scholarly journals Differences in myeloperoxidase activity from neutrophilic polymorphonuclear leukocytes of differing density: relationship to selective exocytosis of distinct forms of the enzyme

Blood ◽  
1983 ◽  
Vol 61 (6) ◽  
pp. 1116-1124
Author(s):  
SO Pember ◽  
JM Jr Kinkade
Keyword(s):  
Polymorphonuclear Leukocytes ◽  
Human Peripheral Blood ◽  
Gradient Centrifugation ◽  
Fold Increase ◽  
Myeloperoxidase Activity ◽  
Percoll Density Gradient Centrifugation ◽  
Density Relationship ◽  
Neutrophilic Polymorphonuclear Leukocytes

Elicited murine neutrophilic polymorphonuclear leukocytes (PMN) were fractionated by Percoll density gradient centrifugation into high density (HD) and intermediate density (ID) populations. As described in the accompanying article HD- and ID-PMN appear to represent “resting” and “activated” cell populations, respectively. Consistent with this possibility, histochemical and biochemical evidence suggested that ID- PMN were degranulated compared to HD-PMN. Myeloperoxidase (MPO) in the ID-PMN population showed increased sensitivity to inhibition by 3-amino- 1,2,4-triazole, and HD-PMN exhibited a 2–3-fold increase in chloride and iodide oxidation per unit of MPO activity compared to ID-PMN. When HD-PMN were induced to degranulate in vitro, the remaining cell- associated MPO displayed enzymatic properties characteristic of the activity associated with ID-PMN. The mechanism of this phenomenon was also investigated in vitro using purified human peripheral blood PMN and the synthetic chemotactic peptide N-formyl-methionyl-leucyl- phenylalanine. Differences in cell-associated MPO activity were shown to be related to selective exocytosis of enzymatically and chromatographically distinct forms of the enzyme. These data indicate that, in addition to the well known selective exocytosis of specific and azurophilic granules induced by various agents, selectivity may also occur at the level of enzymatically distinct forms of a particular granule enzyme. Moreover, our observations provide further evidence that density differences may be utilized to fractionate and study the generation of functionally distinct subpopulations of PMN that arise in vivo as well as in vitro following exposure to various stimuli.

scholarly journals Differences in myeloperoxidase activity from neutrophilic polymorphonuclear leukocytes of differing density: relationship to selective exocytosis of distinct forms of the enzyme

Blood ◽  
1983 ◽  
Vol 61 (6) ◽  
pp. 1116-1124 ◽  
Author(s):  
SO Pember ◽  
JM Jr Kinkade
Keyword(s):  
Polymorphonuclear Leukocytes ◽  
Human Peripheral Blood ◽  
Gradient Centrifugation ◽  
Fold Increase ◽  
Myeloperoxidase Activity ◽  
Percoll Density Gradient Centrifugation ◽  
Density Relationship ◽  
Neutrophilic Polymorphonuclear Leukocytes

Abstract Elicited murine neutrophilic polymorphonuclear leukocytes (PMN) were fractionated by Percoll density gradient centrifugation into high density (HD) and intermediate density (ID) populations. As described in the accompanying article HD- and ID-PMN appear to represent “resting” and “activated” cell populations, respectively. Consistent with this possibility, histochemical and biochemical evidence suggested that ID- PMN were degranulated compared to HD-PMN. Myeloperoxidase (MPO) in the ID-PMN population showed increased sensitivity to inhibition by 3-amino- 1,2,4-triazole, and HD-PMN exhibited a 2–3-fold increase in chloride and iodide oxidation per unit of MPO activity compared to ID-PMN. When HD-PMN were induced to degranulate in vitro, the remaining cell- associated MPO displayed enzymatic properties characteristic of the activity associated with ID-PMN. The mechanism of this phenomenon was also investigated in vitro using purified human peripheral blood PMN and the synthetic chemotactic peptide N-formyl-methionyl-leucyl- phenylalanine. Differences in cell-associated MPO activity were shown to be related to selective exocytosis of enzymatically and chromatographically distinct forms of the enzyme. These data indicate that, in addition to the well known selective exocytosis of specific and azurophilic granules induced by various agents, selectivity may also occur at the level of enzymatically distinct forms of a particular granule enzyme. Moreover, our observations provide further evidence that density differences may be utilized to fractionate and study the generation of functionally distinct subpopulations of PMN that arise in vivo as well as in vitro following exposure to various stimuli.

