Direct Activation Of Neutrophils Research Articles

Immune Thrombosis: Exploring the Significance of Immune Complexes and NETosis.

Neutrophil extracellular traps (NETs) are major contributors to inflammation and autoimmunity, playing a key role in the development of thrombotic disorders. NETs, composed of DNA, histones, and numerous other proteins serve as scaffolds for thrombus formation and promote platelet activation, coagulation, and endothelial dysfunction. Accumulating evidence indicates that NETs mediate thrombosis in autoimmune diseases, viral and bacterial infections, cancer, and cardiovascular disease. This article reviews the role and mechanisms of immune complexes in NETs formation and their contribution to the generation of a prothrombotic state. Immune complexes are formed by interactions between antigens and antibodies and can induce NETosis by the direct activation of neutrophils via Fc receptors, via platelet activation, and through endothelial inflammation. We discuss the mechanisms by which NETs induced by immune complexes contribute to immune thrombotic processes and consider the potential development of therapeutic strategies. Targeting immune complexes and NETosis hold promise for mitigating thrombotic events and reducing the burden of immune thrombosis.

Differential activation of human neutrophils by SARS-CoV-2 variants of concern.

The emerging SARS-CoV-2 virus has affected the entire world with over 600 million confirmed cases and 6.5 million deaths as of September 2022. Since the beginning of the pandemic, several variants of SARS-CoV-2 have emerged, with different infectivity and virulence. Several studies suggest an important role of neutrophils in SARS-Cov-2 infection severity, but data about direct activation of neutrophils by the virus is scarce. Here, we studied the in vitro activation of human neutrophils by SARS-CoV-2 variants of concern (VOCs). In our work, we show that upon stimulation with SARS-Cov-2 infectious particles, human healthy resting neutrophils upregulate activation markers, degranulate IL-8, produce Reactive Oxygen Species and release Neutrophil Extracellular Traps. Neutrophil activation was dependent on TLR7/8 and IRF3/STING. We then compared the activation potential of neutrophils by SARS-CoV-2 variants and showed a significantly increased activation by the Delta variant and a decreased activation by the Omicron variant as compared to the initial strain. In this study, we demonstrate that the SARS-Cov-2 virus can directly activate neutrophils in COVID-19 and that the different VOCs had differences in neutrophil activation intensity that mirror the differences of clinical severity. These data highlight the need to address neutrophil-virus interactions as a potential target for therapeutic intervention in SARS-CoV-2 infection.

Anti-HNA-3a Alloantibodies Can Cause Differential Endothelial Damage through a Direct Neutrophil Activation Pathway

INTRODUCTIONThe human neutrophil antigen (HNA) 3a/b is associated with a single amino acid substitution (R 154Q polymorphism) on the first extracellular loop of choline transporter-like protein 2 (CTL2). Antibodies against HNA-3a have been associated with severe and fatal transfusion-related acute lung injury (TRALI). Epitope mapping suggests the existence of two types of HNA-3a antibodies. Type I antibodies only require first extracellular loop of the CTL2 for expression of the antigen, type II antibodies require at least the first 3 extracellular loops of CTL2 in a native configuration (i.e. expressed on a cell membrane). This study aimed to evaluate the activity of type I and type II anti-HNA-3a antibodies in an in vitro TRALI model.STUDY DESIGN & METHODS: The granulocyte agglutination test (GAT) and granulocyte immunofluorescence test (GIFT) were used to confirm anti-HNA-3a activity in two donor sera (Q49 and Q50). Flow cytometry was used to confirm antibody binding to freshly isolated human or mouse neutrophils. A rabbit polyclonal antibody against an epitope in the third extracellular loop of CTL2's was coated onto the wells of an ELISA plate and neutrophil HNA-3a antigen was captured and sera tested. For the TRALI model, human lung microvascular endothelial cells (HLMVECs) were grown to confluence before being treated with lipopolysaccharide (LPS). Freshly isolated neutrophils from HNA-3aa homozygote donors were then added with 10% Q49 or Q50. Microscopic counting of Trypan blue staining was used to measure rate HLMVEC death. Significance was determined using a one-way ANOVA (p<0.05), followed by Dunnett's post-hoc test. RESULTS AND DISCUSSIONGAT and GIFT confirmed that both Q49 and Q50 contained an anti-HNA-3a antibody, and that neither contained antibodies against any other HNA or HLA molecules. In flow cytometry, both Q49 and Q50 bound to human neutrophils, but only Q49 bound to mouse neutrophils. In the ELISA, signal was detected only for Q49. These data provided evidence that Q49 was a type I antibody and Q50 was a type II antibody. Within the TRALI model, HLVMEC death was observed with both Q49 and Q50 only in the presence of LPS and neutrophils (Table 1). Previously reported HLMVEC permeability in the absence of neutrophils (Bayat et al. Arterioscler Thromb Vasc Biol. 2013) was not observed as in this study the outcome measured was HLMVEC death. In the present study, the effect for Q49 (type I) was significantly larger than for Q50 (type II; Table 1; p-value <0.0001). CONCLUSIONSIn this model, LPS and neutrophils were required for anti-HNA-3a mediated HLMVEC death thought to reflect events involved in the initiation of TRALI. The observation that type I antibodies elicited a stronger response provides a direction for further research. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

