Heparin induces neutrophil elastase-dependent vital and lytic NET formation

2019 ◽  
Vol 32 (5) ◽  
pp. 359-368 ◽  
Author(s):  
Patrick M Lelliott ◽  
Masatoshi Momota ◽  
Takayuki Shibahara ◽  
Michelle S J Lee ◽  
Nicholas I Smith ◽  
...  
Keyword(s):  
Neutrophil Elastase ◽  
Biological Effects ◽  
Extracellular Dna ◽  
Low Molecular Weight ◽  
Oxygen Species ◽  
Heparin Induced Thrombocytopenia ◽  
Extracellular Traps ◽  
Dna Strands ◽  
Life Threatening ◽  
Dose Dependent

Abstract Heparin is used extensively as an anticoagulant in a broad range of diseases and procedures; however, its biological effects are not limited to coagulation and remain incompletely understood. Heparin usage can lead to the life-threatening complication known as heparin-induced thrombocytopenia (HIT), caused by the development of antibodies against heparin/PF4 complexes. Here, we demonstrate the ability of heparin to induce neutrophil extracellular traps (NETs). NETs occurred with cell lysis and death, but live neutrophils releasing extracellular DNA strands, known as vital NETs, also occurred abundantly. Formation of NETs was time and dose dependent, and required reactive oxygen species and neutrophil elastase. Other compounds related to heparin such as low molecular weight heparin, fondaparinux and heparan sulfate either failed to induce NETs, or did so to a much lesser extent. Our findings suggest the ability of heparin to directly induce NET formation should be considered in the context of heparin treatment and HIT pathogenesis.

scholarly journals Significance of Mast Cell Formed Extracellular Traps in Microbial Defense

2021 ◽  
Author(s):  
Daniel Elieh Ali Komi ◽  
Wolfgang M. Kuebler
Keyword(s):  
State Of The Art ◽  
Extracellular Dna ◽  
Cellular Mechanisms ◽  
Extracellular Traps ◽  
Synthetic Chemicals ◽  
Dna Strands ◽  
Associated Proteins ◽  
Microbial Defense ◽  
And Function ◽  
Insight Into

AbstractMast cells (MCs) are critically involved in microbial defense by releasing antimicrobial peptides (such as cathelicidin LL-37 and defensins) and phagocytosis of microbes. In past years, it has become evident that in addition MCs may eliminate invading pathogens by ejection of web-like structures of DNA strands embedded with proteins known together as extracellular traps (ETs). Upon stimulation of resting MCs with various microorganisms, their products (including superantigens and toxins), or synthetic chemicals, MCs become activated and enter into a multistage process that includes disintegration of the nuclear membrane, release of chromatin into the cytoplasm, adhesion of cytoplasmic granules on the emerging DNA web, and ejection of the complex into the extracellular space. This so-called ETosis is often associated with cell death of the producing MC, and the type of stimulus potentially determines the ratio of surviving vs. killed MCs. Comparison of different microorganisms with specific elimination characteristics such as S pyogenes (eliminated by MCs only through extracellular mechanisms), S aureus (removed by phagocytosis), fungi, and parasites has revealed important aspects of MC extracellular trap (MCET) biology. Molecular studies identified that the formation of MCET depends on NADPH oxidase-generated reactive oxygen species (ROS). In this review, we summarize the present state-of-the-art on the biological relevance of MCETosis, and its underlying molecular and cellular mechanisms. We also provide an overview over the techniques used to study the structure and function of MCETs, including electron microscopy and fluorescence microscopy using specific monoclonal antibodies (mAbs) to detect MCET-associated proteins such as tryptase and histones, and cell-impermeant DNA dyes for labeling of extracellular DNA. Comparing the type and biofunction of further MCET decorating proteins with ETs produced by other immune cells may help provide a better insight into MCET biology in the pathogenesis of autoimmune and inflammatory disorders as well as microbial defense.

scholarly journals Neutrophil elastase and myeloperoxidase regulate the formation of neutrophil extracellular traps

2010 ◽  
Vol 191 (3) ◽  
pp. 677-691 ◽  
Author(s):  
Venizelos Papayannopoulos ◽  
Kathleen D. Metzler ◽  
Abdul Hakkim ◽  
Arturo Zychlinsky
Keyword(s):  
Immune Deficiency ◽  
Neutrophil Elastase ◽  
Serine Proteases ◽  
Neutrophil Extracellular Traps ◽  
Oxygen Species ◽  
Chromatin Decondensation ◽  
Unknown Mechanism ◽  
Extracellular Traps ◽  
Decondensed Chromatin ◽  
Chromatin Density

