Kinetics of chemo-attraction of polymorphonuclear leukocytes towards N-formyl peptide studied with a novel polycarbonate (Nuclepore) membrane in the Boyden chamber

The effects of piroxicam, tenoxicam, diclofenac sodium, acetylsalicylic acid and tiaprofenic acid on the chemotaxis and random migration of human polymorphonuclear leukocytes were investigated, using zymosan-activated serum as chemo-attractant, with a modified Boyden chamber technique. All five compounds significantly reduced chemotaxis. The random migration of polymorphonuclear leukocytes was inhibited by piroxicam, diclofenac sodium and tiaprofenic acid but not by tenoxicam or acetylsalicylic acid. The inhibitory effect of these non-steroidal anti-inflammatory drugs on polymorphonuclear leukocyte chemotaxis and on random migration was generally dose-dependent. The results suggest that the drugs studied may have a direct effect on polymorphonuclear leukocyte chemotaxis and that this activity may contribute to their anti-inflammatory properties.

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Uptake and intracellular activity of NM394, a new quinolone, in human polymorphonuclear leukocytes.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.40.3.739 ◽

1996 ◽

Vol 40 (3) ◽

pp. 739-742 ◽

Cited By ~ 15

Author(s):

M Ozaki ◽

K Komori ◽

M Matsuda ◽

R Yamaguchi ◽

T Honmura ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Bacterial Infection ◽

Polymorphonuclear Leukocytes ◽

Concentration Ratio ◽

Extracellular Concentration ◽

Intracellular Activity ◽

Subsequent Elution ◽

Active Transport System ◽

Human Polymorphonuclear Leukocytes ◽

Kinetics Of

The uptake of NM394, a new quinolone, by and its subsequent elution from human polymorphonuclear leukocytes were studied and compared with those of ofloxacin and ciprofloxacin. The kinetics of the uptake of NM394 was similar to that of ciprofloxacin. The maximum intracellular-to-extracellular concentration ratio was 12.3, compared with 8.6 for ciprofloxacin and 4.9 for ofloxacin at the extracellular concentration of 20 micrograms/ml. The elution of NM394 from human polymorphonuclear leukocytes occurs relatively slowly; 5 min after the removal of extracellular NM394, nearly 100% still remained in polymorphonuclear leukocytes, compared with ofloxacin, which was so rapidly eluted that only 12% remained. The uptake of NM394 was significantly decreased at 4 degrees C and by the presence of NaCN but was not affected by the presence of L-glycine, L-leucine, L-serine, adenosine, or NaF. NM394 showed intracellular activity at a concentration of 0.1 microgram/ml that significantly reduced the number of phagocytosed Pseudomonas aeruginosa cells with 2 h of incubation. These results suggest that uptake of NM394 by human polymorphonuclear leukocytes occurs via an active transport system differing from that of ofloxacin, whose uptake is affected by the presence of L-glycine and L-leucine, and that once accumulated, NM394 remains intracellularly active and participates in protection against bacterial infection.

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

The Journal of Cell Biology ◽

10.1083/jcb.119.5.1261 ◽

1992 ◽

Vol 119 (5) ◽

pp. 1261-1270 ◽

Cited By ~ 104

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.

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Active Motion of Polymorphonuclear Leukocytes in Response to Chemoattractant in a Micropipette

Journal of Biomechanical Engineering ◽

10.1115/1.2895531 ◽

1993 ◽

Vol 115 (4B) ◽

pp. 503-509 ◽

Cited By ~ 3

Author(s):

B. A. Skierczynski ◽

S. Usami ◽

S. Chien ◽

R. Skalak

Keyword(s):

