scholarly journals Measurement of free and membrane-bound cathepsin G in human neutrophils using new sensitive fluorogenic substrates

2002 ◽  
Vol 366 (3) ◽  
pp. 965-970 ◽  
Author(s):  
Sylvie ATTUCCI ◽  
Brice KORKMAZ ◽  
Luiz JULIANO ◽  
Eric HAZOUARD ◽  
Catherine GIRARDIN ◽  
...  
Keyword(s):  
Cathepsin G ◽  
Polymorphonuclear Neutrophils ◽  
Human Neutrophils ◽  
Proteinase 3 ◽  
Protein Targets ◽  
Peptide Substrates ◽  
Loop Sequence ◽  
Membrane Bound

Activated human polymorphonuclear neutrophils at inflammatory sites release the chymotrypsin-like protease cathepsin G, together with elastase and proteinase 3 (myeloblastin), from their azurophil granules. The low activity of cathepsin G on synthetic substrates seriously impairs studies designed to clarify its role in tissue inflammation. We have solved this problem by producing new peptide substrates with intramolecularly quenched fluorescence. These substrates were deduced from the sequence of putative protein targets of cathepsin G, including the reactive loop sequence of serpin inhibitors and the N-terminal domain of the protease-activated receptor of thrombin, PAR-1. Two substrates were selected, Abz-TPFSGQ-EDDnp and Abz-EPFWEDQ-EDDnp, that are cleaved very efficiently by cathepsin G but not by neutrophil elastase or proteinase 3, with specificity constants (kcat/Km) in the 105M-1·s-1 range. They can be used to measure subnanomolar concentrations of free enzyme in vitro and at the surface of neutrophils purified from fresh human blood. Purified neutrophils express 0.02—0.7pg of cathepsin G/cell (n = 15) at their surface. This means that about 104 purified cells may be enough to record cathepsin G activity within minutes. This may be most important for investigating the role of cathepsin G as an inflammatory agent, especially in bronchoalveolar lavage fluids from patients with pulmonary inflammatory disorders.

Leukocyte antibacterial functions are not impaired by perfluorocarbon exposure in vitro

2008 ◽  
Vol 295 (1) ◽  
pp. L134-L142 ◽  
Author(s):  
Dirk Haufe ◽  
Eva Koenigshausen ◽  
Lilla Knels ◽  
Martina Wendel ◽  
Sebastian N. Stehr ◽  
...  
Keyword(s):  
Host Defense ◽  
In Vitro Model ◽  
Polymorphonuclear Neutrophils ◽  
Human Neutrophils ◽  
Intracellular Uptake ◽  
E Coli ◽  
Transmission Electron ◽  
Membrane Bound ◽  
Vitro Model

Application of liquid, aerosolized, and vaporized perfluorocarbons (PFC) in acute lung injury has shown anti-inflammatory effects. Although this may be beneficial in states of pulmonary hyperinflammation, it also could increase susceptibility to nosocomial lung infection. We hypothesized that PFC impair cellular host defense and therefore investigated in an in vitro model the influence of perfluorohexane (PFH) on crucial mechanisms of bacterial elimination in human neutrophils and monocytes. Using scanning and transmission electron microscopy, we could show membrane-bound and ingested PFH particles that morphologically did not alter adherence and phagocytosis of Escherichia coli or leukocyte viability. The amount of adherent and phagocytosed bacteria as determined by flow cytometry was not influenced in cells only pretreated with PFH for 1 and 4 h. When PFH was present during E. coli challenge, bacterial adherence was decreased in polymorphonuclear neutrophils, but respective intracellular uptake was not impaired and was even significantly promoted in monocytes. Overall, E. coli-induced respiratory burst capacity was not reduced by PFH. Our findings provide evidence that key functions of innate host defense are not compromised by PFH treatment in vitro.