NMDA receptor modulation of glutamate release in activated neutrophils.

BackgroundNeutrophil depletion improves neurologic outcomes in experimental sepsis/brain injury. We hypothesized that neutrophils may exacerbate neuronal injury through the release of neurotoxic quantities of the neurotransmitter glutamate.MethodsReal-time glutamate release by primary human neutrophils was determined using enzymatic biosensors. Bacterial and direct protein-kinase C (Phorbol 12-myristate 13-acetate; PMA) activation of neutrophils in human whole blood, isolated neutrophils or human cell lines were compared in the presence/absence of N-Methyl-d-aspartic acid receptor (NMDAR) antagonists. Bacterial and direct activation of neutrophils from wild-type and transgenic murine neutrophils deficient in NMDAR-scaffolding proteins were compared using flow cytometry (phagocytosis, reactive oxygen species (ROS) generation) and real-time respirometry (oxygen consumption).FindingsBoth glutamate and the NMDAR co-agonist d-serine are rapidly released by neutrophils in response to bacterial and PMA-induced activation. Pharmacological NMDAR blockade reduced both the autocrine release of glutamate, d-serine and the respiratory burst by activated primary human neutrophils. A highly specific small-molecule inhibitor ZL006 that limits NMDAR-mediated neuronal injury also reduced ROS by activated neutrophils in a murine model of peritonitis, via uncoupling of the NMDAR GluN2B subunit from its' scaffolding protein, postsynaptic density protein-95 (PSD-95). Genetic ablation of PSD-95 reduced ROS production by activated murine neutrophils. Pharmacological blockade of the NMDAR GluN2B subunit reduced primary human neutrophil activation induced by Pseudomonas fluorescens, a glutamate-secreting Gram-negative bacillus closely related to pathogens that cause hospital-acquired infections.InterpretationThese data suggest that release of glutamate by activated neutrophils augments ROS production in an autocrine manner via actions on NMDAR expressed by these cells.FundGLA: Academy Medical Sciences/Health Foundation Clinician Scientist. AVG is a Wellcome Trust Senior Research Fellow.

Choline Transporter-Like Protein-2: New von Willebrand Factor-Binding Partner Involved in Antibody-Mediated Neutrophil Activation and Transfusion-Related Acute Lung Injury.

