The uncovering of ESE-1 in human neutrophils: implication of its role in neutrophil function and survival

Abstract The first immune cell to arrive at the site of infection is the neutrophil. Upon arrival, neutrophils quickly initiate microbicidal functions, including the production of antimicrobial products and proinflammatory cytokines that serve to contain infection. This allows the acquired immune system enough time to generate sterilizing immunity and memory. Neutrophils detect the presence of a pathogen through germ line-encoded receptors that recognize microbe-associated molecular patterns. In vertebrates, the best characterized of these receptors are Toll-like receptors (TLRs). We have determined the expression and function of TLRs in freshly isolated human neutrophils. Neutrophils expressed TLR1, 2, 4, 5, 6, 7, 8, 9, and 10—all the TLRs except TLR3. Granulocyte-macrophage colony-stimulating factor (GM-CSF) treatment increased TLR2 and TLR9 expression levels. The agonists of all TLRs expressed in neutrophils triggered or primed cytokine release, superoxide generation, and L-selectin shedding, while inhibiting chemotaxis to interleukin-8 (IL-8) and increasing phagocytosis of opsonized latex beads. The response to the TLR9 agonist nonmethylated CpG-motif-containing DNA (CpG DNA) required GM-CSF pretreatment, which also enhanced the response to the other TLR agonists. Finally, using quantitative polymerase chain reaction (QPCR), we demonstrate a chemokine expression profile that suggests that TLR-stimulated neutrophils recruit innate, but not acquired, immune cells to sites of infection. (Blood. 2003;102:2660-2669)

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Neuraminidase inhibitors rewire neutrophil function in vivo in murine sepsis and ex vivo in COVID-19

10.1101/2020.11.12.379115 ◽

2020 ◽

Author(s):

Rodrigo O. Formiga ◽

Flávia C. Amaral ◽

Camila F. Souza ◽

Daniel A. G. B. Mendes ◽

Carlos W. S. Wanderley ◽

...

Keyword(s):

Sialic Acid ◽

Ex Vivo ◽

Neutrophil Function ◽

Human Neutrophils ◽

Data Sets ◽

Neuraminidase Inhibitors ◽

Severe Infections ◽

Whole Blood Cells ◽

Neutrophil Dysfunction

ABSTRACTNeutrophil overstimulation plays a crucial role in tissue damage during severe infections. Neuraminidase (NEU)-mediated cleavage of surface sialic acid has been demonstrated to regulate leukocyte responses. Here, we report that antiviral NEU inhibitors constrain host NEU activity, surface sialic acid release, ROS production, and NETs released by microbial-activated human neutrophils. In vivo, treatment with Oseltamivir results in infection control and host survival in peritonitis and pneumonia models of sepsis. Single-cell RNA sequencing re-analysis of publicly data sets of respiratory tract samples from critical COVID-19 patients revealed an overexpression of NEU1 in infiltrated neutrophils. Moreover, Oseltamivir or Zanamivir treatment of whole blood cells from severe COVID-19 patients reduces host NEU-mediated shedding of cell surface sialic acid and neutrophil overactivation. These findings suggest that neuraminidase inhibitors can serve as host-directed interventions to dampen neutrophil dysfunction in severe infections.

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Receptor expression and oxidase activity in human neutrophils: Regulation by granulocyte-macrophage colony-stimulating factor and dependence upon protein biosynthesis

Bioscience Reports ◽

10.1007/bf01117239 ◽

1990 ◽

Vol 10 (4) ◽

pp. 393-401 ◽

Cited By ~ 17

Author(s):

Steven W. Edwards ◽

Fiona Watson ◽

Ronald MacLeod ◽

John Davies

Keyword(s):

