIII. We found that this loss of Fc gamma RIII was not the result of cell activation but correlated strongly with apoptosis. The Fc gamma RIII-negative subpopulation exhibited typical morphologic changes, such as nuclear condensation and DNA fragmentation. Furthermore, this subpopulation appeared to have acquired the property of binding Annexin V, a calcium-dependent, phospholipid-binding protein with high affinity for phosphatidylserine. The external exposure of this phospholipid by cells has been reported to occur during apoptosis. The property of Annexin V binding was not shared by the nonapoptotic, Fc gamma RIII-positive subpopulation. In this respect, we identified binding of Annexin V as an convenient marker for apoptotic cells. Our results indicate that soluble Fc gamma RIII in body fluids might be derived for a large part from neutrophils undergoing apoptosis in the tissues.