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

Blood ◽  
2005 ◽  
Vol 106 (8) ◽  
pp. 2871-2878 ◽  
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)

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

Blood ◽  
2010 ◽  
Vol 116 (20) ◽  
pp. 4288-4296 ◽  
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.

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

1989 ◽  
Vol 67 (5) ◽  
pp. 456-464 ◽  
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.

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

1982 ◽  
Vol 95 (2) ◽  
pp. 519-526 ◽  
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.

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

1988 ◽  
Vol 44 (3) ◽  
pp. 215-229 ◽  
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

Thyroid Hormone and Thyrotropin Regulate Intracellular Free Calcium Concentrations in Human Polymorphonuclear Leukocytes: In Vivo and in vitro Studies

2006 ◽  
Vol 19 (1) ◽  
pp. 205873920601900 ◽  
Author(s):  
F. Marino ◽  
L. Guasti ◽  
M. Cosentino ◽  
D. DE Piazza ◽  
C. Simoni ◽  
...  
Keyword(s):  
Polymorphonuclear Leukocytes ◽  
Differentiated Thyroid Carcinoma ◽  
Intracellular Free Calcium ◽  
Suppressive Therapy ◽  
Free Calcium ◽  
Carbonyl Cyanide ◽  
Intracellular Ca ◽  
Human Polymorphonuclear Leukocytes

Intracellular free calcium concentrations ([Ca++]1) were studied in polymorphonuclear leukocytes (PMNs) from 13 athyreotic patients who had been previously treated by total thyroidectomy and radioiodine therapy for differentiated thyroid carcinoma, and from age- and sex-matched euthyroid healthy controls. Patients were studied twice, when hypothyroid (visit 1) and after restoration of euthyroidism by L-T4 TSH-suppressive therapy (visit 2). PMNs from patients at visit 1 had significantly lower resting [Ca++]1 levels compared to both visit 2 and controls. Values at visit 2 did not differ from those of the controls. Stimulus-induced [Ca++]1 rise was also significantly blunted at visit 1 and normalized at visit 2, possibly through a differential contribution of distinct intracellular Ca++ stores, as suggested by the response pattern to the chemotactic agent, N-formyl-Met-Leu-Phe (fMLP), to the selective SERCA pump inhibitor, thapsigargine, and to the mitochondrial uncoupler, carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone (FCCP). In vitro treatment of PMNs from healthy subjects with high TSH concentrations impaired intracellular Ca++ store function. Both resting [Ca++]1 levels and fMLP-induced [Ca++]1 rise increased in the presence either of low-concentration TSH or of T4, but effects of TSH and T4 were not additive. T3, rT3, and TRIAC had no effect. In conclusion, this study provides evidence for a direct relationship between thyroid status and [Ca[Ca++]1 homeostasis in human PMNs, mainly related to direct actions of TSH and T4 on these cells.

Defibrotide Inhibits Platelet Activation by Cathepsin G Released From Stimulated Polymorphonuclear Leukocytes

1992 ◽  
Vol 67 (06) ◽  
pp. 660-664 ◽  
Author(s):  
Virgilio Evangelista ◽  
Paola Piccardoni ◽  
Giovanni de Gaetano ◽  
Chiara Cerletti
Keyword(s):  
Platelet Activation ◽  
Polymorphonuclear Leukocytes ◽  
Direct Interaction ◽  
Cathepsin G ◽  
In Vivo Test ◽  
Cell Functions ◽  
Test Models ◽  
Antithrombotic Effects

SummaryDefibrotide is a polydeoxyribonucleotide with antithrombotic effects in experimental animal models. Most of the actions of this drug have been observed in in vivo test models but no effects have been reported in in vitro systems. In this paper we demonstrate that defibrotide interferes with polymorphonuclear leukocyte-induced human platelet activation in vitro. This effect was not related to any direct interaction with polymorphonuclear leukocytes or platelets, but was due to the inhibition of cathepsin G, the main biochemical mediator of this cell-cell cooperation. Since cathepsin G not only induces platelet activation but also affects some endothelial cell functions, the anticathepsin G activity of defibrotide could help to explain the antithrombotic effect of this drug.