De Novo ◽

Protein Biosynthesis ◽

Neutrophil Function ◽

Membrane Receptors ◽

Receptor Expression ◽

Human Neutrophils ◽

Colony Stimulating Factor ◽

Macrophage Colony Stimulating Factor ◽

Macrophage Colony ◽

Stimulating Factor

Incubation of human bloodstream neutrophils with 50 u/ml recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF) “primed” the respiratory burst (as assessed by fMet-Leu-Phe stimulated luminol-dependent chemiluminescence) and resulted in a rapid (within 15 min) upregulation of expression of CD11b and CD18 (as measured by FACS analysis). This rapid “priming” and modulation of receptor expression was not inhibited by cycloheximide and hence appeared to be independent of de novo protein biosynthesis. When neutrophils were incubated for up to 5 h in culture, the fluorescence distributions of CD11b and CD18 declined indicating the loss of expression of these receptors as the neutrophils aged, but in rGM-CSF treated suspensions receptor expression was maintained. When neutrophils were incubated in the presence of cycloheximide, they progressively lost their ability to generate reactive oxidants in response to fMet-Leu-Phe so that by 5 h incubation with this inhibitor they could only generate about 25% of the oxidative response stimulated in untreated cells, and the expression of CD16 and CD18 was grossly impaired. Similar effects were observed in rGM-CSF treated suspensions except that cycloheximide required longer incubation times (typically 4–5 h) before impairment of function or receptor expression occurred. These data show that de novo protein biosynthesis is required for both the maintenance of neutrophil function and also for the continued expression of some plasma membrane receptors.

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Microarray analysis of lipopolysaccharide-treated human neutrophils

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00094.2002 ◽

2003 ◽

Vol 284 (4) ◽

pp. L663-L670 ◽

Cited By ~ 51

Author(s):

Kenneth C. Malcolm ◽

Patrick G. Arndt ◽

Elizabeth J. Manos ◽

David A. Jones ◽

G. Scott Worthen

Keyword(s):

Gene Expression ◽

Growth Regulation ◽

Molecular Mechanisms ◽

Neutrophil Function ◽

Interferon Response ◽

Human Neutrophils ◽

Oxygen Intermediates ◽

Microarray Gene Expression ◽

Monocyte Chemoattractant Protein 1 ◽

Cytokines And Chemokines

Neutrophils respond to infection by degranulation, release of reactive oxygen intermediates, and secretion of chemokines and cytokines; however, activation of neutrophil transcriptional machinery has been little appreciated. Recent findings suggest that gene expression may represent an additional neutrophil function after exposure to lipopolysaccharide (LPS). We performed microarray gene expression analysis of 4,608 mostly nonredundant genes on LPS-stimulated human neutrophils. Analysis of three donors indicated some variability but also a high degree of reproducibility in gene expression. Twenty-eight verifiable, distinct genes were induced by 4 h of LPS treatment, and 13 genes were repressed. Genes other than cytokines and chemokines are regulated; interestingly, genes involved in cell growth regulation and survival, transcriptional regulation, and interferon response are among those induced, whereas genes involved in cytoskeletal regulation are predominantly repressed. In addition, we identified monocyte chemoattractant protein-1 as a novel LPS-regulated chemokine in neutrophils. Included in these lists are five clones with no defined function. These data suggest molecular mechanisms by which neutrophils respond to infection and indicate that the transcriptional potential of neutrophils is greater than previously thought.

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Correlation between the stimulation of human neutrophil function by monoclonal antibody and by colony-stimulating factor

Blood ◽

10.1182/blood.v66.3.738.738 ◽

1985 ◽

Vol 66 (3) ◽

pp. 738-741 ◽

Cited By ~ 4

Author(s):

MA Vadas ◽

C Clarke ◽

NA Nicola ◽

AF Lopez

Keyword(s):

Monoclonal Antibody ◽

Neutrophil Function ◽

Common Mechanism ◽

Human Neutrophils ◽

Target Cells ◽

Colony Stimulating Factor ◽

Positive Correlation ◽

Stimulating Factor ◽

Stimulation Of

Abstract Purified human neutrophils from 48 individuals were tested for their capacity to kill antibody-coated target cells in vitro in the absence or presence of stimulating agents. The agents used to stimulate cytotoxic capacity were the monoclonal antibody (MAb) WEM-G1, colony- stimulating factor (CSF-alpha), or mononuclear cell supernatant (MNC- SN). There existed an heterogeneity among the neutrophils of different individuals in the capacity to kill target cells both in the unstimulated (“resting”) or the stimulated state. A positive correlation was found between the ability of neutrophils to kill in the “resting” state and their capacity to be stimulated by MAb WEM-G1, CSF- alpha, or MNC-SN. Furthermore, a strong positive correlation in the ability of neutrophils to be stimulated by the MAb WEM-G1 and either CSF-alpha (r = .76) or MNC-SN (r = .68), as well as between CSF-alpha and MNC-SN (r = .79) was demonstrated. No correlation was seen, however, between stimulation of neutrophil function in vitro and total blood leukocyte counts, neutrophil counts, monocyte counts, or intensity of binding of MAb WEM-G1. The observation that neutrophils respond to a similar extent to different types of stimulators, -such as cytokines (CSF-alpha and MNC-SN) and MAb, suggests that these two factors may be operating through a common mechanism and the degree of stimulation may reflect an intrinsic responsiveness of neutrophils that differs among individuals. Our results also suggest a potential clinical use of WEM-G1 in measuring neutrophil functional capacity in vitro and predicting the capacity to respond to CSF-like cytokines.