Neutrophils release decondensed chromatin termed neutrophil extracellular traps (NETs) to trap and kill pathogens extracellularly. Reactive oxygen species are required to initiate NET formation but the downstream molecular mechanism is unknown. We show that upon activation, neutrophil elastase (NE) escapes from azurophilic granules and translocates to the nucleus, where it partially degrades specific histones, promoting chromatin decondensation. Subsequently, myeloperoxidase synergizes with NE in driving chromatin decondensation independent of its enzymatic activity. Accordingly, NE knockout mice do not form NETs in a pulmonary model of Klebsiella pneumoniae infection, which suggests that this defect may contribute to the immune deficiency of these mice. This mechanism provides for a novel function for serine proteases and highly charged granular proteins in the regulation of chromatin density, and reveals that the oxidative burst induces a selective release of granular proteins into the cytoplasm through an unknown mechanism.

scholarly journals Bullous Hemorrhagic Dermatosis Induced By Heparin: An Indonesian Case Report

2021 ◽  
pp. 8-12
Author(s):  
Syahfori Widiyani ◽  
Irsalina Rahmawati ◽  
W. Yohannes Widodo ◽  
Dian Zamroni ◽  
Fajar L. Gultom ◽  
...  
Keyword(s):  
Skin Lesions ◽  
Low Molecular Weight ◽  
Heparin Induced Thrombocytopenia ◽  
Clinical Disorder ◽  
Heparin Treatment ◽  
Life Threatening ◽  
Topical Treatments ◽  
Lesion Regression ◽  
The Right ◽  
Related Reaction

Introduction: Bullous haemorrhagic dermatosis is a rare clinical disorder which is usually related to a treatment with unfractionated heparin (UFH) or low molecular weight heparin (LMWH), characterized by multiple intra-epidermal haemorrhages distant from the site of injection. Presentation of Case: A 62-year-old male patient with coronary heart disease who received heparin treatment experienced several tense, haemorrhagic bullae located on the right arm area, close to the injection site, and followed by the formation of several hematomas on his back trunk 2 days after he had received UFH. The lesions regressed after discontinuation of heparin and supportive topical treatments. Discussion: The lesions in this patient have similar characteristic with heparin-induced skin necrosis and demonstrate thrombocytopenia probably related to heparin. There are some proposed hypotheses of pathophysiology which include hypersensitivity reaction and idiosyncratic dose-related reaction. Given the clinically course, the discontinuation of heparin treatment was essential for lesion regression in addition other supportive measures. Conclusion: Heparin-induced skin lesions may indicate the presence of life-threatening heparin-induced thrombocytopenia. An early diagnosis is crucial to enable discontinuation of heparin if required.

Neutrophil Extracellular Traps (NETs) in Patients with Congenital Neutropenia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 15-15
Author(s):  
Christine Happle ◽  
Manuela Germeshausen ◽  
Cornelia Zeidler ◽  
Karl Welte ◽  
Julia Skokowa
Keyword(s):  
Peripheral Blood ◽  
Neutrophil Elastase ◽  
Myeloid Cells ◽  
Neutrophil Extracellular Traps ◽  
Extracellular Dna ◽  
Lower Amount ◽  
Congenital Neutropenia ◽  
Aberrant Expression ◽  
Extracellular Traps ◽  
Activated Neutrophils

