Extracellular Vesicles of Neutrophils

한국조명·전기설비학회 2018 한국조명·전기설비학회 학술대회논문집 Vol. No.

<P>Extracellular vesicles (EVs) are membrane-derived vesicles that mediate intercellular communications. As professional phagocytes, neutrophils also produce EVs in response to various inflammatory stimuli during inflammatory processes. Neutrophil-derived EVs can be categorized into 2 subtypes according to the mechanism of generation. Neutrophil-derived trails (NDTRs) are generated from migrating neutrophils. The uropods of neutrophils are elongated by adhesion to endothelial cells, and small parts of the uropods are detached, leaving submicrometer-sized NDTRs. Neutrophil-derived microvesicles (NDMVs) are generated from neutrophils which arrived at the inflammatory foci. Membrane blebbing occurs in response to various stimuli at the inflammatory foci, and small parts of the blebs are detached from the neutrophils, leaving NDMVs. These 2 subtypes of neutrophil-derived EVs share common features such as membrane components, receptors, and ligands. However, there are substantial differences between these 2 neutrophil-derived EVs. NDTRs exert pro-inflammatory functions by guiding subsequent immune cells through the inflammatory foci. On the other hand, NDMVs exert anti-inflammatory functions by limiting the excessive immune responses of nearby cells. This review outlines the current understanding of the different subtypes of neutrophil-derived EVs and provides insights into the clinical relevance of neutrophil-derived EVs.</P>

Extracellular Vesicles of Neutrophils

홍장원 대한면역학회 2018 Immune Network Vol.18 No.6

Extracellular vesicles (EVs) are membrane-derived vesicles that mediate intercellular communications. As professional phagocytes, neutrophils also produce EVs in response to various inflammatory stimuli during inflammatory processes. Neutrophil-derived EVs can be categorized into 2 subtypes according to the mechanism of generation. Neutrophil-derived trails (NDTRs) are generated from migrating neutrophils. The uropods of neutrophils are elongated by adhesion to endothelial cells, and small parts of the uropods are detached, leaving submicrometer-sized NDTRs. Neutrophil-derived microvesicles (NDMVs) are generated from neutrophils which arrived at the inflammatory foci. Membrane blebbing occurs in response to various stimuli at the inflammatory foci, and small parts of the blebs are detached from the neutrophils, leaving NDMVs. These 2 subtypes of neutrophil-derived EVs share common features such as membrane components, receptors, and ligands. However, there are substantial differences between these 2 neutrophil-derived EVs. NDTRs exert pro-inflammatory functions by guiding subsequent immune cells through the inflammatory foci. On the other hand, NDMVs exert anti-inflammatory functions by limiting the excessive immune responses of nearby cells. This review outlines the current understanding of the different subtypes of neutrophil-derived EVs and provides insights into the clinical relevance of neutrophil-derived EVs.

Current Understanding in Neutrophil Differentiation and Heterogeneity

한국조명·전기설비학회 2017 한국조명·전기설비학회 학술대회논문집 Vol. No.

<P>Neutrophils are professional phagocytes that conduct effectors functions in the innate immune systems. They are differentiated in the bone marrow (BM) and terminally differentiated neutrophils are then released into systemic circulation. Neutrophils migrate into inflammatory foci through extravasation, reverse transmigration, and chemotaxis. As neutrophils arrive at a target site, they actively participate in eliminating pathogens. They phagocytose bacteria, and eliminate them through the generation of reactive oxygen species (ROS), release of protease-enriched granules, and formation of neutrophil extracellular traps (NETs). Since neutrophils are equipped with toxic arsenals, the activation of neutrophils is tightly controlled. Priming is the process of unlocking safety mechanisms before complete activation of neutrophils. Since the first discovery of neutrophils, they were considered as a homogeneous population with an inflammatory phenotype. However, heterogenous populations of neutrophils were discovered under physiological and pathological conditions. This review outlines the normal differentiation of neutrophils in the BM, and discusses the current understandings of neutrophil heterogeneity.</P>

Neutrophil Recruitment in Arterial Thrombus and Characteristics of Stroke Patients with Neutrophil-Rich Thrombus

차명진,하지민,이형우,권일,김성은,김영대,남효석,이혜선,송태진,최현정,허지회 연세대학교의과대학 2022 Yonsei medical journal Vol.63 No.11