In vitro Complement-Independent Activation of Human Neutrophils by Hemodialysis Membranes: Role of the Net Electric Charge

1987 ◽  
Vol 10 (2) ◽  
pp. 83-88 ◽  
Author(s):  
C. Tetta ◽  
G. Segoloni ◽  
G. Camussi ◽  
S. Neumann ◽  
S. Griva ◽  
...  
Keyword(s):  
Electric Charge ◽  
Platelet Activating Factor ◽  
Lactic Dehydrogenase ◽  
Cathepsin G ◽  
Polymorphonuclear Neutrophils ◽  
Human Neutrophils ◽  
Reactive Polymer ◽  
Intracellular Content ◽  
Before And After

Polymethylmethacrylate (PMMA) membranes with different net electric charges and percentage water contents (anionic 71%, neutral 70%, cationic 75%) were evaluated for their ability to stimulate plasma-free human polymorphonuclear neutrophils (PMN), and compared for potency to cuprophan (Cu), already described as being a potent trigger of PMN. The release of lysozyme, β-glucuronidase, lactic dehydrogenase (LDH), and the generation of a platelet aggregating activity were studied in the supernatants from plasma-free human PMN incubated with different membranes. The PMN intracellular content of neutrophil cationic proteins (NCP), elastase, and cathepsin G were also studied by immunofluorescence using specific antisera on smears of PMN before and after incubation with each membrane. Only cationic, but not anionic or neutral PMMA induced a marked release of lysozyme (range 20-25% of the sonicated control, assumed as 100%), and β-glu-coronidase (40-43%), and marked depletion of the intracellular content of NCP, elastase, and cathepsin G, suggesting a degranulation process. Platelet aggregating activity was generated and referred to the release of platelet activating factor (PAF) only in the supernatants from PMN incubated with cationic, but not with anionic, or neutral PMMA membranes. These results indicate that modification of the net electric charge can per se turn PMMA, commonly recognized as inert, into a material with marked PMN activating effects, comparable to those of Cu, a highly reactive polymer.

The Signaling Pathways in Nitric Oxide Production by Neutrophils Exposed to N-nitrosodimethylamine

2018 ◽  
Vol 16 (2) ◽  
pp. 194-199
Author(s):  
Wioletta Ratajczak-Wrona ◽  
Ewa Jablonska
Keyword(s):  
Nitric Oxide ◽  
Signaling Pathways ◽  
Molecular Mechanisms ◽  
Polymorphonuclear Neutrophils ◽  
Microbial Pathogens ◽  
Human Neutrophils ◽  
No Production ◽  
Oxide Synthase ◽  
Inducible Nitric Oxide

Background: Polymorphonuclear neutrophils (PMNs) play a crucial role in the innate immune system’s response to microbial pathogens through the release of reactive nitrogen species, including Nitric Oxide (NO). </P><P> Methods: In neutrophils, NO is produced by the inducible Nitric Oxide Synthase (iNOS), which is regulated by various signaling pathways and transcription factors. N-nitrosodimethylamine (NDMA), a potential human carcinogen, affects immune cells. NDMA plays a major part in the growing incidence of cancers. Thanks to the increasing knowledge on the toxicological role of NDMA, the environmental factors that condition the exposure to this compound, especially its precursors- nitrates arouse wide concern. Results: In this article, we present a detailed summary of the molecular mechanisms of NDMA’s effect on the iNOS-dependent NO production in human neutrophils. Conclusion: This research contributes to a more complete understanding of the mechanisms that explain the changes that occur during nonspecific cellular responses to NDMA toxicity.

scholarly journals Sodium Channel Nav1.3 Is Expressed by Polymorphonuclear Neutrophils during Mouse Heart and Kidney Ischemia InVivo and Regulates Adhesion, Transmigration, and Chemotaxis of Human and Mouse Neutrophils In Vitro

2018 ◽  
Vol 128 (6) ◽  
pp. 1151-1166 ◽  
Author(s):  
Marit Poffers ◽  
Nathalie Bühne ◽  
Christine Herzog ◽  
Anja Thorenz ◽  
Rongjun Chen ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Sodium Channel ◽  
Sodium Channels ◽  
Polymorphonuclear Neutrophils ◽  
Human Neutrophils ◽  
Mouse Heart ◽  
Voltage Gated ◽  
Voltage Gated Sodium Channels