Abstract Abstract 15 Activated neutrophils kill microbes by phagocytosis and extracellular mechanisms, including neutrophil extracellular traps (NETs), which are composed of decondensed chromatin and granular proteins such as neutrophil elastase (NE) and cathepsin G. Enzymatic generation of reactive oxygen species (ROS) by NADPH oxidase and the release of serine proteases such as NE have been shown to be essential factors for NET formation. Patients with chronic granulmatous disease (CGD), who lack a normal generation of ROS, show a defective NET formation. Since myeloid cells of patients with severe congenital neutropenia (CN) show an aberrant expression pattern of granular proteases such as neutrophil elastase (NE) or myeloperoxidase, we aimed to analyse NET formation in activated neutrophils of these patients. CN is a heterogeneous hematological disorder, characterized by peripheral blood neutrophil counts below 0,2×109/l and a maturation arrest of myelopoiesis at the promyelocytic/myelocytic stage. 60% of CN patients harbor autosomal dominant mutations within the ELA2 gene encoding for NE, but also mutations in other genes (e.g. HAX1, G6PC3, WAS, GFI1, p14) have been found to be disease-causing. Previously, we described severely diminished levels of NE in myeloid cells of CN patients. Here, we aimed to explore NET formation in neutrophils of CN patients. Moreover, we intended to analyze the effects of a reduced ELA2 expression and gene mutations as seen in CN patients on NET formation in vitro. Granulocytes of CN patients undergoing G-CSF therapy were extracted by density centrifugation, stimulated with phorbol myristate acetate (PMA, 50 nM, up to 240 min) and then tested for NET formation. NETs were stained with an extracellular DNA dye or DAPI. Our analyses showed normal NET formation in peripheral blood granulocytes of two patients with HAX1-related neutropenia, whereas there was a significantly lower amount of NETs in two patients with ELA2 mutations. One further patient out of three CN patients with unknown mutations showed a reduced amount of NETs in bone marrow PMNs. To further evaluate the possible effect of downregulated ELA2 expression on NET formation, we transduced primary human CD34+ cells with a lentiviral-based shRNA construct downregulating the expression of NE. Subsequently, these cells were differentiated into granulocytes with a cytokine cocktail containing G-CSF and tested for their ability to form NETs. We found an almost completely abolished NET formation in cells transduced with ELA2 shRNA as compared to control cells. Hitherto, CGD is the only immunodeficiency with a clearly defective NET formation. Our results point to an impaired formation of NETs also in CN patients carrying ELA2 mutations. This supports the recent finding of a central role for NE in NET formation. Two patients with HAX1 related CN showed a normal ability to form NETs. Our further work will aim to better define the subgroup of CN patients defective in NET formation. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The mercurial nature of neutrophils: still an enigma in ARDS?

2014 ◽  
Vol 306 (3) ◽  
pp. L217-L230 ◽  
Author(s):  
Andrew E. Williams ◽  
Rachel C. Chambers
Keyword(s):  
Clinical Evidence ◽  
Distress Syndrome ◽  
Host Cells ◽  
Oxygen Species ◽  
Pathogenic Microorganisms ◽  
Extracellular Traps ◽  
Pulmonary Cells ◽  
Life Threatening ◽  
Lung Condition ◽  
Excessive Accumulation

The acute respiratory distress syndrome (ARDS) is a life-threatening lung condition resulting from direct and indirect insults to the lung. It is characterized by disruption of the endothelial-epithelial barrier, alveolar damage, pulmonary edema, and respiratory failure. A key feature of ARDS is the accumulation of neutrophils in the lung microvasculature, interstitium, and alveolar space. Despite a clear association between neutrophil influx into the lung and disease severity, there is some debate as to whether neutrophils directly contribute to disease pathogenesis. The primary function of neutrophils is to provide immediate host defense against pathogenic microorganisms. Neutrophils release numerous antimicrobial factors such as reactive oxygen species, proteinases, and neutrophil extracellular traps. However, these factors are also toxic to host cells and can result in bystander tissue damage. The excessive accumulation of neutrophils in ARDS may therefore contribute to disease progression. Central to neutrophil recruitment is the release of chemokines, including the archetypal neutrophil chemoattractant IL-8, from resident pulmonary cells. However, the chemokine network in the inflamed lung is complex and may involve several other chemokines, including CXCL10, CCL2, and CCL7. This review will therefore focus on the experimental and clinical evidence supporting neutrophils as key players in ARDS and the chemokines involved in recruiting them into the lung.

