scholarly journals Quantification of the locomotive behavior of polymorphonuclear leukocytes in clot preparations

Blood ◽  
1982 ◽  
Vol 59 (5) ◽  
pp. 946-951 ◽  
Author(s):  
TH Howard
Keyword(s):  
Polymorphonuclear Leukocytes ◽  
Normal Subjects ◽  
Time Lapse ◽  
Turning Behavior ◽  
Multiple Parameters ◽  
Human Pmns ◽  
Direction Distance ◽  
Active Locomotion ◽  
Locomotive Behavior

Abstract Time-lapse videotape recordings of polymorphonuclear leukocytes (PMNs) from clot preparations were used to quantify the locomotive behavior of individual PMNs from normal subjects. Tracings derived from the videotapes allow one to quantify multiple parameters of the locomotive behavior of PMNs--direction, distance, rate, and angle of turn. The results obtained are reproducible from subject-to-subject and from preparation-to-preparation. The method allows the investigator to record the locomotive behavior of 100 cells simultaneously within a 5- min period and analyze the recording as time permits. We utilized this technique to compare the locomotive behavior of slow and fast PMNs (arbitrarily defined as cells that move less than or equal to 7.0 micrometer/min and greater than 7.0 micrometer/min mean rate of locomotion, respectively). The studies show that slow and fast PMNs, thus defined, differ not only in mean rate of locomotion but also in their rate of locomotion during periods of active locomotion, in the number of periods of inactivity/PMN/5 min (slow = 1.65 +/- 0.31; fast = 0.36 +/- 0.12), and in their turning behavior as measured by angle of turn (slow = 92 degrees +/- 39 degrees; fast = 39 degrees +/- 35 degrees). These results show that human PMNs from clot preparations are remarkably heterogeneous in their locomotive behavior, and the results suggest this heterogeneity is due to endogenous differences within cells.

scholarly journals Quantification of the locomotive behavior of polymorphonuclear leukocytes in clot preparations

Blood ◽  
1982 ◽  
Vol 59 (5) ◽  
pp. 946-951
Author(s):  
TH Howard
Keyword(s):  
Polymorphonuclear Leukocytes ◽  
Normal Subjects ◽  
Time Lapse ◽  
Turning Behavior ◽  
Multiple Parameters ◽  
Human Pmns ◽  
Direction Distance ◽  
Active Locomotion ◽  
Locomotive Behavior

Time-lapse videotape recordings of polymorphonuclear leukocytes (PMNs) from clot preparations were used to quantify the locomotive behavior of individual PMNs from normal subjects. Tracings derived from the videotapes allow one to quantify multiple parameters of the locomotive behavior of PMNs--direction, distance, rate, and angle of turn. The results obtained are reproducible from subject-to-subject and from preparation-to-preparation. The method allows the investigator to record the locomotive behavior of 100 cells simultaneously within a 5- min period and analyze the recording as time permits. We utilized this technique to compare the locomotive behavior of slow and fast PMNs (arbitrarily defined as cells that move less than or equal to 7.0 micrometer/min and greater than 7.0 micrometer/min mean rate of locomotion, respectively). The studies show that slow and fast PMNs, thus defined, differ not only in mean rate of locomotion but also in their rate of locomotion during periods of active locomotion, in the number of periods of inactivity/PMN/5 min (slow = 1.65 +/- 0.31; fast = 0.36 +/- 0.12), and in their turning behavior as measured by angle of turn (slow = 92 degrees +/- 39 degrees; fast = 39 degrees +/- 35 degrees). These results show that human PMNs from clot preparations are remarkably heterogeneous in their locomotive behavior, and the results suggest this heterogeneity is due to endogenous differences within cells.

PLATELET-DEPENDENT ACTIVATION AND AMPLIFICATION OF THE POLYMORPHONUCLEAR LEUKOCYTES LYSOSOMAL ENZYME RELEASE

1987 ◽  
Author(s):  
A Del Maschio ◽  
E Corvazier ◽  
F Maillet ◽  
M Kazatchkine ◽  
J Maclouf
Keyword(s):  
Lysosomal Enzyme ◽  
Polymorphonuclear Leukocytes ◽  
Maximal Effect ◽  
Enzyme Release ◽  
Maximal Intensity ◽  
Platelet Concentration ◽  
Human Pmns ◽  
Stimulation Of

The degranulatlon of human PMNs by opsonlsed zymosan (OpZ) was studied In the presence or In the absence of platelet alone or after stimulation by thrombin. Evidence Is presented that the presence of platelets Increased the extent of the liberation of lysozyme from PMNs stimulated by OpZ with a maximal intensity when they were stimulated by thrombin. The extent of the amplification was higher when the PMNs trigger was lower (i.e. 0.5 x 108 particles/ml as compared to 3.0 x 108 particles). This effect was dependent on the platelet concentration (from 10-80 platelets/PMN). Platelets stimulated by thrombin could alsoactivate the resting PMNs with a maximum obtained ata thrombin concentration of 0.1 U/ml, corresponding to the maximal release by these cells of products stored In their granules. However, the substitution ofplatelet suspensions by the released products found In their supernatant after stimulation by thrombin, resulted In a comparable stimulation only at platelet concentrations above the ones for coincubation experiments. These findings suggest that the presence of platelets themselves or In combination with their released products are responsible for this amplification. The use of zymosan alone or coated with IgG, C3b1, C3b or OpZ did not reveal any specificity of the Inducer for this amplification suggesting that platelets and/or platelet products acted by enhancing acommon step of PMNs activation Independent of the stimulus carried by the particles. Additionally, It could be noted that the maximal effect of the amplification by platelets occurred when the level of stimulation of the PMNs alone was the weakest.