Abstract Background Voltage-gated sodium channels generate action potentials in excitable cells, but they have also been attributed noncanonical roles in nonexcitable cells. We hypothesize that voltage-gated sodium channels play a functional role during extravasation of neutrophils. Methods Expression of voltage-gated sodium channels was analyzed by polymerase chain reaction. Distribution of Nav1.3 was determined by immunofluorescence and flow cytometry in mouse models of ischemic heart and kidney injury. Adhesion, transmigration, and chemotaxis of neutrophils to endothelial cells and collagen were investigated with voltage-gated sodium channel inhibitors and lidocaine in vitro. Sodium currents were examined with a whole cell patch clamp. Results Mouse and human neutrophils express multiple voltage-gated sodium channels. Only Nav1.3 was detected in neutrophils recruited to ischemic mouse heart (25 ± 7%, n = 14) and kidney (19 ± 2%, n = 6) in vivo. Endothelial adhesion of mouse neutrophils was reduced by tetrodotoxin (56 ± 9%, unselective Nav-inhibitor), ICA121431 (53 ± 10%), and Pterinotoxin-2 (55 ± 9%; preferential inhibitors of Nav1.3, n = 10). Tetrodotoxin (56 ± 19%), ICA121431 (62 ± 22%), and Pterinotoxin-2 (59 ± 22%) reduced transmigration of human neutrophils through endothelial cells, and also prevented chemotactic migration (n = 60, 3 × 20 cells). Lidocaine reduced neutrophil adhesion to 60 ± 9% (n = 10) and transmigration to 54 ± 8% (n = 9). The effect of lidocaine was not increased by ICA121431 or Pterinotoxin-2. Conclusions Nav1.3 is expressed in neutrophils in vivo; regulates attachment, transmigration, and chemotaxis in vitro; and may serve as a relevant target for antiinflammatory effects of lidocaine.

scholarly journals Medawar’s PostEra: Galectins Emerged as Key Players During Fetal-Maternal Glycoimmune Adaptation

2021 ◽  
Vol 12 ◽  
Author(s):  
Ellen Menkhorst ◽  
Nandor Gabor Than ◽  
Udo Jeschke ◽  
Gabriela Barrientos ◽  
Laszlo Szereday ◽  
...  
Keyword(s):  
Immune Cell ◽  
Successful Pregnancy ◽  
Fetal Health ◽  
The Past ◽  
Mammalian Embryogenesis ◽  
Carbohydrate Ligands ◽  
Membrane Bound

Lectin-glycan interactions, in particular those mediated by the galectin family, regulate many processes required for a successful pregnancy. Over the past decades, increasing evidence gathered from in vitro and in vivo experiments indicate that members of the galectin family specifically bind to both intracellular and membrane bound carbohydrate ligands regulating angiogenesis, immune-cell adaptations required to tolerate the fetal semi-allograft and mammalian embryogenesis. Therefore, galectins play important roles in fetal development and placentation contributing to maternal and fetal health. This review discusses the expression and role of galectins during the course of pregnancy, with an emphasis on maternal immune adaptions and galectin-glycan interactions uncovered in the recent years. In addition, we summarize the galectin fingerprints associated with pathological gestation with particular focus on preeclampsia.

Interleukin-8 induces neutrophil accumulation but not protease secretion in the canine trachea

1992 ◽  
Vol 263 (6) ◽  
pp. L708-L713 ◽  
Author(s):  
P. G. Jorens ◽  
J. B. Richman-Eisenstat ◽  
B. P. Housset ◽  
P. D. Graf ◽  
I. F. Ueki ◽  
...  
Keyword(s):  
Interleukin 8 ◽  
Cathepsin G ◽  
Human Neutrophils ◽  
Secretory Cells ◽  
Neutrophil Accumulation ◽  
High Concentrations ◽  
Granule Secretion ◽  
Protease Secretion

The neutrophil enzyme elastase is a potent secretagogue of airway secretory cells, and elastase is present in high concentrations in sputum of patients with hypersecretion (e.g., cystic fibrosis, bronchiectasis). Interleukin-8 (IL-8), a recently discovered cytokine with potent neutrophil chemotactic properties in vitro, is also found in the sputum of these patients. We used an isolated tracheal segment in dogs in vivo to study the effect of IL-8 in causing neutrophil accumulation, elastase release, and secretion (by measuring lysozyme concentrations) in the luminal superfusate. IL-8 caused a potent time-dependent neutrophil accumulation at between 3 and 6 h. The effect was significant at 10(-9) and maximum at 10(-8) M. No increase in free elastase, cathepsin G, or lysozyme was detected in the superfusate. Thus, in contrast to previous studies showing that ragweed antigen causes the accumulation of neutrophil elastase which in turn causes lysozyme secretion, IL-8 causes neutrophil accumulation without granule secretion (or subsequent secretagogue activity). The findings were confirmed with dog and human neutrophils in vitro.