In contrast to other antibodies involved in transfusion-related acute lung injury, anti-HNA-3a antibodies are incapable of inducing direct neutrophil activation and seem to interact with endothelial cells (ECs) primarily. In animal studies, anti-HNA-3a-mediated transfusion-related acute lung injury could be precipitated in the absence of neutrophils, but was stronger when neutrophils were present. In a different context the target protein of these antibodies, choline transporter-like protein-2 (CTL-2), was reported to interact with a protein of the inner ear carrying 2 von Willebrand factor (VWF) A-domains. These observations prompted us to investigate whether VWF might be involved in anti-HNA-3a-mediated neutrophil activation, and whether signaling via CD11b/CD18 is involved, as in various other experimental settings. Cell adhesion demonstrated specific binding of CTL-2 to VWF. Immunoprecipitation analysis of CTL-2/CD11b/CD18 coexpressing cells indicated that anti-HNA-3a colocalizes CTL-2 and CD11b/CD18 when VWF is present. Functional studies revealed that anti-HNA-3a-mediated neutrophil agglutination is an active, protein kinase C-dependent and partially Fc-dependent process. Agglutination and the production of reactive oxygen species seem to require the formation of a trimolecular complex between the target antigen (CTL-2), CD11b/CD18 and VWF. In line with these observations, anti-HNA-3a induced less severe transfusion-related acute lung injury and less neutrophil recruitment to the alveolar space in VWF knockout mice. We introduce CTL-2 as a new binding partner for VWF. Interaction of neutrophils with VWF via CTL-2 allows anti-HNA-3a to induce signal transduction via CD11b/CD18, which leads to neutrophil activation and agglutination. In transfusion-related acute lung injury, this mechanism may further aggravate endothelial leakage.

Poly(vinyl alcohol)-coated silver nanoparticles: Activation of neutrophils and nanotoxicology effects in human hepatocarcinoma and mononuclear cells

Silver nanoparticles (AgNps) have been described as important for their excellent biocompatibility, biomedical applications. Nevertheless, AgNps can interact with the immune system which is essential to analyze human exposure to assess their potential risk to health and environment. In general, the primary site for accumulation of nanoparticles has been demonstrated to be the liver. Furthermore, the direct activation of neutrophils or oxidative burst by a given nanoparticle is poorly documented. In this paper, we investigated the cell uptake, apoptosis, necrosis, DNA damage in human hepatocarcinoma cells (HepG2), primary normal human peripheral blood mononuclear cells (PBMC) and the direct activation of primary isolated neutrophils through the oxidative burst on exposure to AgNps coated with Polyvinyl-alcohol (PVA). All cell types were incubated in the presence of 1.0 and 50.0μM of AgNps-PVA for 24h. A significant cyto- and genotoxic-response and the activation of human neutrophils were induced by AgNps-PVA (p<0.05). Our results revealed that AgNps can interact with the normal isolated neutrophils, PBMC and HepG2 cells in vitro, which opens the way for further studies on the toxicological effects of AgNps in the human immune system response and cancer cells.

Emerging Roles for Platelets in Inflammation and Disease

Platelets and their interaction with cells of the immune system contribute through a variety of molecular mechanisms to support hemostasis and inflammation. These simple yet essential cells exert their effects in lymphocytes, monocytes, and neutrophils, both recruiting and modulating their function after activation. Emerging evidence is starting to define the mechanisms that allow platelets to also play pivotal roles in host defense. For example, platelet cell-surface expression of toll-like receptors allows platelets to direct neutrophil activation toward extracellular trap formation and facilitate the elimination of blood pathogens. In addition to these well-known receptors, two of the most recently discovered platelet receptors, C-type lectin receptor 2 (CLEC-2), and TREM-like transcript-1 (TLT-1), have been shown to modulate hemostatic and inflammation-related roles in platelets. This review will discuss the evolution of our understanding of platelet functions from hemostasis to inflammation, and highlight novel mechanisms that platelets use to mediate hemostasis under inflammatory pressure.

Activation of Neutrophils by Nanoparticles

The use of nanoparticles (NPs) has increased in the past few years in various fields, including defence, aerospace, electronics, biology, medicine, and so forth. and in applications such as diagnostic technology, bioimaging, and drug/gene delivery. Thus, human exposure to NPs and nanomaterials is unavoidable and will certainly expand in the future resulting in a growing interest in nanotoxicology, the study of toxicity of nanomaterials. A number of studies have reported the effects of NPs in respect to pulmonary inflammation by investigating in vitro activation of pulmonary cells with NPs and in vivo in a variety of models in which neutrophils appear to be the predominant leukocyte cell type in lungs and in bronchoalveolar lavages following inhalation or intratracheal instillation of NPs. Despite the fact that several studies have reported an increased number of neutrophils, the literature dealing with the direct activation of neutrophils by a given NP is poorly documented. This paper will summarize the current literature in this latter area of research and will end with a perspective view in which our laboratory will be involved in the following years.