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Metabolic abnormalities in G6PC3-deficient human neutrophils result in severe functional defects

Blood Advances ◽

10.1182/bloodadvances.2020002225 ◽

2020 ◽

Vol 4 (23) ◽

pp. 5888-5901

Author(s):

Christopher McKinney ◽

Michael Ellison ◽

Natalie J. Briones ◽

Angelina Baroffio ◽

John Murphy ◽

...

Keyword(s):

Missense Mutation ◽

Bacterial Infections ◽

Neutrophil Function ◽

Functional Improvement ◽

Human Neutrophils ◽

Compound Heterozygous ◽

Cd11b Expression ◽

Exon 2 ◽

Metabolic Defects ◽

Functional Defects

Abstract Severe congenital neutropenia type 4 (SCN-4) is an autosomal recessive condition in which mutations in the G6PC3 gene encoding for the catalytic 3 subunit of glucose-6-phosphatase-β result in neutropenia, neutrophil dysfunction, and other syndromic features. We report a child with SCN-4 caused by compound heterozygous mutations in G6PC3, a previously identified missense mutation in exon 6 (c.758G>A[p.R235H]), and a novel missense mutation in exon 2 (c.325G>A[p.G109S]). The patient had recurrent bacterial infections, inflammatory bowel disease, neutropenia, and intermittent thrombocytopenia. Administration of granulocyte colony–stimulating factor (G-CSF) resolved the neutropenia and allowed for detailed evaluation of human neutrophil function. Random and directed migration by the patient’s neutrophils was severely diminished. Associated with this were defects in CD11b expression and F-actin assembly. Bactericidal activity at bacteria/neutrophil ratios >1:1 was also diminished and was associated with attenuated ingestion. Superoxide anion generation was <25% of control values, but phox proteins appeared quantitatively normal. Extensive metabolomics analysis at steady state and upon incubation with stable isotope–labeled tracers (U-13C-glucose, 13C,15N-glutamine, and U-13C-fructose) demonstrated dramatic impairments in early glycolysis (hexose phosphate levels), hexosemonophosphate shunt (required for the generation of the NADPH), and the total adenylate pool, which could explain the dramatic cell dysfunction displayed by the patient’s neutrophils. Preliminary experiments with fructose supplementation to bypass the enzyme block demonstrated that the metabolic profile could be reversed, but was not sustained long enough for functional improvement. In human deficiency of G6PC3, metabolic defects resulting from the enzyme deficiency account for diverse neutrophil functional defects and present a major risk of infection.

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Src kinase inhibition with dasatinib impairs neutrophil function and clearance of Escherichia coli infection in a murine model of acute lung injury

Journal of Inflammation ◽

10.1186/s12950-020-00261-5 ◽

2020 ◽

Vol 17 (1) ◽

Author(s):

James G. Macfarlane ◽

David A. Dorward ◽

Marie-Hélène Ruchaud-Sparagano ◽

Jonathan Scott ◽

Christopher D. Lucas ◽

...

Keyword(s):