scholarly journals Peptide inhibition of neutrophil-mediated injury after in vivo challenge with supernatant of Pseudomonas aeruginosa and immune-complexes

PLoS ONE ◽  
2021 ◽  
Vol 16 (7) ◽  
pp. e0254353
Author(s):  
Adrianne Enos ◽  
Parvathi Kumar ◽  
Brittany Lassiter ◽  
Alana Sampson ◽  
Pamela Hair ◽  
...  
Keyword(s):  
Immune Complexes ◽  
Hypochlorous Acid ◽  
Inflammatory Diseases ◽  
Fold Increase ◽  
Neutrophil Extracellular Trap ◽  
Myeloperoxidase Activity ◽  
Free Dna ◽  
Dna 2

Neutrophils are recognized for their role in host defense against pathogens as well as inflammatory conditions mediated through many mechanisms including neutrophil extracellular trap (NET) formation and generation of reactive oxygen species (ROS). NETs are increasingly appreciated as a major contributor in autoimmune and inflammatory diseases such as cystic fibrosis. Myeloperoxidase (MPO), a key neutrophil granule enzyme mediates generation of hypochlorous acid which, when extracellular, can cause host tissue damage. To better understand the role played by neutrophils in inflammatory diseases, we measured and modulated myeloperoxidase activity and NETs in vivo, utilizing a rat peritonitis model. RLS-0071 is a 15 amino acid peptide that has been shown to inhibit myeloperoxidase activity and NET formation in vitro. The rat model of inflammatory peritonitis was induced with intraperitoneal injection of either P. aeruginosa supernatant or immune-complexes. After euthanasia, a peritoneal wash was performed and measured for myeloperoxidase activity and free DNA as a surrogate for measurement of NETs. P. aeruginosa supernatant caused a 2-fold increase in MPO activity and free DNA when injected IP. Immune-complexes injected IP increased myeloperoxidase activity and free DNA 2- fold. RLS-0071 injection decreased myeloperoxidase activity and NETs in the peritoneal fluid generally to baseline levels in the presence of P. aeruginosa supernatant or immune-complexes. Taken together, RLS-0071 demonstrated the ability to inhibit myeloperoxidase activity and NET formation in vivo when initiated by different inflammatory stimuli including shed or secreted bacterial constituents as well as immune-complexes.

scholarly journals Density heterogeneity of neutrophilic polymorphonuclear leukocytes: gradient fractionation and relationship to chemotactic stimulation

Blood ◽  
1983 ◽  
Vol 61 (6) ◽  
pp. 1105-1115 ◽  
Author(s):  
SO Pember ◽  
KC Barnes ◽  
SJ Brandt ◽  
JM Jr Kinkade
Keyword(s):  
Human Peripheral Blood ◽  
Gradient Centrifugation ◽  
Buoyant Density ◽  
Peritoneal Exudate ◽  
Polymorphonuclear Neutrophils ◽  
Mean Cell Volume ◽  
Peritoneal Exudate Cells ◽  
Percoll Density Gradient Centrifugation ◽  
Induced Aggregation