Neutrophilic Extracellular Traps (NETs); A Subset of Smudge Cells Identifiable by Peripheral Smear Autoanalyzers in the Rising Era of Artificial Intelligence

2020 ◽  
Vol 154 (Supplement_1) ◽  
pp. S10-S11
Author(s):  
Mohammad Barouqa ◽  
Kenji Ikemura ◽  
Henny Billett ◽  
Margarita Kushnir ◽  
Kateryna Fedorov ◽  
...  
Keyword(s):  
Artificial Intelligence ◽  
Flow Cytometry ◽  
Neutrophil Elastase ◽  
Morphological Changes ◽  
Extracellular Dna ◽  
Control Group ◽  
Microbial Infection ◽  
Sample Collection ◽  
Extracellular Traps ◽  
Morphologic Characteristics

Abstract Background Digitized microscopy such as CellaVision® technology has revolutionized the laboratory. Smudge cells, also called basket cells, are usually seen in lymphoproliferative disorders representing remnants from degenerated lymphocytes (DLs). CellaVision® classifies DLs and web-like remnants as smudge cells. The morphology of the web-like remnants is compatible with Neutrophil Extracellular Traps (NETs) where extracellular decondensed DNA chromatin network is formed as one of several neutrophilic reactions to stress. Currently, we lack clinical tests that reliably identify and quantify NETs. Aims To develop an in-vitro model for NETs formation in blood, create a library of their morphological changes at different maturation stages; correlate their presence to infections in absence of leukocytosis and develop an artificial intelligence platform (AI-Heme-1) for their detection. Methods A library was built to develop AI-Heme-1 where NETs were induced with classic triggers (phorbol-myristate-acetate, lipopolysaccharide and ionomycin) in EDTA whole blood from normal subjects. Smears were prepared at 30 minutes intervals for 24 hours to identify NETs by Immunofluorescence and immunohistochemistry. WBC differentials were performed by CellaVision® to capture different stages of NETs. AI-Heme-1 was modified from Python online convolutional neural network. For the clinical correlation, smears with >20% smudge cells were classified morphologically as NETs vs. DLs compared to a control group, < 5% smudge cells. We used morphologic characteristics, immunohistochemistry, immunofluorescence and flow cytometry to differentiate NETs from DLs. Medical chart review performed by blinded investigators, included patient demographics, CBC and presence of microbial infection occurring < 1 week of sample collection. Statistical analyses included two sided t-test and chi square. Results The classical triggers for Netosis showed consistent morphological changes following a canonical order: vacuolation, nuclear decondensation, degranulation and chromatin ejection. These cell remnants were positive for citrullinated histones, myeloperoxidase, leukocyte alkaline phosphatase and neutrophil elastase by immunofluorescence. On Wright Giemsa stain, web-like remnants resembling NETs stained for SytoxGreen. On flow cytometry, NETs were large with extracellular DNA and MPO. For the clinical study group of >20% smudge cells, 88 were morphologically designated as NETs, 8 as DL vs. 59 as control group. A random sampling from >20% smudge cells showed cases with NET subclassification stained strongly with myeloperoxidase, neutrophil elastase and SytoxGreen while DLs were negative. Comparing patients with >20% smudge and NET sub-classification to <5% smudge cells, the formers had higher incidence of bacterial and viral infections (p=0.009/0.005 and p=0.008/0.007). Conclusions Our study was able to identify NETs on peripheral smears performed by a routine Hematology Autoanalyzer using a reliable set of morphologic characteristics, immunohistochemical stains and flow cytometry. It supports data that associate NETs with infections in the absence of leukocytosis. AI-Heme-1 was able to identify NETs on blood smears. This approach can provide a rapid, early and accurate tool to screen patients with infections.

scholarly journals Anti-inflammatory activity of soluble chito-oligosaccharides (CHOS) on VitD3-induced human THP-1 monocytes

PLoS ONE ◽  
2021 ◽  
Vol 16 (2) ◽  
pp. e0246381
Author(s):  
Paiboon Jitprasertwong ◽  
Munthipha Khamphio ◽  
Phornsiri Petsrichuang ◽  
Vincent G. H. Eijsink ◽  
Wanangkan Poolsri ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Vitamin D3 ◽  
Biological Effects ◽  
Water Soluble ◽  
Inflammatory Activity ◽  
Low Molecular Weight ◽  
Dependent Manner ◽  
Human Immune ◽  
Anti Inflammatory ◽  
Dose Dependent

