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

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 Vivo and In Vitro Effects of Glucocorticoids on Glucose Transport in Human Polymorphonuclear Leukocytes

Hormone and Metabolic Research ◽

10.1055/s-2007-1002069 ◽

1993 ◽

Vol 25 (03) ◽

pp. 165-169

Author(s):

Y. Okuno ◽

Y. Nishizawa ◽

T. Kawagishi ◽

H. Morii

Keyword(s):

Glucose Transport ◽

Polymorphonuclear Leukocytes ◽

In Vitro Effects ◽

Human Polymorphonuclear Leukocytes

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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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A monoclonal antibody to oestradiol potentiates the stimulation of the specific activity of the brain type creatine kinase by oestrogen in vivo and in vitro

The Journal of Steroid Biochemistry and Molecular Biology ◽

10.1016/s0960-0760(97)00198-2 ◽

1998 ◽

Vol 64 (5-6) ◽

pp. 297-304 ◽

Cited By ~ 1

Author(s):

D. Sömjen ◽

Y. Amir-Zaltsman ◽

G. Mor ◽

B. Gayer ◽

S. Lichter ◽

...

Keyword(s):

Monoclonal Antibody ◽

Creatine Kinase ◽

Specific Activity ◽

The Brain ◽

Stimulation Of

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Role of microtubules in granulocyte adherence

Blood ◽

10.1182/blood.v51.6.1045.bloodjournal5161045 ◽

1978 ◽

Vol 51 (6) ◽

pp. 1045-1050

Author(s):

LA Boxer ◽

JM Allen ◽

AM Watanabe ◽

HR Jr Besch ◽

RL Baehner

Keyword(s):

Ascorbic Acid ◽

Cyclic Nucleotides ◽

Polymorphonuclear Leukocytes ◽

Microtubule Assembly ◽

Dose Dependent Fashion ◽

Chediak Higashi Syndrome ◽

Human Polymorphonuclear Leukocytes ◽

Dose Dependent

The adherence of human polymorphonuclear leukocytes (PMN) to nylon fibers is inhibited in a dose-dependent fashion by exposure in vitro of these cells to either colchicine or VM-26, both of which agents prevent microtubule assembly. Mean adherence of human PMN was 48% +/- 2%, following treatment with 10(-5) M colchicine or 10(-4) M VM-26 it was reduced to 31% +/- 2% and 7%, respectively. Peritoneal PMN obtained from mice and mink with Chediak-Higashi syndrome (CHS) thought to have a microtubule-membrane disorder affecting the PMN had a mean adherence of 29% +/- 3% and 40% +/- 8% compared to control values of 46% +/- 5% and 73% +/- 8%, respectively, from the mice and mink. Both ascorbic acid and bethanechol, shown previously to enhance microtubule assembly in humans with CHS, normalized granulocyte adherence in PMN obtained from mice with CHS. Cyclic nucleotide levels were not altered by treatment of human PMN with colchicine, nor did they differ between normal and CHS animals. Thus it appears that the state of microtubule assembly may directly affect the properties of the PMN plasma membrane without requiring alterations of cyclic nucleotides as an intermediary.

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Stimulation of creatine kinase activity in rat skeletal tissue in vivo and in vitro by protease-resistant variants of parathyroid hormone fragments

Biochemical Journal ◽

10.1042/bj3090085 ◽

1995 ◽

Vol 309 (1) ◽

pp. 85-90 ◽

Cited By ~ 3

Author(s):

D Sömjen ◽

V Vargas ◽

A Waisman ◽

E Wingender ◽

W Tegge ◽

...

Keyword(s):