Brucella abortus activates human neutrophils

Human brucellosis is caused by infection with certain species of the genus Brucella and is characterized by bacterial persistence and inflammation of many host tissues. Neutrophils are one of the predominant cell types present in the infiltrate of these inflamed tissues, and due to their potential effect on the inflammatory response and tissue damage, direct activation of neutrophils by Brucella abortus might contribute to the pathology associated with human brucellosis. B. abortus expresses outer membrane lipoproteins (Omp) with inflammatory properties on a variety of cell types. This study examines the effect of B. abortus and its lipoproteins on neutrophil functions. B. abortus induced an increment in CD35 and CD11b expression and a decline in CD62L accompanied by IL-8 secretion, a response compatible with neutrophil activation. B. abortus lipoprotein Omp19 (L-Omp19), but not its unlipidated form, mimicked the changes associated with neutrophil activation induced by B. abortus. L-Omp19 primed neutrophils for oxidative burst as well as promoted neutrophil migration and prolonged neutrophil survival. Thus, Brucella lipoproteins possess pro-inflammatory properties that could contribute to the localize tissue injury and inflammation by direct activation of neutrophils. Data presented here, together with our previous results implicate Brucella lipoproteins in the pathogenesis of human brucellosis.

Selective and direct activation of human neutrophils but not eosinophils by Toll-like receptor 8

Granulocytes represent the largest fraction of immune cells in peripheral blood and are directly exposed to circulating Toll-like receptor (TLR) ligands. Although highly relevant for TLR-based therapies, because of the technical challenge, activation of the granulocyte subsets of neutrophils and eosinophils by TLR ligands is less well studied than activation of other immune cell subsets. The aim of this work was to study direct versus indirect neutrophil and eosinophil activation by TLR7 and TLR8 ligands. We used a new whole-blood assay, single cell-based cytokine detection, and highly purified primary human neutrophils and eosinophils to separate direct and indirect effects on these blood cell subsets. We found indirect but not direct activation of neutrophils but not eosinophils in whole blood by using unmodified immunostimulatory RNA (isRNA; TLR7/8 ligand). In contrast, direct activation and stimulation of the respiratory burst and degranulation was seen with nuclease-stable isRNA and with the small-molecule TLR8 agonist 3M002 but not 3M001 (TLR7). Neutrophils expressed TLR8 but none of the other 2 RNA-detecting TLRs (TLR3 and TLR7). Together, these results demonstrate that neutrophils are directly and fully activated through TLR8 but not TLR7. Furthermore, the results predict that the clinical utility of small-molecule TLR8 ligands or nuclease-stable RNA ligands for TLR8 might be limited because of neutrophil-mediated toxicity and that no such limitation applies for unmodified isRNA, which is known to induce desired T(H)1 activities in other immune cell subsets.

Acute inflammatory response induced by Helicobacter pylori in the rat air pouch

Infection by Helicobacter pylori elicits persistent neutrophil infiltration in the gastric mucosa and stimulates the release of substances that may contribute to the establishment of gastritis. In this study, we used the rat air pouch model to evaluate the acute inflammatory response to H. pylori, in vivo. A pronounced neutrophil infiltration was observed 6 h and 12 h after the injection of H. pylori into the air pouch. Strains with different genotypes were able to induce cellular influx. This response was dependent upon the amount of bacteria injected and still occurred when heat-killed bacteria were employed. An increase in prostaglandin E 2 levels was observed, indicating that H. pylori induced cyclooxygenase 2 in this model. The production of interleukin-1β and tumor necrosis factor-α by leukocytes was also enhanced, suggesting that this model may be useful for studying the direct activation of neutrophils by H. pylori in vivo.

Meconium aspiration injury: Uncoupling between the in vivo physiologic and in vitro inflammatory responses.