Chito-oligosaccharides (CHOS) are oligomers of D-glucosamine and N-acetyl-glucosamine. Anti-inflammatory activities of a wide variety of CHOS mixtures have previously been reported, mainly based on studies with mouse models and murine macrophages. Since the mouse and human immune systems are quite different, gaining insight into the activity of CHOS on human cell lines, using well-characterized CHOS mixtures, is of considerable interest. Bacillus subtilis chitosanase (BsCsn46A) can efficiently convert chitosan to mixtures of water soluble low molecular weight CHOS. Here, the anti-inflammatory activity of a properly characterized CHOS mixture was studied, using human THP-1 cells that were differentiated to mature monocytes using vitamin D3. Addition of CHOS reduced the production of multiple pro-inflammatory cytokines associated with bacterial lipopolyssacharide (LPS)-stimulated inflammation, in a dose-dependent manner and without affecting cell viability. Interestingly, only minimal effects of CHOS were observed in similar experiments with phorbol 12-myristate 13-acetate- (PMA-) differentiated, macrophage-like, THP-1 cells. Altogether, in addition to showing promising biological effects of well-characterized low molecular weight soluble CHOS in a human system, the present study also points at Vitamin D3-stimulated THP-1 cells as a favorable system for assessing the anti-inflammatory activity of bioactive compounds.

scholarly journals Clinical Appearance and Diagnosis of Heparin Induced Thrombocytopenia

2021 ◽  
pp. 466-471
Author(s):  
Gunduz T ◽  
Cakir M ◽  
Bakirci EM ◽  
DEGIRMENCI H
Keyword(s):  
Molecular Weight ◽  
Unfractionated Heparin ◽  
Low Molecular Weight Heparin ◽  
Low Molecular Weight ◽  
Heparin Induced Thrombocytopenia ◽  
Clinical Appearance ◽  
Treatment Management ◽  
Life Threatening ◽  
Life Threatening Complication

Heparin-İnduced Thrombocytopenia (HIT) is a life-threatening complication that occurs in a small percentage of exposed patients (e.g. unfractionated heparin, Low Molecular Weight Heparin [LMWH]) regardless of dose and treatment management.

Abstract 481: Downregulation of MicroRNA-143 Induces Proximal Aorta Dilatation in C57BL6/J Mice

2014 ◽  
Vol 34 (suppl_1) ◽  
Author(s):  
Emanuela Branchetti ◽  
Bartosz Rylski ◽  
Paolo Poggio ◽  
Eric K Lai ◽  
Robert C Gorman ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Aortic Wall ◽  
Wall Structure ◽  
Oxygen Species ◽  
Phenotypic Switch ◽  
Down Regulation ◽  
Threatening Condition ◽  
Life Threatening ◽  
Aorta Dilatation ◽  
Dose Dependent

Background: Thoracic Aortic Aneurysm (TAA) is a silent life-threatening condition which leads to death if not surgically treated. TAA development is secondary to the remodeling of the aortic wall structure due to pathological changes in both vascular smooth muscle cells (VSMC) and extracellular components. The phenotypic switch of VSMC from a contractile to a pathological synthetic state by inducing excessive production of matrix components is associated with the aberrant remodeling of the aorta. MicroRNA (miR)-143 and -145 are critical in controlling VSMC phenotype and are highly expressed in the aorta. In investigating the molecular mechanisms controlling VSMC phenotype we found that accumulation of reactive oxygen species (ROS) linearly correlates with synthetic VSMC accumulation as well as dilatation of the ascending aorta in humans. Furthermore ROS contribute to the onset of TAA in a murine model based on Angiotensin II (AngII) infusion. Interestingly we found that miR-143 is down regulated in patients with aneurysmal aorta. We hypothesized that miR-143 may be regulated by ROS and its down regulation could contribute to TAA development. Methods: MiR expression, MMP-2, MMP-9, and VSMC markers of phenotype (Vimentin, Elk-1, Myocardin) were tested in human isolated aortic VSMC treated with ROS. C57BL6/J mice were subjected to AngII for 28 days in the presence or absence of LNA-anti-miR-143. Two-dimensional M Mode echocardiography was performed to measure aortic diameter. Results: ROS treatment of human VSMC induces a dose dependent down regulation of miR-143 and -145 together with up regulation of MMP-2, MMP-9 and VSMC synthetic markers Vimentin and Elk-1. Both miR-143/145 are down regulated in the aneurysmal proximal aorta of C57BL6/J mice treated with AngII. Injection of LNA-anti-miR-143 alone induces dilatation of the proximal aorta but has no effect on AngII induced TAA development. Conclusion: ROS induced down regulation of miR-143 is involved in the pathological remodeling of the proximal aorta leading to TAA. Understanding the mechanism behind ROS-mediated regulation of miR-143 may unveil new targets for the development of therapeutic strategies against TAA.

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