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 Membrane fluidity changes accompanying phagocytosis in normal and in chronic granulomatous disease polymorphonuclear leukocytes

Blood ◽  
1981 ◽  
Vol 58 (4) ◽  
pp. 830-835 ◽  
Author(s):  
LM Ingraham ◽  
LA Boxer ◽  
RA Haak ◽  
RL Baehner
Keyword(s):  
Membrane Fluidity ◽  
Polymorphonuclear Leukocytes ◽  
Direct Reduction ◽  
Normal Subjects ◽  
Esr Spectra ◽  
Resting Cells ◽  
Opsonized Zymosan ◽  
Acid Analog ◽  
Spin Resonance ◽  
By Products

Abstract We have studied membrane fluidity changes in polymorphonuclear leukocytes (PMN) during phagocytosis. Membrane fluidity was assessed by electron spin resonance (ESR) using a nitroxide-substituted stearic acid analog (5DS) as a spin probe. PMN from normal subjects and from 3 CGD patients (2 males, 1 female) were incubated in Kreb's Ringers phosphate with or without opsonized zymosan. ESR spectra were obtained and the order parameter (S), which is inversely related to membrane fluidity, was calculated. Without zymosan addition, S for normal (0.638) and for CGD (0.635) were not significantly different (p less than 0.35). The S values indicate that under resting conditions the molecular environment of the CGD membrane is similar to that of normal PMN membranes. However, with addition of opsonized zymosan, the normal, but not the CGD, PMN showed a significant increase (CGD, S = 0.638; normal, S = 0.647; p less than 0.001). This change in S for the normals is consistent with a more restricted movement of 5DS. Treatment of normal PMN with a mixture of scavengers specific for H2O2 (catalase, 1600 U/ml), O2-.(superoxide dismutase, 100 micrograms/ml), and for HO., (sodium benzoate, 1mM) during zymosan stimulation gave S values similar to those of resting cells. Catalase alone also lowered S value, suggesting that H2O2 was instrumental in causing the initial S value increase. This idea was supported by studies in which CGD cells were incubated with zymosan in the presence of glucose oxidase, an enzyme that catalyzes glucose oxidation resulting in the direct reduction of molecular oxygen to H2O2. Our results indicate that reduced O2 by- products, particularly H2O2, can cause altered biophysical properties of PMN membrane during phagocytosis.

scholarly journals Mechanisms for actin reorganization in chemotactic factor-activated polymorphonuclear leukocytes

Blood ◽  
1993 ◽  
Vol 81 (10) ◽  
pp. 2750-2757
Author(s):  
RG Watts ◽  
TH Howard
Keyword(s):  
Polymorphonuclear Leukocytes ◽  
Actin Polymerization ◽  
Insoluble Fraction ◽  
Chemotactic Factor ◽  
Actin Dynamics ◽  
Cross Linking ◽  
Actin Reorganization ◽  
Cytoskeletal Structure ◽  
Cross Links ◽  
Human Pmns

Cytoskeletal structure in polymorphonuclear leukocytes (PMNs) is thought to reflect a simple equilibrium between two actin pools (globular [G]- and filamentous [F] actin). Recent description of two distinct F-actin pools in PMNs (Triton-insoluble [stable] and Triton- soluble [labile] F-actin pools) (Watts and Howard, Cell Motil Cytoskeleton, 21:25, 1992) suggest a tripartite equilibrium between these F-actin pools and G-actin and multiple possible mechanisms for polymerization. To study the contribution of each actin pool to actin dynamics in PMNs, changes in actin content of the Triton-soluble and - insoluble F-actin pools and G-actin in chemotactic factor (CTF)- activated PMNs were measured by NBDphallacidin binding and by gel scans of Triton-lysed PMNs. From 0 to 30 seconds after CTF activation, PMNs rapidly increase total (Triton-soluble + Triton-insoluble) F-actin content (maximum = 1.7- +/- 0.10-fold basal at 30 seconds). Concurrent measures of the actin content of individual actin pools (Triton-soluble and -insoluble F-actin and G-actin) show that at all times (0 to 30 seconds) only the Triton-insoluble F-actin pool grows (maximum = 2.81- +/- 0.73-fold basal at 30 seconds), whereas both the Triton-soluble and G-actin pools simultaneously decrease (50% decrease at 30 seconds). Concurrent growth of one F-actin pool (Triton-insoluble) and loss of another F-actin pool (Triton-soluble) emphasize the functional uniqueness of the F-actin pools and can occur only if the Triton- soluble F-actin anneals or cross-links filament-to-filament with the Triton-insoluble fraction or if the Triton-insoluble F-actin pool first depolymerizes to monomer, which is then added to the Triton-insoluble pool. Because from 0 to 30 seconds after FMLP activation G-actin never increases, but, like the Triton-soluble F-actin progressively decreases, the results suggest that F-actin growth results from simultaneous new filament growth by monomer addition to the Triton- insoluble F-actin and cytoskeletal remodelling by Triton-soluble F- actin annealing or cross-linking to Triton-insoluble F-actin. These findings offer important new insights into the mechanism(s) of actin polymerization in CTF-activated human PMNs.

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.

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.

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 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.

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