Purpose: Neutrophils contribute to thrombosis. However, there is limited information on the temporal course of neutrophil re cruitment in thrombosis, the contribution of neutrophils to thrombus growth, and the characteristics of stroke patients with neu trophil-rich thrombi. Materials and Methods: After inducing carotid artery thrombosis in Institute of Cancer Research mice using ferric chloride, aged thrombi were produced by ligating the distal portion of the carotid artery in mice for 0.5, 1, 2, 3, 6, or 24 h. For thrombus analysis in stroke patients, we used registry data and thrombi that were obtained during intra-arterial thrombectomy. Immunohisto chemistry was performed to determine thrombus composition. Results: In the thrombi of 70 mice, Ly6G positive cell counts (neutrophils) and histone H3-positive cell counts increased in a time-dependent manner (both p<0.001). Ly6G-positive cell count was strongly correlated with histone H3-positive cell counts (r=0.910, p<0.001), but not with thrombus size (p=0.320). In 75 stroke patients, atrial fibrillation and cardioembolism were more fre quent in the higher neutrophil group (32/37, 86.5%) than in the lower neutrophil group (19/38, 50%) (p=0.002). The median erythro cyte fraction was higher [52.0 (interquartile range 39.9−57.8)] in the higher neutrophil group than in the lower neutrophil group [40.3 (interquartile range 23.5−53.2)]. The fraction of neutrophils was positively correlated with that of erythrocytes (R=0.35, p=0.002). Conclusion: Neutrophils were recruited and increased in arterial thrombosis in a time-dependent manner; however, they were not associated with the growth of formed thrombi. Neutrophil fractions in the thrombi of stroke patients appeared to be associat ed with atrial fibrillation and erythrocyte fraction.

Current Understanding in Neutrophil Differentiation and Heterogeneity

홍장원 대한면역학회 2017 Immune Network Vol.17 No.5

Neutrophils are professional phagocytes that conduct effectors functions in the innate immune systems. They are differentiated in the bone marrow (BM) and terminally differentiated neutrophils are then released into systemic circulation. Neutrophils migrate into inflammatory foci through extravasation, reverse transmigration, and chemotaxis. As neutrophils arrive at a target site, they actively participate in eliminating pathogens. They phagocytose bacteria, and eliminate them through the generation of reactive oxygen species (ROS), release of protease-enriched granules, and formation of neutrophil extracellular traps (NETs). Since neutrophils are equipped with toxic arsenals, the activation of neutrophils is tightly controlled. Priming is the process of unlocking safety mechanisms before complete activation of neutrophils. Since the first discovery of neutrophils, they were considered as a homogeneous population with an inflammatory phenotype. However, heterogenous populations of neutrophils were discovered under physiological and pathological conditions. This review outlines the normal differentiation of neutrophils in the BM, and discusses the current understandings of neutrophil heterogeneity.

Human Neutrophil의 수명연장과 Superoxide 생산에 관여하는 미지의 Monocyte 생성물질

허억,배진우 大韓免疫學會 1993 大韓免疫學會誌 Vol.15 No.-

It has long been known that neutrophils are quickly infilterated and recruited to infected sites and then kill invaders by phagocytic action. Unfortunatly it is not yet revealed which molecule or cytokine is involved in the phagocytic action and viability sustaining activity of neutrophils. The aim of this study was whether lipopolysaccharide (LPS)-stimulated monocyte may control those neutrophil actions. Human peripheral blood monocytes and neutrophils were isolated by Ficollpaque density sedimentation from heparin anti-coagulated blood of healthy adult donors. After preparation of these cells, the purity of both was more than 90%. Monocytes stimulated in various dose(0.1-10pug/ml) of LPS for various times of incubation(0-3 days). and then LPS-stimulated monocyte conditioned medium was collected in order to find an optimal dose and incubation time for the neutrophil viability. It was found out that 3,ug/ml of LPS in 24 hours incubation was maximal effective condition for the activity of neutrophil sustaining viability. Monocyte conditioned medium (MCM) under this condition was used for the comparison with LPS-nonstimulated monocyte conditioned medium or enriched medium alone. When neutrophils were stimulated with each medium for 1-3 days, the activity of neutrophil sustaining viability with MCM was significantly higher than the activity with other medium (in 1 day of culture, 72-1:8 vs 4311:7 vs 17 ±10; p <O. 01). The superoxide production of neutrophil stimulated with MCM for 24 hours incubation was significantly higher than that with other medium under fMLP doses of 0.1-100,uM (p <0.01). Under fMLP l,uM, the superoxide production is predominantly different between them(23.8±2 vs 10.3±3 vs 7.8±1.6). The maximal effective dose of GM-CSF(granulocyte/macrophage colony stimulating factor ; 10pM) enhanced the neutrophil viability in 1 day of culture (50 ± 4%) . In the study to assess whether MCM contains GM-CSF, anti-GM-CSF antibody slightly blocked the MCM-dependent neutrophil viability(73±9 vs 50±12; p <0.07), indicating that MCM might not contain GM-CSF. These data indicate that LPS-stimulated monocyte surely product a factor for the neutrophil sustaining viability and the enhancement of superoxide production, suggesting that a factor is not GMCSF. If more than one factor were producted form LPS-stimulated monocyte, one minor factor might be GM-CSF.