Creatine Kinase ◽

Parathyroid Hormone ◽

Double Mutant ◽

Bone Cells ◽

Specific Activity ◽

Control Level ◽

Maximal Stimulation ◽

Stimulation Of

We have reported that mid-region fragments of human parathyroid hormone (hPTH), exemplified by hPTH-(28-48), stimulated [3H]thymidine incorporation into DNA and increased the specific activity of the brain-type isoenzyme of creatine kinase (CK) in both skeletal-derived cell cultures (ROS 17/2.8 cells) and immature rat epiphyseal cartilage and diaphyseal bone, without stimulating cyclic AMP synthesis which is a prerequisite for bone resorption. In the present study, substitution of amino acids in hPTH-(28-48), which resulted in increased resistance to proteolysis, produced variants that stimulated skeletal systems at two orders of magnitude lower concentration than the wild-type fragment. We modified hPTH-(28-48) at Leu-37 by replacement with Met, Thr or Val. Under conditions in which 20% of the native hPTH-(28-48) resisted proteolysis by cathepsin D for 6 h, approx. 40% of the L37V mutant and 70% of the L37T mutant remained intact. Substitution of Met for Phe-34 in addition to Thr for Leu-37, or the substitution of Met for Phe-34 alone, produced 100%-resistant fragments. These variants at residue 34 caused maximal stimulation of CK in ROS 17/2.8 cells at 0.24 nM compared with 24 nM for hPTH-(28-48). The double mutant stimulated CK activity significantly in immature rats, at a minimum dose of 12.5 ng/rat, and caused maximal stimulation at 125 ng/rat, a 10-fold lower dose than for hPTH-(28-48). The effect of the double mutant lasted up to 24 h which differs from the stimulation by hPTH-(28-48) in which CK specific activity returns to the control level at 24 h. This same dose also significantly stimulated CK activity in gonadectomized rats. These results show the advantage of using protease-resistant mid-region variants of hPTH-(28-48) to stimulate bone cells, in terms of lower doses and longer duration of effectiveness, both in vitro and in vivo.

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Role of microtubules in granulocyte adherence

Blood ◽

10.1182/blood.v51.6.1045.1045 ◽

1978 ◽

Vol 51 (6) ◽

pp. 1045-1050 ◽

Cited By ~ 24

Author(s):

LA Boxer ◽

JM Allen ◽

AM Watanabe ◽

HR Jr Besch ◽

RL Baehner

Keyword(s):

Ascorbic Acid ◽

Cyclic Nucleotides ◽

Polymorphonuclear Leukocytes ◽

Microtubule Assembly ◽

Dose Dependent Fashion ◽

Chediak Higashi Syndrome ◽

Human Polymorphonuclear Leukocytes ◽

Dose Dependent

Abstract The adherence of human polymorphonuclear leukocytes (PMN) to nylon fibers is inhibited in a dose-dependent fashion by exposure in vitro of these cells to either colchicine or VM-26, both of which agents prevent microtubule assembly. Mean adherence of human PMN was 48% +/- 2%, following treatment with 10(-5) M colchicine or 10(-4) M VM-26 it was reduced to 31% +/- 2% and 7%, respectively. Peritoneal PMN obtained from mice and mink with Chediak-Higashi syndrome (CHS) thought to have a microtubule-membrane disorder affecting the PMN had a mean adherence of 29% +/- 3% and 40% +/- 8% compared to control values of 46% +/- 5% and 73% +/- 8%, respectively, from the mice and mink. Both ascorbic acid and bethanechol, shown previously to enhance microtubule assembly in humans with CHS, normalized granulocyte adherence in PMN obtained from mice with CHS. Cyclic nucleotide levels were not altered by treatment of human PMN with colchicine, nor did they differ between normal and CHS animals. Thus it appears that the state of microtubule assembly may directly affect the properties of the PMN plasma membrane without requiring alterations of cyclic nucleotides as an intermediary.

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Fluorimetric Measurement of Exogenous and Endogenous Epinephrine and Norepinephrine in Peripheral Blood

American Journal of Physiology-Legacy Content ◽

10.1152/ajplegacy.1958.194.3.476 ◽

1958 ◽

Vol 194 (3) ◽

pp. 476-480 ◽

Cited By ~ 6

Author(s):

George F. Mangan ◽

John W. Mason

Keyword(s):

Peripheral Blood ◽

Splanchnic Nerve ◽

Adrenergic Nerve ◽

Blood Levels ◽

In Vivo Experiments ◽

Significant Difference ◽

Adrenergic Nerve Endings ◽

Stimulation Of

The reliability of the Weil-Malherbe and Bone method for the measurement of epinephrine and norepinephrine fluctuations in peripheral blood has been investigated. Epinephrine and norepinephrine injected intravenously (20 µg/kg) produced sharp elevations in appropriate blood levels but disappeared very rapidly, returning to the preinjection range in 2.5–5 minutes. No significant difference was observed in the disappearance rate of epinephrine as compared to norepinephrine. In one animal, evisceration and hepatectomy produced relatively little change in the disappearance pattern, suggesting that peripheral mechanisms, perhaps at adrenergic nerve endings or in skeletal muscle, are involved in removal of these hormones from the circulation. Added epinephrine does not disappear appreciably from plasma in whole blood maintained at body temperature in vitro in time periods comparable to these in the in vivo experiments. Stimulation of the splanchnic nerve in a dog and monkey produced readily detectable changes in both plasma epinephrine and norepinephrine levels with rather characteristic temporal patterns of response for each hormone. It is concluded that the Weil-Malherbe procedure is suitable for further application to investigations of sympathetic nervous system-adrenal medullary function.

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