Activation of a Plasma Membrane–Associated Neutral Sphingomyelinase and Concomitant Ceramide Accumulation During IgG-Dependent Phagocytosis in Human Polymorphonuclear Leukocytes

Blood ◽  
1998 ◽  
Vol 91 (12) ◽  
pp. 4761-4769 ◽  
Author(s):  
Vania Hinkovska-Galcheva ◽  
Lars Kjeldsen ◽  
Pamela J. Mansfield ◽  
Laurence A. Boxer ◽  
James A. Shayman ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Polymorphonuclear Leukocytes ◽  
Fold Increase ◽  
Acid Sphingomyelinase ◽  
Functional Responses ◽  
Neutral Sphingomyelinase ◽  
Sphingomyelin Cycle ◽  
Human Pmns ◽  
Ceramide Accumulation ◽  
Human Polymorphonuclear Leukocytes

Abstract The sphingomyelin cycle, which plays an important role in regulation of cell growth, differentiation, and apoptosis, involves the formation of ceramide by the action of a membrane-associated, Mg2+-dependent, neutral sphingomyelinase and/or a lysosomal acid sphingomyelinase. In human polymorphonuclear leukocytes (PMNs), ceramide production correlates with and plays a role in the regulation of functional responses such as oxidant release and Fcγ receptor-mediated phagocytosis. To increase our understanding of the sphingomyelin cycle in human PMNs, the cellular location of neutral and acid sphingomyelinases was investigated in resting, formylmethionylleucylphenylalanine (FMLP)-activated, and FMLP-activated PMNs engaged in phagocytosis. In resting PMNs, a Mg2+-dependent, neutral sphingomyelinase was the predominant activity and was localized to the plasma membrane fractions along with the majority of ceramide. Upon FMLP-activation, there was a 1.9-fold increase in this neutral, Mg2+-dependent sphingomyelinase activity, which increased to 2.7-fold subsequent to phagocytosis of IgG opsonized targets. This increase in sphingomyelinase activity was restricted to the plasma membrane fractions, which were also the site of increased ceramide levels. Phospholipase D (PLD) activity, which is a target of ceramide action and is required for phagocytosis, was also found primarily in the plasma membrane fractions of FMLP-activated and phagocytosing PMNs. Our findings indicate that in human PMNs engaged in phagocytosis, the sphingomyelin cycle is restricted to the plasma membrane where intracellular targets of ceramide action, such as PLD, are localized.

Activation of a Plasma Membrane–Associated Neutral Sphingomyelinase and Concomitant Ceramide Accumulation During IgG-Dependent Phagocytosis in Human Polymorphonuclear Leukocytes

Blood ◽  
1998 ◽  
Vol 91 (12) ◽  
pp. 4761-4769 ◽  
Author(s):  
Vania Hinkovska-Galcheva ◽  
Lars Kjeldsen ◽  
Pamela J. Mansfield ◽  
Laurence A. Boxer ◽  
James A. Shayman ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Polymorphonuclear Leukocytes ◽  
Fold Increase ◽  
Acid Sphingomyelinase ◽  
Functional Responses ◽  
Neutral Sphingomyelinase ◽  
Sphingomyelin Cycle ◽  
Human Pmns ◽  
Ceramide Accumulation ◽  
Human Polymorphonuclear Leukocytes