Anti-inflammatory Effects of Quercetin and Vitexin on Activated Human Peripheral Blood Neutrophils - The effects of quercetin and vitexin on human neutrophils -

Nikfarjam, Bahareh Abd,Hajiali, Farid,Adineh, Mohtaram,Nassiri-Asl, Marjan KOREAN PHARMACOPUNCTURE INSTITUTE 2017 Journal of pharmacopuncture Vol.20 No.2

Objectives: Polymorphonuclear neutrophils (PMNs) constitute the first line of defense against invading microbial pathogens. Early events in inflammation involve the recruitment of neutrophils to the site of injury or damage where changes in intracellular calcium can cause the activation of pro-inflammatory mediators from neutrophils including superoxide generation, degranulation and release of myeloperoxidase (MPO), productions of interleukin (IL)-8 and tumor necrosis factor ${\alpha}$ ($TNF-{\alpha}$), and adhesion to the vascular endothelium. To address the anti-inflammatory role of flavonoids, in the present study, we investigated the effects of the flavonoids quercetin and vitexin on the stimulus-induced nitric oxide (NO), $TNF-{\alpha}$, and MPO productions in human neutrophils. Methods: Human peripheral blood neutrophils were isolated, and their viabilities were determined by using the Trypan Blue exclusion test. The polymorphonuclear leukocyte (PMNL) preparations contained more than 98% neutrophils as determined by morphological examination with Giemsa staining. The viabilities of cultured neutrophils with various concentrations of quercetin and vitexin ($1-100{\mu}M$) were studied using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays. Neutrophils were cultured in complete Roswell Park Memorial Institute (RPMI) medium, pre-incubated with or without quercetin and vitexin ($25{\mu}M$) for 45 min, and stimulated with phorbol 12-myristate 13-acetate (PMA) ($10^{-7}M$). NO production was carried out through nitrite determination by using the Griess method. Also, the $TNF-{\alpha}$ and the MPO productions were measured using enzyme-linked immunosorbent assay (ELISA) kits and MPO assay kits. Results: Neutrophil viability was not affected up to a concentration of $100{\mu}M$ of quercetin or vitexin. Both quercetin and vitexin significantly inhibited $TNF-{\alpha}$, NO, and MPO productions in human neutrophils (P < 0.001). Conclusion:The present study showed that both quercetin and vitexin had significant anti-inflammatory effects. Thus, treatment with either quercetin or vitexin may be considered as a therapeutic strategy for treating patients with neutrophil-mediated inflammatory diseases.

Treatment with Rutin - A Therapeutic Strategy for Neutrophil-Mediated Inflammatory and Autoimmune Diseases - Anti-inflammatory Effects of Rutin on Neutrophils -

Nikfarjam, Bahareh Abd,Adineh, Mohtaram,Hajiali, Farid,Nassiri-Asl, Marjan KOREAN PHARMACOPUNCTURE INSTITUTE 2017 Journal of pharmacopuncture Vol.20 No.1

