Human Polymorphonuclear Leukocytes Derived from Chronically Inflamed Tissue Express Inflammatory Cytokines in Vivo

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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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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L-663,536 (MK-886) (3-[1-(4-chlorobenzyl)-3-t-butyl-thio-5-isopropylindol-2-yl]-2,2-dimethylpropanoic acid), a novel, orally active leukotriene biosynthesis inhibitor

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y89-073 ◽

1989 ◽

Vol 67 (5) ◽

pp. 456-464 ◽

Cited By ~ 242

Author(s):

J. Gillard ◽

A. W. Ford-Hutchinson ◽

C. Chan ◽

S. Charleson ◽

D. Denis ◽

...

Keyword(s):

Polymorphonuclear Leukocytes ◽

Potent Inhibitor ◽

Human Platelets ◽

Ionophore A23187 ◽

Lipoxygenase Inhibitor ◽

12 Lipoxygenase ◽

Rat Paw ◽

Human Polymorphonuclear Leukocytes

L-663,536 (3-[1-(4-chlorobenzyl)-3-t-butyl-thio-5-isopropylindol-2-yl]-2,2-dimethylpropanoic acid) is a potent inhibitor of leukotriene (LT) biosynthesis in intact human polymorphonuclear leukocytes (PMN) (IC50, 2.5 nM). Similarly, L-663,536 inhibited A23187-induced LTB4 formation by rat peripheral blood and elicited PMN. At concentrations where inhibition of leukotriene biosynthesis occurred in human whole blood (1.1 μM), no effect was seen on cyclooxygenase or 12-lipoxygenase, an effect also observed in washed human platelets. The compound had no effect on rat or porcine 5-lipoxygenase indicating that L-663,536 is not a direct 5-lipoxygenase inhibitor. When administered in vivo L-663,536 was a potent inhibitor of antigen-induced dyspnea in inbred rats pretreated with methysergide (ED50, 0.036 mg/kg p.o.) and of Ascaris-induced bronchoconstriction in squirrel monkeys (1 mg/kg p.o.). The compound inhibited leukotriene biosynthesis in vivo in a rat pleurisy model (ED50, 0.2 mg/kg p.o.), an inflamed rat paw model (ED50, 0.8 mg/kg), a model of leukotriene excretion in rat bile following antigen provocation, and a model in the guinea-pig ear where leukotriene synthesis was induced by topical challenge with ionophore A23187 (ED50, 2.5 mg/kg p.o. and 0.6 μg topically). The results indicate that L-663,536 is a potent inhibitor of leukotriene biosynthesis both in vitro and in vivo indicating that the compound is suitable for studying the role of leukotrienes in a variety of pathological situations.Key words: leukotriene, 5-lipoxygenase, polymorphonuclear leukocytes, leukotriene B4, leukotriene inhibitor.

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Quiescent and activated mouse granulocytes do not express granzyme A and B or perforin: similarities or differences with human polymorphonuclear leukocytes?

Blood ◽

10.1182/blood-2005-04-1522 ◽

2005 ◽

Vol 106 (8) ◽

pp. 2871-2878 ◽

Cited By ~ 23

Author(s):

Praxedis Martin ◽

Reinhard Wallich ◽

Julian Pardo ◽

Arno Müllbacher ◽

Markus Munder ◽

...

Keyword(s):

Polymorphonuclear Leukocytes ◽

Human Neutrophils ◽

Enzymatic Assays ◽

Effector Functions ◽

Healthy Control ◽

Polymerase Chain ◽

Human Polymorphonuclear Leukocytes ◽

Cytotoxic Effector

AbstractPolymorphonuclear leukocytes have been shown to use a multitude of effector functions to combat pathogens and tumors, including enzymes, defensins, and toxic products such as oxygen radicals and nitrogen oxides. Recent studies provided evidence for the expression of granzymes (gzms) and perforin (perf) within the cytotoxic arsenal of human neutrophils, the validity of which was questioned by 2 subsequent studies. We have now used cytology, intracellular flow cytometry, enzymatic assays, immunoelectron microscopy, and quantitative reverse transcriptase-polymerase chain reaction to obtain evidence of the presence of gzms and/or perf in mouse Gr-1+ granulocyte populations. The data obtained clearly demonstrate that neither in vitro- nor in vivo-derived mouse granulocytes synthesize gzmA and gzmB or perf, even following infection/immunization with pathogens or pathogen-derived material. A parallel comparable analysis on the expression of gzmB in human neutrophils from 3 healthy control subjects and 4 patients with diverse diseases failed to detect gzmB expression. The data indicate that polymorphonuclear leukocytes from mice and humans lack the 3 cytotoxic effector molecules, gzmA, gzmB, and perf, generally associated with natural killer and cytotoxic T lymphocytes. (Blood. 2005;106:2871-2878)