OBJECTIVE: To correlate the in vivo physiologic changes that occur with meconium aspiration injury to an associated in vitro cellular response to meconium. DESIGN: Experimental, prospective, randomized, controlled study. SETTING: University research laboratory. SUBJECTS: Eighteen adult Sprague-Dawley rats with meconium aspiration injury. INTERVENTIONS: Rats were given 3 mL/kg of a 25% meconium solution and were treated with conventional gas ventilation; nine rats were given exogenous surfactant therapy (Survanta, 4 mL/kg), and nine rats were not treated (control). Bronchoalveolar lavages were collected for total cell counts. Histologic samples also were taken for analysis. In addition, the in vitro effect of meconium on granulocytic elastase release from human neutrophils was determined. MEASUREMENTS AND MAIN RESULTS: Meconium caused significant morbidity in vivo, including poor oxygenation, elevated Paco(2), diminished compliance, and elevated white cell count in the bronchial lavages. Lung white cell count was significantly less in the surfactant group (p <.01). Meconium did not cause elastase release from human neutrophils in vitro. CONCLUSIONS: This study demonstrated uncoupling between in vivo physiologic responses to meconium injury in rats and the in vitro effect of meconium on human neutrophils. Surfactant therapy alleviated some of the perturbations associated with meconium injury, including a reduction in the inflammatory cell count in lung lavages. The absence of direct neutrophil activation by meconium suggests the requirement of an intermediary in the pathogenesis of meconium aspiration injury.

Borrelia burgdorferi outer surface protein A (OspA) activates and primes human neutrophils.

Lyme disease is caused by infection with the spirochete Borrelia burgdorferi and is characterized by bacterial persistence and inflammation of many host tissues. B. burgdorferi express outer surface lipoproteins, including OspA, with inflammatory properties that could contribute to the localized tissue inflammation. Neutrophils are the predominant infiltrate into the inflamed arthritic joints, and are crucial for controlling the spirochete infection. They may also contribute to the joint pathology associated with Lyme arthritis. This study examines the effect of OspA on the activities of the neutrophil. Picomolar concentrations of OspA induce surface markers associated with neutrophil activation: increased CD10 and CD11b expression; decreased CD62-L expression; and an increased adherence to extracellular matrix. These events were similar in kinetics and magnitude to those induced by the strong activators LPS and FMLP. Like LPS, OspA could prime neutrophils for FMLP-induced release of lysosomal granules and production of superoxide. Thus, models of Lyme arthritis should include the possible contribution of direct activation of neutrophils to both defense and disease.

Neutrophil Activation by Helicobacter pylori Lipopolysaccharides

Helicobacter pylori-induced release of toxic substances by neutrophils could be of potential importance in the pathogenesis of gastroduodenal inflammatory diseases. Lipopolysaccharide (LPS) has the ability to induce neutrophil activation at very low concentrations. Neutrophil oxidative metabolism and enzyme release were assessed after stimulation of neutrophils with various preparations of LPS from H. pylori and compared with that obtained with Salmonella typhimurium LPS. No direct activation of neutrophils by LPS was observed. Preincubation with LPS showed a significant priming for increased activity on subsequent stimulation, particularly with rough-form LPS. The potency of H. pylori LPS was 10-fold lower than that of S. typhimurium LPS, probably reflecting the unique biochemical structure of H. pylori LPS. Chronic low-grade stimulation by H. pylori LPS in the gastric mucosa may render neutrophils primed for the excessive release of detrimental substances into the tissue on stimulation by other mediators.

1,3-Dioctanoylglycerol modulates arachidonate mobilization in human neutrophils and its inhibition by PGBx: Evidence of a protein-kinase-C-independent role for diacylglycerols in signal transduction

Preincubation of human neutrophils with 1-oleoyl-2-acetylglycerol (OAG) enhances subsequent f-Met-Leu-Phe (fMLP)-stimulated arachidonate mobilization. We have recently demonstrated that preincubation of neutrophils with OAG also reverses inhibition of A23187 stimulated [ 3H]arachidonate mobilization by the phospholipase A 2 inhibitors, PGBx and aristolochic acid. The present study has compared the effects of 1,2- sn-dioctanoylglycerol (1,2-diC8) and 1,3-dioctanoylglycerol (1,3-diC8) on these cellular events. Dose-dependent priming ( ED 50 < 2.5 μ M ) of fMLP-stimulated [ 3H]arachidonate mobilization is obtained with both 1,2-diC8 and 1,3-diC8. Both diC8s also enhance fMLP-stimulated synthesis of leukotriene B4, 5-hydroxyeicosatetraenoic acid and platelet-activating factor, and generation of superoxide. Furthermore, both 1,2-diC8 and 1,3-diC8 reverse the effects of PGBx on A23187-stimulated [ 3H]arachidonate mobilization and platelet-activating factor synthesis. By contrast, higher concentrations (5–10 μM) of 1,2-diC8, but not 1,3-diC8, directly stimulate both [ 3H]arachidonate mobilization and superoxide generation. Since 1,3-diC8 does not activate protein kinase C (PKC), these results suggest that PKC is involved in direct activation of neutrophils by diacylglycerols but not in priming. Furthermore, reversal of the inhibitory effects of PGBx by diacylglycerols also appears to involve a PKC-independent mechanism.