Objectives: Neutrophils represent the front line of human defense against infections. Immediately after stimulation, neutrophilic enzymes are activated and produce toxic mediators such as pro-inflammatory cytokines, nitric oxide (NO) and myeloperoxidase (MPO). These mediators can be toxic not only to infectious agents but also to host tissues. Because flavonoids exhibit antioxidant and anti-inflammatory effects, they are subjects of interest for pharmacological modulation of inflammation. In the present study, the effects of rutin on stimulus-induced NO and tumor necrosis factor $(TNF)-{\alpha}$ productions and MPO activity in human neutrophils were investigated. Methods: Human peripheral blood neutrophils were isolated using Ficoll-Hypaque density gradient centrifugation coupled with dextran T500 sedimentation. The cell preparations containing > 98% granulocytes were determined by morphological examination through Giemsa staining. Neutrophils were cultured in complete Roswell Park Memorial Institute (RPMI) medium, pre-incubated with or without rutin ($25{\mu}M$) for 45 minutes, and stimulated with phorbol 12-myristate 13-acetate (PMA). Then, the $TNF-{\alpha}$, NO and MPO productions were analyzed using enzyme-linked immunosorbent assay (ELISA), Griess Reagent, and MPO assay kits, respectively. Also, the viability of human neutrophils was assessed using tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), and neutrophils were treated with various concentrations of rutin ($1-100{\mu}M$), after which MTT was appended and incubated at $37^{\circ}C$ for 4 hour. Results: Rutin at concentrations up to $100{\mu}M$ did not affect neutrophil viability during the 4-hour incubation period. Rutin significantly decreased the NO and $TNF-{\alpha}$ productions in human peripheral blood neutrophils compared to PMA-control cells (P < 0.001). Also, MPO activity was significantly reduced by rutin (P < 0.001). Conclusion: In this in vitro study, rutin had an anti-inflammatory effect due to its inhibiting NO and $TNF-{\alpha}$ productions, as well as MPO activity, in activated human neutrophils. Treatment with rutin may be considered as a therapeutic strategy for neutrophil-mediated inflammatory/autoimmune diseases.

Autophagy in neutrophils

Sanjeeb Shrestha,Jae Man Lee,Chang-Won Hong 대한생리학회-대한약리학회 2020 The Korean Journal of Physiology & Pharmacology Vol.24 No.1

Autophagy is a highly conserved intracellular degradation and energyrecycling mechanism that contributes to the maintenance of cellular homeostasis. Extensive researches over the past decades have defined the role of autophagy innate immune cells. In this review, we describe the current state of knowledge regarding the role of autophagy in neutrophil biology and a picture of molecular mechanism underlying autophagy in neutrophils. Neutrophils are professional phagocytes that comprise the first line of defense against pathogen. Autophagy machineries are highly conserved in neutrophils. Autophagy is not only involved in generalized function of neutrophils such as differentiation in bone marrow but also plays crucial role effector functions of neutrophils such as granule formation, degranulation, neutrophil extracellular traps release, cytokine production, bactericidal activity and controlling inflammation. This review outlines the current understanding of autophagy in neutrophils and provides insight towards identification of novel therapeutics targeting autophagy in neutrophils.

Roles of White Blood Cells and Subtypes as Inflammatory Markers in Skin Cancer

Baykan, Halit,Cihan, Yasemin Benderli,Ozyurt, Kemal Asian Pacific Journal of Cancer Prevention 2015 Asian Pacific journal of cancer prevention Vol.16 No.6

Objective: Skin tumors are the most commonly seen cancer type worldwide. Regarding pathogenesis, it is thought that disruption of kinetics through T lymphocyte-mediated development of chronic inflammation may be involved. The present study was intended to identify role of inflammatory cells such as neutrophils, monocytes and lymphocytes in the determination of risk for skin cancer. Materials and Methods: We retrospectively reviewed charts of 569 cases diagnosed as having primary skin tumors. Data regarding age, gender and histopathological subtype were recorded. Blood parameters studied on the day before surgery including WBCs, neutrophils, and lymphocyte counts, neutrophil:lymphocyte and neutrophil:monocyte ratios were also recorded. Two-hundred and two healthy individuals presented for check-up in an outpatient clinic were selected as the control group. Parameters studied in cases with skin cancer were compared to those healthy individuals. Findings: Of the cases with skin cancer, 401 were basal cell carcinoma (BCC) while 144 were squamous cell carcinoma (SCC) and 13 were malignant melanoma (MM). WBC, neutrophil and monocyte counts and the neutrophil:lymphocyte ratio were found to be lower in the patient group than in the healthy control group (p<0.001) while no significant difference was found in other parameters reviewed (p>0.05). No significant difference was found in WBC, neutrophil, neutrophil: monocyte ratio according to gender (p>0.05). Monocyte count was found to be $0.68{\pm}0.61$ in men and $0.55{\pm}0.25$ in women, indicating strong statistical significance (p<0.001). WBC, neutrophil and monocyte values were highest in control group while lowest in BCC. When BCC and SCC groups were compared to controls, significant differences found (p<0.001). There were no significant differences in lymphocyte counts among groups (p=0.976). Neutrophil:lymphocyte ratios were 3.24 in BCC, 3.59 in SCC, 3.44 in MM and 5.06 in control group (p<0.001). Conclusions: In our study, it was found that there were significant differences in complete blood count, neutrophil, monocyte and neutrophil:lymphocyte levels among groups. Neutrophil: lymphocyte ratio was found to be lowest in BCC among skin cancers.