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Polymorphonuclear leukocyte phagocytosis of Capnocytophaga ochracea in three-dimensional plasma clots

Journal of Cell Science ◽

10.1242/jcs.95.3.487 ◽

1990 ◽

Vol 95 (3) ◽

pp. 487-498

Author(s):

T.J. Hurst ◽

J.M. Wilton

Keyword(s):

Polymorphonuclear Leukocyte ◽

Polymorphonuclear Leukocytes ◽

Three Dimensional ◽

Human Polymorphonuclear Leukocytes

We have studied the ability of human polymorphonuclear leukocytes (PMN) to phagocytose Capnocytophaga ochracea in three-dimensional fibrin meshworks. Phagocytosis was assessed in three systems: (1) the PMN and bacteria were mixed together with plasma and clotted; 60 +/− 13% phagocytosis occurred after 60 min; (2) PMN were overlaid on clots containing bacteria; the PMN migrated into the clot and after 60 min 52 +/− 7% phagocytosis was seen; (3) PMN had to migrate from within one clot into a second containing bacteria; phagocytosis after 60 min was 54 +/− 3%. In the clots, PMN released lysozyme but this was not significantly enhanced by phagocytosis. These findings indicate that PMN are capable of phagocytosing in each of the three-dimensional systems tested and that they are capable of both migration into and subsequent phagocytosis in a model that more closely mimics the in vivo structure in which PMN would normally perform.

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Autooxidation as a basis for altered function by polymorphonuclear leukocytes

Blood ◽

10.1182/blood.v50.2.327.327 ◽

1977 ◽

Vol 50 (2) ◽

pp. 327-335 ◽

Cited By ~ 146

Author(s):

RL Baehner ◽

LA Boxer ◽

JM Allen ◽

J Davis

Keyword(s):

Vitamin E ◽

Polymorphonuclear Leukocytes ◽

Cell Movement ◽

Membrane Lipid ◽

Alpha Tocopherol ◽

Directed Movement ◽

Aerobic Conditions ◽

Ingestion Rates ◽

Human Polymorphonuclear Leukocytes

Abstract To investigate the possibility that human polymorphonuclear leukocytes (PMN) elaborate sufficient amounts of hydrogen peroxide (H2O2) and other radicals of reduced oxygen to be autotoxic and retard directed cell movement and phagocytosis, the rate of ingestion of opsonized lipopolysaccharide-paraffin oil particles and movement through Nuclepore filters were studied. Ingestion rates were increased under anaerobic conditions and in normal aerobic conditions in the presence of extracellular catalase but not superoxide dismutase (SOD) or scavengers of singlet oxygen or hydroxyl radicals. Conversely, ingestion rates were decreased when cells were exposed to H2O2 or a superoxide anion (O2-)-H2O2 generating system of xanthine-xanthine oxidase. Catalase, but not SOD, prevented the effect and also enhanced the directed movement of PMN in normal aerobic conditions. PMN from volunteers administered 1600 U/day of the membrane lipid antioxidant alpha-tocopherol were hyperphagocytic but killed Staphylococcus aureus 502A less effectively than controls, suggesting that less H2O2 was available to damage PMN or kill bacteria. H2O2-dependent stimulation of the hexose monophosphate shunt, H2O2 release from phaogytizing PMN, and fluoresceinated concanavalin A cap formation promoted by H2O2 damage to microtubules were all diminished, but the release of O2- from phagocytizing PMN was not diminished in the vitamin E group. These results support the hypothesis that directed movement and phagocytosis by PMN are attenuated by autooxidative damage to the cell membrane by endogenously derived H2O2 and that the administration in vivo of vitamin E may prevent this damage by scavenging H2O2.