Activation of human neutrophils by Mycobacterium tuberculosis-derived sulfolipid-1.

The principal sulfatide of a group of acidic lipids from virulent Mycobacterium tuberculosis, sulfolipid-1 (SL-1), stimulates neutrophil superoxide (O2-) generation and, at lower concentrations, primes neutrophil response to several other metabolic agonists including FMLP, and PMA. These responses to SL-1 were examined in relation to diacylglycerol (DAG) generation, Ca2+ availability and activation of guanine nucleotide binding proteins to clarify the signal transduction pathways involved. Pertussis toxin inhibited the ability of SL-1 to both stimulate neutrophils directly and to prime neutrophils for subsequent responses induced by PMA, suggesting a role for one or more guanine nucleotide regulating proteins in both responses. SL-1 induced a rise in neutrophil DAG levels. DAG generation was inhibited by pretreatment of cells with pertussis toxin. Depletion of extracellular Ca2+ ablated O2- release induced by stimulatory levels of SL-1 but did not inhibit the priming effect induced by substimulatory concentrations of the lipid. Investigation of the activation of the neutrophil NADPH oxidase in a cell-free system revealed that the SL-1-priming effect was associated with translocation of the soluble cytosolic factors required for activation of the enzyme. Cytosolic factor translocation was not observed in pertussis toxin pretreated cells. Our results provide evidence for the role of a guanine nucleotide binding protein in both priming and direct activation of neutrophils by SL-1. This G protein regulates both SL-1-induced DAG generation and cytosolic cofactor translocation involved in neutrophil activation and priming. The multiplicity of effects of SL-1 on signal transduction pathways leading to phagocyte activation and priming may exert a profound influence on the pathogenicity of M. tuberculosis.

Enhancement of neutrophil adherence to isolated rat liver sinusoidal endothelial cells by supernatants of lipopolysaccharide-activated monocytes: Role of tumor necrosis factor

Supernatants of endotoxin-activated monocytes have been shown to stimulate human neutrophil adherence to rat liver sinusoidal endothelial cells 3-4-fold. Evidence will be presented that tumor necrosis factor (TNF) is responsible for this phenomenon: (a) in high-performance gel filtration of supernatants of lipopolysaccharide-activated monocytes, neutrophil adhesion-inducing activity coeluted with TNF activity measured in the L929 cell-lysing assay at 25-45 kDa; (b) anti-TNF antibody treatment of supernatants of activated macrophages abolished their adhesion-inducing activity; (c) human recombinant TNF alpha stimulated neutrophil adhesion to sinusoidal endothelial cells in a dose-dependent manner. In addition, polymyxine B sulfate, which was capable of neutralizing direct effects of lipopolysaccharide on neutrophil adhesion, could abolish neither the neutrophil-adhesion-inducing activity of the supernatants of endotoxin-activated monocytes nor the effect of human recombinant TNF itself. The neutrophil-adhesion-inducing activity was due both to a direct activation of neutrophils and to an influence of the sinusoidal endothelium itself by TNF: pretreatment of sinusoidal endothelial cells with TNF followed by thorough washing resulted in an increased neutrophil attachment. Protein synthesis by endothelial cells was not required. However, incubation of sinusoidal endothelium with TNF followed by anti-TNF antibody treatment abrogated the increased neutrophil adhesion. This suggests that TNF bound to sinusoidal endothelial cell surfaces was responsible for neutrophil adhesion. It is concluded that TNF by increasing granulocyte sticking to the endothelial lining of the liver sinusoids may play a significant role in endotoxin-induced inflammation of the liver as it is found in the septic state.