The sphingomyelin cycle, which plays an important role in regulation of cell growth, differentiation, and apoptosis, involves the formation of ceramide by the action of a membrane-associated, Mg2+-dependent, neutral sphingomyelinase and/or a lysosomal acid sphingomyelinase. In human polymorphonuclear leukocytes (PMNs), ceramide production correlates with and plays a role in the regulation of functional responses such as oxidant release and Fcγ receptor-mediated phagocytosis. To increase our understanding of the sphingomyelin cycle in human PMNs, the cellular location of neutral and acid sphingomyelinases was investigated in resting, formylmethionylleucylphenylalanine (FMLP)-activated, and FMLP-activated PMNs engaged in phagocytosis. In resting PMNs, a Mg2+-dependent, neutral sphingomyelinase was the predominant activity and was localized to the plasma membrane fractions along with the majority of ceramide. Upon FMLP-activation, there was a 1.9-fold increase in this neutral, Mg2+-dependent sphingomyelinase activity, which increased to 2.7-fold subsequent to phagocytosis of IgG opsonized targets. This increase in sphingomyelinase activity was restricted to the plasma membrane fractions, which were also the site of increased ceramide levels. Phospholipase D (PLD) activity, which is a target of ceramide action and is required for phagocytosis, was also found primarily in the plasma membrane fractions of FMLP-activated and phagocytosing PMNs. Our findings indicate that in human PMNs engaged in phagocytosis, the sphingomyelin cycle is restricted to the plasma membrane where intracellular targets of ceramide action, such as PLD, are localized.

scholarly journals Fodrin and band 4.1 in a plasma membrane-associated fraction of human neutrophils

Blood ◽  
1989 ◽  
Vol 74 (6) ◽  
pp. 2136-2143 ◽  
Author(s):  
KB Stevenson ◽  
RA Clark ◽  
WM Nauseef
Keyword(s):  
Plasma Membrane ◽  
Actin Filaments ◽  
Polymorphonuclear Leukocytes ◽  
Membrane Vesicles ◽  
Human Neutrophils ◽  
Plasma Membrane Vesicles ◽  
Associated Proteins ◽  
Human Pmns ◽  
Discontinuous Percoll Gradient ◽  
Human Polymorphonuclear Leukocytes

Abstract Erythrocytes possess a well-characterized submembranous filamentous network which interacts with transmembrane glycoproteins and is composed primarily of spectrin, ankyrin, band 4.1, and short actin filaments. An analogous structure was recently described in platelets. Human polymorphonuclear leukocytes (PMNs) were examined for the presence and plasma membrane association of similar proteins. Isolated PMNs, free of contamination with erythrocytes or platelets, were disrupted by nitrogen cavitation and separated into subcellular organelles on a discontinuous Percoll gradient. Detergent lysates of plasma membrane vesicles, but not azurophilic or specific granules, contained insoluble actin filaments and associated proteins. Immunoblots of detergent-insoluble plasma membrane fractions contained proteins recognized by antibodies to brain fodrin and erythrocyte band 4.1, whereas blots probed with antibodies to erythrocyte spectrin and ankyrin were negative. Fodrin and band 4.1 were not detected in granule fractions, but some fodrin was present in the cytosol. The association of proteins related to fodrin and band 4.1 with the plasma membrane suggests that PMNs contain a submembranous skeleton structurally analogous to that of erythrocytes and platelets. The specific function of these proteins and their structural organization in human PMNs await further study.

scholarly journals Fodrin and band 4.1 in a plasma membrane-associated fraction of human neutrophils

Blood ◽  
1989 ◽  
Vol 74 (6) ◽  
pp. 2136-2143
Author(s):  
KB Stevenson ◽  
RA Clark ◽  
WM Nauseef
Keyword(s):  
Plasma Membrane ◽  
Actin Filaments ◽  
Polymorphonuclear Leukocytes ◽  
Membrane Vesicles ◽  
Human Neutrophils ◽  
Plasma Membrane Vesicles ◽  
Associated Proteins ◽  
Human Pmns ◽  
Discontinuous Percoll Gradient ◽  
Human Polymorphonuclear Leukocytes

Erythrocytes possess a well-characterized submembranous filamentous network which interacts with transmembrane glycoproteins and is composed primarily of spectrin, ankyrin, band 4.1, and short actin filaments. An analogous structure was recently described in platelets. Human polymorphonuclear leukocytes (PMNs) were examined for the presence and plasma membrane association of similar proteins. Isolated PMNs, free of contamination with erythrocytes or platelets, were disrupted by nitrogen cavitation and separated into subcellular organelles on a discontinuous Percoll gradient. Detergent lysates of plasma membrane vesicles, but not azurophilic or specific granules, contained insoluble actin filaments and associated proteins. Immunoblots of detergent-insoluble plasma membrane fractions contained proteins recognized by antibodies to brain fodrin and erythrocyte band 4.1, whereas blots probed with antibodies to erythrocyte spectrin and ankyrin were negative. Fodrin and band 4.1 were not detected in granule fractions, but some fodrin was present in the cytosol. The association of proteins related to fodrin and band 4.1 with the plasma membrane suggests that PMNs contain a submembranous skeleton structurally analogous to that of erythrocytes and platelets. The specific function of these proteins and their structural organization in human PMNs await further study.