In vitro effect of elastase and cathepsin G from human neutrophils on creatine kinase and lactate dehydrogenase isoenzymes

1993 ◽  
Vol 39 (6) ◽  
pp. 986-992
Author(s):  
J A Stark ◽  
A R Henderson
Keyword(s):  
Creatine Kinase ◽  
Lactate Dehydrogenase ◽  
Hypochlorous Acid ◽  
Proteolytic Enzymes ◽  
Sodium Dodecyl ◽  
Cathepsin G ◽  
Human Neutrophils ◽  
Sample Treatment ◽  
Vitro Effect

Abstract The polymorphonuclear granulocyte, or neutrophil, has been implicated as a mediator of tissue-destructive events because it releases the preformed proteolytic enzymes elastase and cathepsin G, and, as a result of myeloperoxidase action, hypochlorous acid. We show that elastase inactivates and fragments creatine kinase isoenzymes CK-2 and CK-3, and, to a lesser extent, lactate dehydrogenase (LD) isoenzyme LD-1, whereas cathepsin G acts only on CK-2. Both neutrophil enzymes act on LD-3. The course of inactivation was followed by measuring the loss of catalytic activity at 37 degrees C. The evidence for fragmentation was obtained by gel filtration; electrophoresis after sample treatment with sodium dodecyl sulfate and 2-mercaptoethanol was less satisfactory for this purpose. Hypochlorous acid inactivates CK activity by about 75% at concentrations as low as 8 mumol/L and totally at concentrations &gt; 140 mumol/L, whereas LD activity is not affected until concentrations exceed 200 mumol/L. After a myocardial infarction, the number of neutrophils increases; they are triggered and concentrate around damaged myocardial tissue. Our data suggest that neutrophils may inactivate and fragment "cardiac" enzymes released from such damaged tissue.

scholarly journals G Protein-Coupled Estrogen Receptor 1 Regulates Human Neutrophil Functions

2017 ◽  
Vol 2 (1) ◽  
pp. 1-13 ◽  
Author(s):  
M. Carmen Rodenas ◽  
Nicola Tamassia ◽  
Isabel Cabas ◽  
Federica Calzetti ◽  
José Meseguer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Estrogen Receptor ◽  
Protein Kinase ◽  
G Protein ◽  
Respiratory Burst ◽  
Human Neutrophils ◽  
G Protein Coupled ◽  
Estrogen Receptor 1

Background: The role of estrogens in immune functioning is relatively well known under both physiological and pathological conditions. Neutrophils are the most abundant circulating leukocytes in humans, and their abundance and function are regulated by estrogens, since they express estrogen receptors (ERs). Traditionally, estrogens were thought to act via classical nuclear ERs, namely ERα and ERβ. However, it was observed that some estrogens induced biological effects only minutes after their application. This rapid, “nongenomic” effect of estrogens is mediated by a membrane-anchored receptor called G protein-coupled estrogen receptor 1 (GPER1). Nevertheless, the expression and role of GPER1 in the immune system has not been exhaustively studied, and its relevance in neutrophil functions remains unknown. Methods: Human neutrophils were incubated in vitro with 10-100 µM of the GPER1-specific agonist G1 alone or in combination with lipopolysaccharide. GPER1 expression and subcellular localization, respiratory burst, life span, gene expression profile, and cell signaling pathways involved were then analyzed in stimulated neutrophils. Results: Human neutrophils express a functional GPER1 which regulates their functions through cAMP/protein kinase A/cAMP response element-binding protein, p38 mitogen-activated protein kinase, and extracellular regulated MAPK signaling pathways. Thus, GPER1 activation in vitro increases the respiratory burst of neutrophils, extends their life span, and drastically alters their gene expression profile. Conclusions: Our results demonstrate that GPER1 activation promotes the polarization of human neutrophils towards a proinflammatory phenotype and point to GPER1 as a potential therapeutic target in immune diseases where neutrophils play a key role.