Concentration Gradient ◽

Polymorphonuclear Leukocytes ◽

Initial Conditions ◽

Front Face ◽

Boyden Chamber ◽

Internal Source ◽

Exact Nature ◽

Active Motion ◽

Cell Locomotion ◽

Sequence Of Events

A novel experimental method of producing and observing the active motion of polymorphonuclear leukocytes (PMNs) using a micropipette technique has been recently developed (Usami et al., 1992). The present paper develops a quantitative theory for the chemoattractant gradients and cell locomotion observed in these experiments. In previous experimental methods (e.g., the Boyden chamber, the Zygmond chamber and the Dunn chamber) for study chemotaxis of leukocytes, fibroblasts, and PMNs, the exact nature of the concentration gradient of the chemoattractant is unknown. The cells may themselves modify the local gradient of the chemoattractant. In experiments using the micropipette, an internal source of chemoattractant provides well-defined boundary and initial conditions which allow the computation of the chemoattractant concentration gradient during the active locomotion of the PMNs. Since the cell completely fills the pipette lumen, convection is limited to the motion of the cells themselves. In coordinates moving with cell, it is assumed that diffusion is the only mechanism of mass transport of the chemoattractant (fMLP). Computations of the fMLP concentration during locomotion of the cell were carried out for a range of rates of fMLP binding by the receptors expressed on the front face of the cell membrane. The results show that the front face of the cell is subjected to increasing fMLP concentration during the cell motion. The sequence of events involve receptor binding of fMLP, signal transduction, polymerization of the cell cytoskeleton at the membrane of the front face, spatially dependent adhesion to the pipette wall, and localized contraction of the cytoskeleton. This sequence of events leads to the steady locomotion of the leukocytes in the micropipette. The computation of the distribution of the fMLP concentration during cell locomotion with constant velocity in micropipette experiments shows that the cell is exposed to increasing concentration of fMLP. This suggests that chemotaxis maybe induced by temporal gradient of an attractant.

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Design and characterization of a cleavage-resistant Annexin A1 mutant to control inflammation in the microvasculature

Blood ◽

10.1182/blood-2010-02-270520 ◽

2010 ◽

Vol 116 (20) ◽

pp. 4288-4296 ◽

Cited By ~ 49

Author(s):

Magali Pederzoli-Ribeil ◽

Francesco Maione ◽

Dianne Cooper ◽

Adam Al-Kashi ◽

Jesmond Dalli ◽

...

Keyword(s):

Polymorphonuclear Leukocytes ◽

Early Stage ◽

Leukocyte Adhesion ◽

Formyl Peptide Receptor ◽

Annexin A1 ◽

Formyl Peptide ◽

Anti Inflammatory ◽

Human Polymorphonuclear Leukocytes

Abstract Human polymorphonuclear leukocytes adhesion to endothelial cells during the early stage of inflammation leads to cell surface externalization of Annexin A1 (AnxA1), an effector of endogenous anti-inflammation. The antiadhesive properties of AnxA1 become operative to finely tune polymorphonuclear leukocytes transmigration to the site of inflammation. Membrane bound proteinase 3 (PR3) plays a key role in this microenvironment by cleaving the N terminus bioactive domain of AnxA1. In the present study, we generated a PR3-resistant human recombinant AnxA1—named superAnxA1 (SAnxA1)—and tested its in vitro and in vivo properties in comparison to the parental protein. SAnxA1 bound and activated formyl peptide receptor 2 in a similar way as the parental protein, while showing a resistance to cleavage by recombinant PR3. SAnxA1 retained anti-inflammatory activities in the murine inflamed microcirculation (leukocyte adhesion being the readout) and in skin trafficking model. When longer-lasting models of inflammation were applied, SAnxA1 displayed stronger anti-inflammatory effect over time compared with the parental protein. Together these results indicate that AnxA1 cleavage is an important process during neutrophilic inflammation and that controlling the balance between AnxA1/PR3 activities might represent a promising avenue for the discovery of novel therapeutic approaches.

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Mitochondrial N-formylmethionyl proteins as chemoattractants for neutrophils.