Abstract When elicited murine peritoneal exudate cells were subjected to Percoll density gradient centrifugation, polymorphonuclear neutrophils (PMN) were found to distribute over a broad spectrum of buoyant densities (1.10–1.06 g/ml). PMN isolated between approximately 1.10 and 1.085 g/ml were referred to as high density PMN (HD-PMN), and those isolated at approximately 1.085–1.06 g/ml were designated intermediate density PMN (ID-PMN). Cells were characterized on the basis of morphology and specific markers: PMN by lactoferrin immunocytofluorescence and macrophages by nicotinamide adenine dinucleotide glycohydrase activity. Macrophages banded near the top of the gradient with a peak at 1.04 g/ml. At increasing times following elicitation, the ratio of HD to ID- PMN decreased. Decreased density of either murine HD-PMN or human peripheral blood PMN could be induced in vitro by exposure of the cells to endotoxin-activated serum. A decrease in buoyant density of human PMN was also demonstrated in vitro using the synthetic chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP). The response was time dependent, related to dose, and appeared to be mediated by the cell membrane receptor for FMLP. A competitive antagonist of FMLP binding, carbobenzoxy-phenylalanyl-methionine, inhibited the density change with a calculated Kd similar to that reported for inhibition of FMLP-induced aggregation, degranulation, locomotion, and superoxide production. The FMLP-induced decrease in PMN density was shown to be directly correlated with increases in relative mean cell volume. The density response is a new measurement of PMN interaction with specific chemotactic factors, which may be important in the generation of PMN heterogeneity observed in elicited peritoneal exudate cells. In addition, this approach offers a means of physically separating “activated” from “resting” PMN and of studying resultant biochemical differences between these cell populations using both in vivo and in vitro systems.

scholarly journals Density heterogeneity of neutrophilic polymorphonuclear leukocytes: gradient fractionation and relationship to chemotactic stimulation

Blood ◽  
1983 ◽  
Vol 61 (6) ◽  
pp. 1105-1115 ◽  
Author(s):  
SO Pember ◽  
KC Barnes ◽  
SJ Brandt ◽  
JM Jr Kinkade
Keyword(s):  
Human Peripheral Blood ◽  
Gradient Centrifugation ◽  
Buoyant Density ◽  
Peritoneal Exudate ◽  
Polymorphonuclear Neutrophils ◽  
Mean Cell Volume ◽  
Peritoneal Exudate Cells ◽  
Percoll Density Gradient Centrifugation ◽  
Induced Aggregation

When elicited murine peritoneal exudate cells were subjected to Percoll density gradient centrifugation, polymorphonuclear neutrophils (PMN) were found to distribute over a broad spectrum of buoyant densities (1.10–1.06 g/ml). PMN isolated between approximately 1.10 and 1.085 g/ml were referred to as high density PMN (HD-PMN), and those isolated at approximately 1.085–1.06 g/ml were designated intermediate density PMN (ID-PMN). Cells were characterized on the basis of morphology and specific markers: PMN by lactoferrin immunocytofluorescence and macrophages by nicotinamide adenine dinucleotide glycohydrase activity. Macrophages banded near the top of the gradient with a peak at 1.04 g/ml. At increasing times following elicitation, the ratio of HD to ID- PMN decreased. Decreased density of either murine HD-PMN or human peripheral blood PMN could be induced in vitro by exposure of the cells to endotoxin-activated serum. A decrease in buoyant density of human PMN was also demonstrated in vitro using the synthetic chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP). The response was time dependent, related to dose, and appeared to be mediated by the cell membrane receptor for FMLP. A competitive antagonist of FMLP binding, carbobenzoxy-phenylalanyl-methionine, inhibited the density change with a calculated Kd similar to that reported for inhibition of FMLP-induced aggregation, degranulation, locomotion, and superoxide production. The FMLP-induced decrease in PMN density was shown to be directly correlated with increases in relative mean cell volume. The density response is a new measurement of PMN interaction with specific chemotactic factors, which may be important in the generation of PMN heterogeneity observed in elicited peritoneal exudate cells. In addition, this approach offers a means of physically separating “activated” from “resting” PMN and of studying resultant biochemical differences between these cell populations using both in vivo and in vitro systems.

scholarly journals Effect of Red Blood Cell Aging In Vivo on Their Aggregation Properties In Vitro: Measurements with Laser Tweezers