Escherichia Coli ◽

Acute Lung Injury ◽

Lung Injury ◽

Murine Model ◽

Acute Inflammation ◽

Neutrophil Function ◽

Mouse Lung ◽

Human Neutrophils ◽

Bacterial Clearance ◽

E Coli

Abstract Background Neutrophils rapidly respond to and clear infection from tissues, but can also induce tissue damage through excessive degranulation, when acute inflammation proceeds unchecked. A number of key neutrophil functions, including adhesion-dependent degranulation, are controlled by src family kinases. Dasatinib is a potent src inhibitor used in treating patients with chronic myeloid leukaemia and treatment-resistant acute lymphoblastic leukaemia. We hypothesized that dasatinib would attenuate acute inflammation by inhibiting neutrophil recruitment, degranulation and endothelial cell injury, without impairing bacterial clearance, in a murine model of bacteria-induced acute lung injury. C57BL/6 mice received intratracheal Escherichia coli, and were treated with intraperitoneal dasatinib or control. Bacterial clearance, lung injury, and markers of neutrophil recruitment and degranulation were measured. Separately, human blood neutrophils were exposed to dasatinib or control, and the effects on a range of neutrophil functions assessed. Results Dasatinib was associated with a dose-dependent significant increase in E. coli in the mouse lung, accompanied by impairment of organ function, reflected in significantly increased protein leak across the alveolar-capillary membrane. However, the number of neutrophils entering the lung was unaffected, suggesting that dasatinib impairs neutrophil function independent of migration. Dasatinib did not cause direct toxicity to human neutrophils, but led to significant reductions in phagocytosis of E. coli, adhesion, chemotaxis, generation of superoxide anion and degranulation of primary and secondary granules. However, no biologically important effect of dasatinib on neutrophil degranulation was observed in mice. Conclusions Contrary to our starting hypothesis, src kinase inhibition with dasatinib had a detrimental effect on bacterial clearance in the mouse lung and therefore does not represent an attractive therapeutic strategy to treat primary infective lung inflammation. Data from human neutrophils suggest that dasatanib has inhibitory effects on a range of neutrophil functions.

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L-selectin function is required for beta 2-integrin-mediated neutrophil adhesion at physiological shear rates in vivo

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1992.263.4.h1034 ◽

1992 ◽

Vol 263 (4) ◽

pp. H1034-H1044 ◽

Cited By ~ 20

Author(s):

U. H. Von Andrian ◽

P. Hansell ◽

J. D. Chambers ◽

E. M. Berger ◽

I. Torres Filho ◽

...

Keyword(s):

Neutrophil Function ◽

Leukotriene B4 ◽

Human Neutrophils ◽

Neutrophil Adhesion ◽

Shear Rates ◽

Sequence Of Events ◽

Multistep Process ◽

Beta 2

In vivo interactions between neutrophils and endothelial cells (EC) follow a multistep process involving two distinct neutrophil adhesion receptors. L-selectin, constitutively functional on resting neutrophils, mediates an activation-independent primary interaction resulting in rolling along the venular wall. Subsequent activation of rolling neutrophils induces upregulation and functional activation of beta 2-integrins (CD11/CD18) leading to firm attachment. Based on previous findings we hypothesized that, under shear force, rolling may be essential for successful neutrophil-EC recognition. Here we report results of our studies of human neutrophil behavior in interleukin (IL)-1-activated rabbit mesentery venules, an interaction that requires both L-selectin and beta 2-integrins. Rolling of human neutrophils is L-selection mediated; it was strongly reduced by monoclonal antibody inhibition or enzymatic removal of L-selectin. Furthermore, activation induced L-selectin shedding and, in a dose- and time-dependent fashion, rendered neutrophils unable to recognize inflamed EC despite expression of active beta 2-integrins, which promoted adhesion in vitro. Neutrophils activated for 5 min or longer lost most of their ability to roll. However, 1-3 min after activation, rolling was reduced (not abolished), and cells that were still able to roll displayed a significant tendency for a CD18-dependent transition from rolling to sticking. The whole sequence of events, rolling, sticking, and transendothelial migration, could be observed if an extravascular chemotactic stimulus was applied by superfusing mesenteries with leukotriene B4. Under such conditions, sticking and emigration was blocked when rolling was inhibited by enzymatic removal of L-selectin. Our results indicate that primary neutrophil interaction with inflamed EC through the L-selectin is a prerequisite for neutrophil function at physiological shear rates in vivo.

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Enhancement of chemotactic factor-stimulated neutrophil oxidative metabolism by leukotriene B4

Blood ◽

10.1182/blood.v64.4.780.bloodjournal644780 ◽

1984 ◽

Vol 64 (4) ◽

pp. 780-785

Author(s):

JC Gay ◽

JK Beckman ◽

AR Brash ◽

JA Oates ◽

JN Lukens

Keyword(s):