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Tubulin tyrosinolation in human polymorphonuclear leukocytes: studies in normal subjects and in patients with the Chediak-Higashi syndrome.

The Journal of Cell Biology ◽

10.1083/jcb.95.2.519 ◽

1982 ◽

Vol 95 (2) ◽

pp. 519-526 ◽

Cited By ~ 24

Author(s):

J Nath ◽

M Flavin ◽

J I Gallin

Keyword(s):

Polymorphonuclear Leukocytes ◽

Specific Activity ◽

Normal Subjects ◽

In Vivo Experiments ◽

Peritoneal Leukocytes ◽

Chediak Higashi Syndrome ◽

Human Polymorphonuclear Leukocytes ◽

Stimulation Of

We have recently reported a specific dose-dependent stimulation of posttranslational incorporation of tyrosine into tubulin alpha-chains of rabbit peritoneal leukocytes as induced by the synthetic peptide chemoattractant formyl-methionyl-leucyl-phenylalanine (FMLP). The present study reports a similar, specific stimulation of tubulin tyrosinolation in human polymorphonuclear leukocytes (PMN). When compared to normal PMN, both the resting and FMLP-stimulated levels of posttranslational tyrosine incorporation were two- to threefold higher in PMN of three patients with the Chediak-Higashi syndrome (CHS). The concentration of cellular tubulin and the specific activity of tubulin tyrosine ligase were similar in PMN of CHS patients and normal donors and resembled that of other non-neuronal cells. The high levels of tyrosine incorporation in PMN of CHS patients were normalized by the administration of ascorbate, both in vitro and in in vivo experiments. In vitro addition of ascorbate also inhibited the FMLP-induced stimulation of tyrosine incorporation in both normal and CHS cells. Normalization of higher levels of tyrosine incorporation in PMN of CHS patients and the inhibition of FMLP-induced stimulation of tubulin tyrosinolation in normal and CHS cells as observed with ascorbate could also be affected by other reducing agents such as reduced glutathione, cysteine, or dithiothreitol. These results suggest a possible relationship between cellular redox and tubulin tyrosinolation in PMN.

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In vitro and in vivo effects produced by the immunomodulating agent ru 41740 on human polymorphonuclear leukocytes in the elderly

Mechanisms of Ageing and Development ◽

10.1016/0047-6374(88)90023-1 ◽

1988 ◽

Vol 44 (3) ◽

pp. 215-229 ◽

Cited By ~ 3

Author(s):

Ahmed El Abbouyi ◽

Monique Roch-Arveiller ◽

Christiane Marchiani ◽

Robert Dahan ◽

François Congy ◽

...

Keyword(s):

Polymorphonuclear Leukocytes ◽

The Elderly ◽

Human Polymorphonuclear Leukocytes ◽

In Vivo Effects

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Secretion of plasminogen activator by human polymorphonuclear leukocytes. Modulation by glucocorticoids and other effectors.

Journal of Experimental Medicine ◽

10.1084/jem.146.6.1693 ◽

1977 ◽

Vol 146 (6) ◽

pp. 1693-1706 ◽

Cited By ~ 159

Author(s):

A Granelli-Piperno ◽

J D Vassalli ◽

E Reich

Keyword(s):

Plasminogen Activator ◽

Polymorphonuclear Leukocytes ◽

Response Pattern ◽

Actinomycin D ◽

Lytic Enzymes ◽

Con A ◽

Low Concentrations ◽

Human Pmns ◽

Human Polymorphonuclear Leukocytes

Purified human PMNs secrete plasminogen activator. This secretion is stimulated by Con A and low concentrations of PMA, and is inhibited by low concentrations of glucocorticoids, and by cAMP, actinomycin D, and cycloheximide. In contrast, the release of granule-bound enzymes, such as elastase, is achieved only at higher concentrations of PMA, and is not affected by any of the inhibitors that block plasminogen activator production. These results show that the production of plasminogen activatory by PMNs is controlled by agents that affect inflammations, and that this control is not shared by other lytic enzymes known to be associated with these cells. This suggests a particular role for plasminogen activator in the response pattern of PMNs and also supports the concept, previously developed for macrophages, that the secretion of this enzyme is correlated with cell migration in vivo.

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