2020 ◽  
Vol 10 (21) ◽  
pp. 7581
Author(s):  
Petr Ermolinskiy ◽  
Andrei Lugovtsov ◽  
François Yaya ◽  
Kisung Lee ◽  
Lars Kaestner ◽  
...  
Keyword(s):  
Blood Cell ◽  
Red Blood Cell ◽  
Single Cells ◽  
Gradient Centrifugation ◽  
Cell Aging ◽  
Laser Tweezers ◽  
Percoll Density Gradient Centrifugation ◽  
Rbc Aggregation

Red blood cell (RBC) aggregation highly influences hemorheology and blood microcirculation in the human body. The aggregation properties of RBCs can vary due to numerous factors, including RBC age. The aim of this work was to estimate in vitro the differences in the RBC aggregation properties of different RBC age populations in single-cell experiments using laser tweezers. RBCs from five healthy volunteers were separated into four subpopulations by Percoll density gradient centrifugation. Each subpopulation of the RBC was separately resuspended in autologous plasma or dextran 70 kDa (50 mg/mL). The aggregation force between the single cells was measured with holographic laser tweezers. The obtained data demonstrated an enhancement of RBC aggregation force in doublets with age: the older the cells, the higher the aggregation force. The obtained data revealed the differences between the aggregation and aggregability of RBC in dependence of the RBC in vivo age.

Heavy endosomes isolated from the rat renal cortex show attributes of intermicrovillar clefts

1994 ◽  
Vol 267 (4) ◽  
pp. F516-F527 ◽  
Author(s):  
T. G. Hammond ◽  
P. J. Verroust ◽  
R. R. Majewski ◽  
K. E. Muse ◽  
T. D. Oberley
Keyword(s):  
Flow Cytometry ◽  
Brush Border ◽  
Renal Cortex ◽  
Gradient Centrifugation ◽  
Membrane Vesicles ◽  
Gamma Glutamyl Transpeptidase ◽  
Percoll Density Gradient Centrifugation ◽  
Enzyme Markers

The endosomal pathway of the rat renal cortex was labeled by intravenous infusion of fluorescent dextran small enough to cross the glomerular ultrafiltration barrier and be taken up by luminal endocytosis. A fraction containing entrapped fluorescein was isolated from a cortical homogenate after differential centrifugation and Percoll density gradient centrifugation. This fraction has been dubbed heavy endosomes. To our surprise, small-particle flow cytometry techniques demonstrated that heavy endosomes are homogeneous for entrapped fluorescein dextran and the presence of H(+)-adenosinetriphosphatase activity. The abundance of heavy endosomes, combined with the findings that true endosomal populations are identifiable in other renal cortical fractions, led us to test whether heavy endosomes had the attributes of intermicrovillar clefts. First, we tested whether heavy endosomes vesiculate in vivo or in vitro. Vesicle-by-vesicle flow cytometry analysis of uptake of fluorescein dextran added to the homogenate demonstrated that virtually all the vesicles form in vitro (99 +/- 2%, n = 4). Second, the fraction contains markers associated with intermicrovillar clefts: clathrin light chains, actin, glycoprotein gp280, and gp330, the "Heymann antigen." The presence of the brush border enzyme markers gamma-glutamyl transpeptidase and leucine aminopeptidase in > 99% of the heavy endosomes confirms that the vesicles are of apical origin. The activity of the enzymes colocalized with entrapped markers but was tenfold less than in brush-border membrane vesicles. Heavy endosomes isolated from the rat renal cortex vesiculate in vitro and contain several intermicrovillar markers.