scholarly journals Entry of Sanfetrinem into Human Polymorphonuclear Granulocytes and Its Cell-Associated Activity against Intracellular, Penicillin-Resistant Streptococcus pneumoniae

1998 ◽  
Vol 42 (7) ◽  
pp. 1745-1750 ◽  
Author(s):  
Anna Maria Cuffini ◽  
Vivian Tullio ◽  
Alessandro Bonino ◽  
Alessandra Allocco ◽  
Angela Ianni Palarchio ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Polymorphonuclear Leukocytes ◽  
Environmental Temperature ◽  
Metabolic Inhibitor ◽  
Intracellular Pathogens ◽  
New Class ◽  
Polymorphonuclear Granulocytes ◽  
Human Pmns ◽  
Energy Dependent ◽  
Human Polymorphonuclear Leukocytes

ABSTRACT The entry of antibiotics into phagocytes is necessary for activity against intracellular pathogens. The ability of sanfetrinem, the first member of a new class of antibiotics, to penetrate human polymorphonuclear granulocytes and its consequences upon subsequent phagocytosis and killing of ingested penicillin-resistantStreptococcus pneumoniae have been evaluated. Sanfetrinem penetrated into human polymorphonuclear leukocytes (PMNs) at all concentrations tested, with cellular concentration/extracellular concentration ratios of 6.6 to 5.03 and 4.21 when sanfetrinem was used at 0.25 to 0.5 and 1 μg/ml, respectively, within 30 min of incubation. The uptake was complete within 5 min and was not energy dependent, since it was not affected by cell viability, environmental temperature, or the addition of a metabolic inhibitor. At a concentration of one-half the MIC, sanfetrinem significantly enhanced human PMN phagocytosis and increased intracellular bactericidal activity against penicillin-resistant S. pneumoniae. Following preexposure of PMNs to a concentration of one-half the MIC of sanfetrinem, there was a significant increase in both phagocytosis and killing compared with that for the controls, indicating the ability of sanfetrinem to interact with biological membranes and remain active within PMNs. Preexposure of streptococci to sanfetrinem made penicillin-resistant S. pneumoniae more susceptible to the bactericidal mechanisms of human PMNs than untreated organisms.

scholarly journals Thymosin beta 4 sequesters the majority of G-actin in resting human polymorphonuclear leukocytes.

1992 ◽  
Vol 119 (5) ◽  
pp. 1261-1270 ◽  
Author(s):  
L Cassimeris ◽  
D Safer ◽  
V T Nachmias ◽  
S H Zigmond
Keyword(s):  
Polymorphonuclear Leukocytes ◽  
Actin Polymerization ◽  
Large Fraction ◽  
Reverse Phase Hplc ◽  
Thymosin Beta 4 ◽  
Rapid Changes ◽  
Human Pmns ◽  
Human Polymorphonuclear Leukocytes ◽  
Kinetics Of