Neutrophil Function ◽

Leukotriene B4 ◽

Human Neutrophils ◽

Neutrophil Chemotaxis ◽

Myristate Acetate ◽

Fmlp Receptor ◽

Dose Dependent ◽

O2 Production ◽

Oxygen Metabolites ◽

Oxidative Responses

Leukotriene B4 (LTB4) is a potent primary stimulator of neutrophil chemotaxis, aggregation, and degranulation and induces superoxide production at higher concentrations. In order to determine whether LTB4 modulates neutrophil responses to oxidative stimuli, human neutrophils (PMNs) were incubated with LTB4 prior to stimulation with f-Met-Leu-Phe (fMLP, 10(-7) mol/L), opsonized zymosan (OZ, 250 micrograms/mL), or phorbol myristate acetate (PMA, 32 nmol/L). Superoxide (O2-) production by stimulated PMNs was assessed by the superoxide dismutase-inhibitable reduction of cytochrome c. LTB4 alone did not stimulate O2- production in concentrations below 10(-7) mol/L and had no effect on the O2- assay. In the concentration range of 10(-12) to 10(-8) mol/L, LTB4 did not alter O2- release induced by OZ or PMA. In contrast, LTB4-treated cells demonstrated enhanced O2- production following exposure to fMLP, and in the presence of 10 nmol/LLTB4, generated 180% +/- 41% of O-2 quantities produced by control cells (n = 23). Enhancement was LTB4 dose-dependent, was maximal in the range of 1 to 10 nmol/L LTB4, was not reversed by removal of the lipid from the medium prior to fMLP stimulation, and was not dependent on the presence of Ca++ or Mg++ in the suspending medium. Chemiluminescence of fMLP-stimulated neutrophils was increased to 323% of controls in neutrophils preincubated with 10 nmol/L LTB4. Unlike augmentation of oxidative responses to fMLP seen with other degranulating stimuli, enhancement by LTB4 was not correlated with an increase in 3H-fMLP receptor binding. These results indicate that, in addition to its primary effects on neutrophil function, LTB4 modulates PMN oxidative responses to the chemotactic peptide and, thus, may amplify the release of oxygen metabolites at inflammatory foci.

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Enhancement of chemotactic factor-stimulated neutrophil oxidative metabolism by leukotriene B4

Blood ◽

10.1182/blood.v64.4.780.780 ◽

1984 ◽

Vol 64 (4) ◽

pp. 780-785 ◽

Cited By ~ 49

Author(s):

JC Gay ◽

JK Beckman ◽

AR Brash ◽

JA Oates ◽

JN Lukens

Keyword(s):

Neutrophil Function ◽

Leukotriene B4 ◽

Human Neutrophils ◽

Neutrophil Chemotaxis ◽

Myristate Acetate ◽

Fmlp Receptor ◽

Dose Dependent ◽

O2 Production ◽

Oxygen Metabolites ◽

Oxidative Responses

Abstract Leukotriene B4 (LTB4) is a potent primary stimulator of neutrophil chemotaxis, aggregation, and degranulation and induces superoxide production at higher concentrations. In order to determine whether LTB4 modulates neutrophil responses to oxidative stimuli, human neutrophils (PMNs) were incubated with LTB4 prior to stimulation with f-Met-Leu-Phe (fMLP, 10(-7) mol/L), opsonized zymosan (OZ, 250 micrograms/mL), or phorbol myristate acetate (PMA, 32 nmol/L). Superoxide (O2-) production by stimulated PMNs was assessed by the superoxide dismutase-inhibitable reduction of cytochrome c. LTB4 alone did not stimulate O2- production in concentrations below 10(-7) mol/L and had no effect on the O2- assay. In the concentration range of 10(-12) to 10(-8) mol/L, LTB4 did not alter O2- release induced by OZ or PMA. In contrast, LTB4-treated cells demonstrated enhanced O2- production following exposure to fMLP, and in the presence of 10 nmol/LLTB4, generated 180% +/- 41% of O-2 quantities produced by control cells (n = 23). Enhancement was LTB4 dose-dependent, was maximal in the range of 1 to 10 nmol/L LTB4, was not reversed by removal of the lipid from the medium prior to fMLP stimulation, and was not dependent on the presence of Ca++ or Mg++ in the suspending medium. Chemiluminescence of fMLP-stimulated neutrophils was increased to 323% of controls in neutrophils preincubated with 10 nmol/L LTB4. Unlike augmentation of oxidative responses to fMLP seen with other degranulating stimuli, enhancement by LTB4 was not correlated with an increase in 3H-fMLP receptor binding. These results indicate that, in addition to its primary effects on neutrophil function, LTB4 modulates PMN oxidative responses to the chemotactic peptide and, thus, may amplify the release of oxygen metabolites at inflammatory foci.

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