Defibrotide Inhibits Platelet Activation by Cathepsin G Released From Stimulated Polymorphonuclear Leukocytes

1992 ◽  
Vol 67 (06) ◽  
pp. 660-664 ◽  
Author(s):  
Virgilio Evangelista ◽  
Paola Piccardoni ◽  
Giovanni de Gaetano ◽  
Chiara Cerletti
Keyword(s):  
Platelet Activation ◽  
Polymorphonuclear Leukocytes ◽  
Direct Interaction ◽  
Cathepsin G ◽  
In Vivo Test ◽  
Cell Functions ◽  
Test Models ◽  
Antithrombotic Effects

SummaryDefibrotide is a polydeoxyribonucleotide with antithrombotic effects in experimental animal models. Most of the actions of this drug have been observed in in vivo test models but no effects have been reported in in vitro systems. In this paper we demonstrate that defibrotide interferes with polymorphonuclear leukocyte-induced human platelet activation in vitro. This effect was not related to any direct interaction with polymorphonuclear leukocytes or platelets, but was due to the inhibition of cathepsin G, the main biochemical mediator of this cell-cell cooperation. Since cathepsin G not only induces platelet activation but also affects some endothelial cell functions, the anticathepsin G activity of defibrotide could help to explain the antithrombotic effect of this drug.

scholarly journals 1592U89, a novel carbocyclic nucleoside analog with potent, selective anti-human immunodeficiency virus activity.

1997 ◽  
Vol 41 (5) ◽  
pp. 1082-1093 ◽  
Author(s):  
S M Daluge ◽  
S S Good ◽  
M B Faletto ◽  
W H Miller ◽  
M H St Clair ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Oral Bioavailability ◽  
Human Peripheral Blood ◽  
Cross Resistance ◽  
Immunodeficiency Virus ◽  
Carbocyclic Nucleoside ◽  
Hiv 1 ◽  
In Cells

1592U89, (-)-(1S,4R)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclo pentene-1-methanol, is a carbocyclic nucleoside with a unique biological profile giving potent, selective anti-human immunodeficiency virus (HIV) activity. 1592U89 was selected after evaluation of a wide variety of analogs containing a cyclopentene substitution for the 2'-deoxyriboside of natural deoxynucleosides, optimizing in vitro anti-HIV potency, oral bioavailability, and central nervous system (CNS) penetration. 1592U89 was equivalent in potency to 3'-azido-3'-deoxythymidine (AZT) in human peripheral blood lymphocyte (PBL) cultures against clinical isolates of HIV type 1 (HIV-1) from antiretroviral drug-naive patients (average 50% inhibitory concentration [IC50], 0.26 microM for 1592U89 and 0.23 microM for AZT). 1592U89 showed minimal cross-resistance (approximately twofold) with AZT and other approved HIV reverse transcriptase (RT) inhibitors. 1592U89 was synergistic in combination with AZT, the nonnucleoside RT inhibitor nevirapine, and the protease inhibitor 141W94 in MT4 cells against HIV-1 (IIIB). 1592U89 was anabolized intracellularly to its 5'-monophosphate in CD4+ CEM cells and in PBLs, but the di- and triphosphates of 1592U89 were not detected. The only triphosphate found in cells incubated with 1592U89 was that of the guanine analog (-)-carbovir (CBV). However, the in vivo pharmacokinetic, distribution, and toxicological profiles of 1592U89 were distinct from and improved over those of CBV, probably because CBV itself was not appreciably formed from 1592U89 in cells or animals (<2%). The 5'-triphosphate of CBV was a potent, selective inhibitor of HIV-1 RT, with Ki values for DNA polymerases (alpha, beta, gamma, and epsilon which were 90-, 2,900-, 1,200-, and 1,900-fold greater, respectively, than for RT (Ki, 21 nM). 1592U89 was relatively nontoxic to human bone marrow progenitors erythroid burst-forming unit and granulocyte-macrophage CFU (IC50s, 110 microM) and human leukemic and liver tumor cell lines. 1592U89 had excellent oral bioavailability (105% in the rat) and penetrated the CNS (rat brain and monkey cerebrospinal fluid) as well as AZT. Having demonstrated an excellent preclinical profile, 1592U89 has progressed to clinical evaluation in HIV-infected patients.

scholarly journals Methylation of rRNA as a host defense against rampant group II intron retrotransposition