Thymosin beta 4 (T beta 4), a 5-kD peptide which binds G-actin and inhibits its polymerization (Safer, D., M. Elzinga, and V. T. Nachmias. 1991. J. Biol. Chem. 266:4029-4032), appears to be the major G-actin sequestering protein in human PMNs. In support of a previous study by Hannappel, E., and M. Van Kampen (1987. J. Chromatography. 397:279-285), we find that T beta 4 is an abundant peptide in these cells. By reverse phase HPLC of perchloric acid supernatants, human PMNs contain approximately 169 fg/cell +/- 90 fg/cell (SD), corresponding to a cytoplasmic concentration of approximately 149 +/- 80.5 microM. On non-denaturing polyacrylamide gels, a large fraction of G-actin in supernatants prepared from resting PMNs has a mobility similar to the G-actin/T beta 4 complex. Chemoattractant stimulation of PMNs results in a decrease in this G-actin/T beta 4 complex. To determine whether chemoattractant induced actin polymerization results from an inactivation of T beta 4, the G-actin sequestering activity of supernatants prepared from resting and chemoattractant stimulated cells was measured by comparing the rates of pyrenyl-actin polymerization from filament pointed ends. Pyrenyl actin polymerization was inhibited to a greater extent in supernatants from stimulated cells and these results are qualitatively consistent with T beta 4 being released as G-actin polymerizes, with no chemoattractant-induced change in its affinity for G-actin. The kinetics of bovine spleen T beta 4 binding to muscle pyrenyl G-actin are sufficiently rapid to accommodate the rapid changes in actin polymerization and depolymerization observed in vivo in response to chemoattractant addition and removal.

scholarly journals Antipseudomonal Agents Exhibit Differential Pharmacodynamic Interactions with Human Polymorphonuclear Leukocytes against Established Biofilms of Pseudomonas aeruginosa

2015 ◽  
Vol 59 (4) ◽  
pp. 2198-2205 ◽  
Author(s):  
Athanasios Chatzimoschou ◽  
Maria Simitsopoulou ◽  
Charalampos Antachopoulos ◽  
Thomas J. Walsh ◽  
Emmanuel Roilides
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Respiratory Tract ◽  
Polymorphonuclear Leukocytes ◽  
Planktonic Cells ◽  
Phagocytic Cells ◽  
Positive Interaction ◽  
Content Type ◽  
Pharmacodynamic Interactions ◽  
Human Pmns ◽  
Human Polymorphonuclear Leukocytes

ABSTRACTPseudomonas aeruginosais the most common pathogen infecting the lower respiratory tract of cystic fibrosis (CF) patients, where it forms tracheobronchial biofilms.Pseudomonasbiofilms are refractory to antibacterials and to phagocytic cells with innate immunity, leading to refractory infection. Little is known about the interaction between antipseudomonal agents and phagocytic cells in eradication ofP. aeruginosabiofilms. Herein, we investigated the capacity of three antipseudomonal agents, amikacin (AMK), ceftazidime (CAZ), and ciprofloxacin (CIP), to interact with human polymorphonuclear leukocytes (PMNs) against biofilms and planktonic cells ofP. aeruginosaisolates recovered from sputa of CF patients. Three of the isolates were resistant and three were susceptible to each of these antibiotics. The concentrations studied (2, 8, and 32 mg/liter) were subinhibitory for biofilms of resistant isolates, whereas for biofilms of susceptible isolates, they ranged between sub-MIC and 2 × MIC values. The activity of each antibiotic alone or in combination with human PMNs against 48-h mature biofilms or planktonic cells was determined by XTT [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] assay. All combinations of AMK with PMNs resulted in synergistic or additive effects against planktonic cells and biofilms ofP. aeruginosaisolates compared to each component alone. More than 75% of CAZ combinations exhibited additive interactions against biofilms ofP. aeruginosaisolates, whereas CIP had mostly antagonistic interaction or no interaction with PMNs against biofilms ofP. aeruginosa. Our findings demonstrate a greater positive interaction between AMK with PMNs than that observed for CAZ and especially CIP against isolates ofP. aeruginosafrom the respiratory tract of CF patients.

scholarly journals Role of microtubule assembly in lysosomal enzyme secretion from human polymorphonuclear leukocytes. A reevaluation.