Proteinase-3 Deficiency Contributes to Thrombotic Risk in PNH

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 406-406
Author(s):  
Anna M. Jankowska ◽  
Hadrian Szpurka ◽  
Andrew E. Schade ◽  
Sanjay R. Mohan ◽  
Jaroslaw P. Maciejewski
Keyword(s):  
Platelet Activation ◽  
Thrombus Formation ◽  
Flow Cytometric Analysis ◽  
Significant Inhibition ◽  
Cathepsin G ◽  
Proteinase 3 ◽  
Membrane Expression ◽  
Hematopoietic Stem ◽  
Thrombotic Risk ◽  
Membrane Bound

Abstract PNH is an acquired clonal disorder of hematopoietic stem cells due to a somatic mutation of the PIG-A gene, resulting in a membrane deficiency of glycosylphosphatidyl inositol-anchored proteins (GPI-AP). The mechanism of thrombosis, the most serious complication of PNH, has not been sufficiently clarified. GPI-AP functions include enzymatic activities and maintenance of lipid rafts (LR) integrity. Some GPI-AP also serve as a scaffold for various proteins. Therefore deficiency of GPI-AP has consequences for the expression of some non-GPI-AP. For example, GPI-anchored NB1 (CD177) glycoprotein co-localizes with membrane-bound proteinase 3 (mPR3) on granulocytes. We demonstrate that anti-CD177 antibody co-precipitated PR3 and the PNH phenotype results in a concomitant loss of mPR3 expression despite the fact that PR3 does not have a GPI tail. Among other functions, PR3 can modulate thrombus formation by cleavage of the thrombin receptor (TR) downstream from the thrombin cleavage site, decreasing thrombin-mediated platelet activation. We show that pre-incubation of platelets with purified PR3 resulted in a significant inhibition of platelet activation in response to thrombin, as measured by flow cytometric analysis of P-selectin and PAC-1 expression, while PR3 itself did not affect platelet function. However, PR3 failed to inhibit platelet activation induced by ADP and/or TRAP42-55 agonist, suggesting that PR3 renders TR inactive to thrombin while thrombin-independent activation is not affected. Membrane deficiency of PR3 on granulocytes may contribute to unopposed platelet activation promoting a procoagulable state inherent to PNH. Consequently, we investigated PR3 expression in PNH; when we applied surface/intracellular cytometry and Western blotting for PR3 in patients (N=20), mPR3 was present on the surface of wild-type but absent on GPI-deficient granulocytes. However, intracellular levels of PR3 were equal in both cell types. Association of CD177 and mPR3 was further supported by analysis of LR purified from PNH granulocytes. Immunoblots preformed following density gradient separation, showed a complete loss of LR-associated PR3 and CD177 in PNH cells while PR3 was present in cytoplasmatic fractions. Similar observation was made when another related neutrophilic protease, cathepsin G (CTSG) was studied and found in cytoplasmic fractions in PNH cells, but was detected only in LR derived from normal cells. When we tested the levels of plasma PR3 in patients with PNH (N=40) and controls (N=69), we demonstrated that secreted PR3 is derived from the membrane bound fractions as circulating levels of PR3 were significantly decreased in PNH patients when compared to healthy and hematologic controls with unrelated conditions (p=.0023 and p&lt;.0001). Thus the decrease in the overall levels of available PR3 can be related not only to lower membrane but also circulating PR3 levels in PNH. However, when we tested either probands with NB1/PR3-null phenotype or patients with lower NB1/PR3 membrane expression, normal or elevated level of sPR3 was found, likely compensating for membrane PR deficiency. Our study demonstrates a novel mechanism that may be contributing to the increased thrombotic risk in PNH. Among PNH patients who experienced thrombotic complications 7/9 showed decreased sPR3 and significantly lower level of mPR3 than patients without history of thrombotic events.

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