Journal of Experimental Medicine ◽

10.1084/jem.155.1.264 ◽

1982 ◽

Vol 155 (1) ◽

pp. 264-275 ◽

Cited By ~ 212

Author(s):

H Carp

Keyword(s):

Polymorphonuclear Leukocytes ◽

Inflammatory Cells ◽

Chemotactic Activity ◽

Mitochondrial Proteins ◽

Mitochondrial Enzymes ◽

Directed Migration ◽

Formyl Peptide ◽

Cultured Human Cells ◽

Chemotactic Factors

Mitochondria synthesize several hydrophobic proteins. Like bacteria, mitochondria initiate protein synthesis with an N-formylmethionine residue. Because N-formylmethionyl peptides have been found to be chemotactic for polymorphonuclear leukocytes (PMN), mitochondria isolated from cultured human cells and purified bovine mitochondrial proteins were tested for PMN chemotactic activity in vitro. Nondisrupted mitochondria were not chemotactic. However, intact mitochondria that had been incubated with a lysosomal lysate did stimulate PMN migration. Antibodies directed against two mitochondrial enzymes, cytochrome oxidase and ATPase, (both of which contain mitochondrially synthesized subunits) but not anti-C3 or anti-C5 decreased mitochondrially derived chemotactic activity. In addition, purified bovine mitochondrial N-formylmethionyl proteins stimulated PMN migration in vitro, whereas nonformylated mitochondrial proteins did not. Furthermore, the chemotactic activity of purified mitochondrial proteins and disrupted mitochondria was decreased by the formyl peptide antagonist butyloxycarbonyl-phenylalanine-leucine-phenylalanine-leucine-phenylalanine. Finally, disrupted mitochondria and purified mitochondrial proteins stimulated PMN-directed migration (chemotaxis), according to accepted criteria. In addition to other chemotactic factors, release of N-formylmethionyl proteins from mitochondria at sites of tissue damage, may play a role in the accumulation of inflammatory cells at these sites.

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Human leukocyte elastase is an endogenous ligand for the integrin CR3 (CD11b/CD18, Mac-1, alpha M beta 2) and modulates polymorphonuclear leukocyte adhesion.

Journal of Experimental Medicine ◽

10.1084/jem.184.4.1213 ◽

1996 ◽

Vol 184 (4) ◽

pp. 1213-1223 ◽

Cited By ~ 123

Author(s):

T Q Cai ◽

S D Wright

Keyword(s):

Cell Adhesion ◽

Polymorphonuclear Leukocytes ◽

Leukocyte Adhesion ◽

Human Leukocyte ◽

Endogenous Ligand ◽

Leukocyte Elastase ◽

Human Leukocyte Elastase ◽

Beta 2 ◽

Coated Surfaces ◽

Kinetics Of

Integrin CR3 (CD11b/CD18, Mac-1, alpha M beta 2) mediates the transient adhesion of polymorphonuclear leukocytes (PMN) to surfaces coated with fibrinogen, C3bi, ICAM-1, and other ligands. Recent studies (Cai, T.-Q., and S.D. Wright 1995. J. Biol. Chem. 270:14358) suggest that adhesion may be favored by stimulus-dependent changes in the kinetics of ligand binding by CR3. Cell detachment, on the other hand, must occur by a different mechanism because binding kinetics cannot affect cell adhesion after binding of ligand has occurred. We have sought a mechanism that would reverse binding of ligand to CR3 and report here that lysates of PMN contain an endogenous ligand that binds CR3 and competes the binding of C3bi. Purification and sequence analysis identified the structurally homologous azurophilic granule proteins, elastase, protease 3, and azurocidin as candidates. Studies with purified elastase and azurocidin showed that each bound specifically to purified, immobilized CR3. Elastase may play a role in modulating integrin-mediated cell adhesion because it is expressed at the cell surface, and the expression level is inversely proportional to cell adhesivity. Furthermore, a monoclonal antibody against elastase prevented detachment of PMN from fibrinogen-coated surfaces and blocked chemotaxis, confirming a role for this protein in regulating integrin-mediated adhesion. These studies suggest a model for release of integrin-mediated cell adhesion in which endogenous ligands such as elastase may release adhesion by "'eluting" substrate-bound ligand. A role for the proteolytic activity of elastase appears likely but is not demonstrated in this study.

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