Mobile DNA ◽  
2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Justin M. Waldern ◽  
Dorie Smith ◽  
Carol Lyn Piazza ◽  
E. Jake Bailey ◽  
Nicholas J. Schiraldi ◽  
...  
Keyword(s):  
Insertional Mutagenesis ◽  
Fold Increase ◽  
Group Ii Intron ◽  
In Vivo Experiments ◽  
Cellular Factors ◽  
Group Ii ◽  
Self Splicing ◽  
Native Host

Abstract Background Group II introns are mobile retroelements, capable of invading new sites in DNA. They are self-splicing ribozymes that complex with an intron-encoded protein to form a ribonucleoprotein that targets DNA after splicing. These molecules can invade DNA site-specifically, through a process known as retrohoming, or can invade ectopic sites through retrotransposition. Retrotransposition, in particular, can be strongly influenced by both environmental and cellular factors. Results To investigate host factors that influence retrotransposition, we performed random insertional mutagenesis using the ISS1 transposon to generate a library of over 1000 mutants in Lactococcus lactis, the native host of the Ll.LtrB group II intron. By screening this library, we identified 92 mutants with increased retrotransposition frequencies (RTP-ups). We found that mutations in amino acid transport and metabolism tended to have increased retrotransposition frequencies. We further explored a subset of these RTP-up mutants, the most striking of which is a mutant in the ribosomal RNA methyltransferase rlmH, which exhibited a reproducible 20-fold increase in retrotransposition frequency. In vitro and in vivo experiments revealed that ribosomes in the rlmH mutant were defective in the m3Ψ modification and exhibited reduced binding to the intron RNA. Conclusions Taken together, our results reinforce the importance of the native host organism in regulating group II intron retrotransposition. In particular, the evidence from the rlmH mutant suggests a role for ribosome modification in limiting rampant retrotransposition.

scholarly journals Monocyte tissue factor induction by lipopolysaccharide (LPS): dependence on LPS-binding protein and CD14, and inhibition by a recombinant fragment of bactericidal/permeability-increasing protein

Blood ◽  
1994 ◽  
Vol 83 (9) ◽  
pp. 2516-2525 ◽  
Author(s):  
K Meszaros ◽  
S Aberle ◽  
R Dedrick ◽  
R Machovich ◽  
A Horwitz ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Binding Protein ◽  
Mononuclear Phagocytes ◽  
Factor Vii ◽  
Peripheral Blood Mononuclear ◽  
Recombinant Fragment

Abstract Mononuclear phagocytes, stimulated by bacterial lipopolysaccharide (LPS), have been implicated in the activation of coagulation in sepsis and endotoxemia. In monocytes LPS induces the synthesis of tissue factor (TF) which, assembled with factor VII, initiates the blood coagulation cascades. In this study we investigated the mechanism of LPS recognition by monocytes, and the consequent expression of TF mRNA and TF activity. We also studied the inhibition of these effects of LPS by rBPI23, a 23-kD recombinant fragment of bactericidal/permeability increasing protein, which has been shown to antagonize LPS in vitro and in vivo. Human peripheral blood mononuclear cells, or monocytes isolated by adherence, were stimulated with Escherichia coli O113 LPS at physiologically relevant concentrations (&gt; or = 10 pg/mL). The effect of LPS was dependent on the presence of the serum protein LBP (lipopolysaccharide-binding protein), as shown by the potentiating effect of human recombinant LBP or serum. Furthermore, recognition of low amounts of LPS by monocytes was also dependent on CD14 receptors, because monoclonal antibodies against CD14 greatly reduced the LPS sensitivity of monocytes in the presence of serum or rLBP. Induction of TF activity and mRNA expression by LPS were inhibited by rBPI23. The expression of tumor necrosis factor showed qualitatively similar changes. Considering the involvement of LPS-induced TF in the potentially lethal intravascular coagulation in sepsis, inhibition of TF induction by rBPI23 may be of therapeutic benefit.

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