1977 ◽  
Vol 73 (1) ◽  
pp. 242-256 ◽  
Author(s):  
S Hoffstein ◽  
I M Goldstein ◽  
G Weissmann
Keyword(s):  
Plasma Membrane ◽  
Dose Response ◽  
Lysosomal Enzyme ◽  
Polymorphonuclear Leukocytes ◽  
Cytochalasin B ◽  
Microtubule Assembly ◽  
Enzyme Release ◽  
Thin Sections ◽  
Human Polymorphonuclear Leukocytes ◽  
In Cells

The dose-related inhibition by colchicine of both lysosomal enzyme release and microtubule assembly was studied in human polymorphonuclear leukocytes (PMN) exposed to the nonphagocytic stimulus, zymosan-treated serum (ZTS). Cells were pretreated with colchicine (60 min, 37 degrees C) with or without cytochalasin B (5 microng/ml, 10 min) and then stimulated with ZTS (10%). Microtubule numbers in both cytochalasin B-treated and untreated PMN were increased by stimulation and depressed below resting levels in a dose-response fashion by colchicine concentrations above 10(-7) M. These concentrations also inhibited enzyme release in a dose-response fashion although the inhibition of microtubule assembly was proportionately greater than the inhibition of enzyme release. Other aspects of PMN morphology were affected by colchicine. Cytochalasin B-treated PMN were rounded, and in thin sections the retracted plasma membrane appeared as invaginations oriented toward centrally located centrioles. Membrane invaginations were restricted to the cell periphery in cells treated with inhibitory concentrations of colchicine, and the centrioles and Golgi apparatus were displaced from their usual position. After stimulation and subsequent degranulation, the size and number of membrane invaginations greatly increased. They remained peripheral in cells pretreated with greater than 10(-7) M colchicine but were numerous in the pericentriolar region in cells treated with less than 10(-7) M. Similarly, untreated PMN that were permitted to phagocytose immune precipitates had many phagosomes adjacent to the centriole. After colchicine treatment, phagosomes were distributed randomly, without any preferential association with the centrioles. These data suggest that microtubules are involved in maintaining the internal organization of cells and the topologic relationships between organelles and the plasma membrane.

scholarly journals Basis for the Failure of Francisella tularensis Lipopolysaccharide To Prime Human Polymorphonuclear Leukocytes

2006 ◽  
Vol 74 (6) ◽  
pp. 3277-3284 ◽  
Author(s):  
Jason H. Barker ◽  
Jerrold Weiss ◽  
Michael A. Apicella ◽  
William M. Nauseef
Keyword(s):  
Francisella Tularensis ◽  
Innate Immune System ◽  
Polymorphonuclear Leukocytes ◽  
Innate Immune ◽  
Mononuclear Leukocytes ◽  
Live Vaccine Strain ◽  
Gram Negative ◽  
Human Pmns ◽  
Human Polymorphonuclear Leukocytes ◽  
Lps Binding

ABSTRACT Francisella tularensis is the intracellular gram-negative coccobacillus that causes tularemia, and its virulence and infectiousness make it a potential agent of bioterrorism. Previous studies using mononuclear leukocytes have shown that the lipopolysaccharide (LPS) of F. tularensis is neither a typical proinflammatory endotoxin nor an endotoxin antagonist. This inertness suggests that F. tularensis LPS does not bind host LPS-sensing molecules such as LPS-binding protein (LBP). Using priming of the polymorphonuclear leukocyte (PMN) oxidase as a measure of endotoxicity, we found that F. tularensis live vaccine strain LPS did not behave like either a classic endotoxin or an endotoxin antagonist in human PMNs, even when the concentration of LBP was limiting. Furthermore, F. tularensis LPS did not compete with a radiolabeled lipooligosaccharide from Neisseria meningitidis for binding to LBP or to the closely related PMN granule protein, bactericidal/permeability-increasing protein. Our results suggest that the inertness of F. tularensis LPS and the resistance of F. tularensis to oxygen-independent PMN killing may result from the inability of F. tularensis LPS to be recognized by these important LPS-sensing molecules of the innate immune system.

scholarly journals Functional activity of enucleated human polymorphonuclear leukocytes.

1983 ◽  
Vol 97 (2) ◽  
pp. 368-377 ◽  
Author(s):  
D Roos ◽  
A A Voetman ◽  
L J Meerhof
Keyword(s):  
Plasma Membrane ◽  
Polymorphonuclear Leukocytes ◽  
Cytochalasin B ◽  
Unit Area ◽  
Chemotactic Activity ◽  
Myristate Acetate ◽  
Intact Cells ◽  
Ficoll Gradient ◽  
Pmn Functions ◽  
Human Polymorphonuclear Leukocytes

Enucleated human polymorphonuclear leukocytes (PMN) were prepared by centrifuging isolated, intact PMN over a discontinuous Ficoll gradient that contained 20 microM cytochalasin B. The enucleated cells (PMN cytoplasts) contained about one-third of the plasma membrane and about one-half of the cytoplasm present in intact PMN. The PMN cytoplasts contained no nucleus and hardly any granules. The volume of the PMN cytoplasts was about one-fourth of that of the original PMN. Greater than 90% of the PMN cytoplasts had an "outside-out" topography of the plasma membrane. Cytoplasts prepared from resting PMN did not generate superoxide radicals (O2-) or hydrogen peroxide. PMN cytoplasts incubated with opsonized zymosan particles or phorbol-myristate acetate induced a respiratory burst that was qualitatively (O2 consumption, O2- and H2O2 generation) and quantitatively (per unit area of plasma membrane) comparable with that of intact, stimulated PMN. Moreover, at low ratios of bacteria/cells, PMN cytoplasts ingested opsonized Staphylococcus aureus bacteria as well as did intact PMN. At higher ratios, the cytoplasts phagocytosed less well. The killing of these bacteria by PMN cytoplasts was slower than by intact cells. The chemotactic activity of PMN cytoplasts was very low. These results indicate that the PMN apparatus for phagocytosis, generation of bactericidal oxygen compounds, and killing of bacteria, as well as the mechanism for recognizing opsonins and activating PMN functions, are present in the plasma membrane and cytosol of these cells.

scholarly journals Ceramide Inhibits IgG-Dependent Phagocytosis in Human Polymorphonuclear Leukocytes

Blood ◽  
1997 ◽  
Vol 89 (6) ◽  
pp. 2139-2147 ◽  
Author(s):  
Suzanne J. Suchard ◽  
Vania Hinkovska-Galcheva ◽  
Pamela J. Mansfield ◽  
Laurence A. Boxer ◽  
James A. Shayman
Keyword(s):  
Polymorphonuclear Leukocytes ◽  
Cell Types ◽  
Short Chain ◽  
Sphingolipid Metabolism ◽  
Sphingomyelin Cycle ◽  
Intracellular Target ◽  
Human Polymorphonuclear Leukocytes ◽  
Kinetics Of ◽  
Hydrolysis Of ◽  
Ceramide Production

Abstract Ceramide is a product of agonist-induced sphingolipid metabolism in several cell types, including polymorphonuclear leukocytes (PMNs). In adherent PMNs, the kinetics of ceramide production correspond with the termination of fMLP-stimulated H2O2 release. Furthermore, short chain ceramides inhibit fMLP-mediated H2O2 release in adherent PMNs. In the present study, we investigated the effects of short chain ceramides and sphingoid bases on phagocytosis of IgG-opsonized erythrocytes (EIgG) by suspended PMNs activated with fMLP. N-Acetylsphingosine, N-acetylphytosphingosine, phytosphingosine, sphingosine, and dihydrosphingosine, but not N-acetyldihydrosphingosine, inhibited phagocytosis of EIgG. In contrast, these same lipids did not inhibit fMLP-mediated chemotaxis. Endogenous ceramide levels increased within the first few minutes of phagocytosis, with a significant (P < .05) accumulation by 30 minutes, the time by which phagocytosis was terminated. Neutral sphingomyelinase activity paralleled the increase in ceramide, consistent with the generation of ceramide by the hydrolysis of sphingomyelin. The N-acetyl-conjugated sphingols (C2 ceramides) blocked phosphatidylethanol formation indicating that phospholipase D (PLD) is an intracellular target of ceramide action. These data suggest that ceramides, generated through activation of the sphingomyelin cycle, act as negative regulators of FcγR-mediated phagocytosis.

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