The Use of the Vitality and Chemotaxis of Human Polymorphonuclear Leukocytes for the In Vitro Estimation of the Acute Toxicity of the First Ten Chemicals from the MEIC List

1993 ◽  
Vol 21 (1) ◽  
pp. 73-80
Author(s):  
Matteo Valentino ◽  
Francesca Monaco ◽  
Maria Antonietta Pizzichini ◽  
Mario Governa
Keyword(s):  
Ethidium Bromide ◽  
Polymorphonuclear Leukocytes ◽  
Rank Correlation ◽  
Fluorescein Diacetate ◽  
Live Cells ◽  
In Vitro Toxicity ◽  
Ic50 Values ◽  
Acute Cytotoxicity ◽  
Human Polymorphonuclear Leukocytes

The acute cytotoxicity of the first ten MEIC chemicals has been estimated by others in various cell lines. In the present investigation, isolated human polymorphonuclear leukocytes (PMN) from ten healthy non-smoking laboratory personnel were used to assess in vitro toxicity of the same chemicals. The cells were treated with different concentrations of the respective chemicals for three hours and their vitality and chemotaxis were tested. Vitality was measured by fluorescence microscopy after the addition of fluorescein diacetate and ethidium bromide. Living cells which took up and hydrolysed fluorescein diacetate, and dead cells, stained by ethidium bromide, were counted and the percentage of live cells was calculated. Locomotion stimulated by the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (F-MLP), was measured in blind-well Boyden chambers and a chemotactic index was calculated. The results were mathematically transformed to produce a linear curve, and then fitted by the linear least squares procedure, from which LC50 and IC50 values were obtained by interpolation. All the chemicals decreased the vitality and inhibited the chemotaxis of the PMN. Obviously the chemotactic test was more sensitive than the vitality one. A correlation (r = 0.933) was found between vitality and chemotaxis inhibition. Spearman rank correlation analysis revealed significant correlations between our results and those from in vitro experiments conducted in other laboratories, as well as with data concerning mouse, rat and human lethal doses.

scholarly journals A sand fly salivary protein acts as a neutrophil chemoattractant

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Anderson B. Guimaraes-Costa ◽  
John P. Shannon ◽  
Ingrid Waclawiak ◽  
Jullyanna Oliveira ◽  
Claudio Meneses ◽  
...  
Keyword(s):  
Inflammatory Responses ◽  
Calcium Influx ◽  
Parasite Burden ◽  
Salivary Protein ◽  
Sand Fly ◽  
Human Neutrophils ◽  
Chemotactic Protein ◽  
The Impact

AbstractApart from bacterial formyl peptides or viral chemokine mimicry, a non-vertebrate or insect protein that directly attracts mammalian innate cells such as neutrophils has not been molecularly characterized. Here, we show that members of sand fly yellow salivary proteins induce in vitro chemotaxis of mouse, canine and human neutrophils in transwell migration or EZ-TAXIScan assays. We demonstrate murine neutrophil recruitment in vivo using flow cytometry and two-photon intravital microscopy in Lysozyme-M-eGFP transgenic mice. We establish that the structure of this ~ 45 kDa neutrophil chemotactic protein does not resemble that of known chemokines. This chemoattractant acts through a G-protein-coupled receptor and is dependent on calcium influx. Of significance, this chemoattractant protein enhances lesion pathology (P < 0.0001) and increases parasite burden (P < 0.001) in mice upon co-injection with Leishmania parasites, underlining the impact of the sand fly salivary yellow proteins on disease outcome. These findings show that some arthropod vector-derived factors, such as this chemotactic salivary protein, activate rather than inhibit the host innate immune response, and that pathogens take advantage of these inflammatory responses to establish in the host.

scholarly journals Evaluation of a Novel Therapeutic Approach to Treating Severe Pneumococcal Infection Using a Mouse Model

2009 ◽  
Vol 16 (6) ◽  
pp. 806-810 ◽  
Author(s):  
Nikkol Melnick ◽  
Gowrisankar Rajam ◽  
George M. Carlone ◽  
Jacquelyn S. Sampson ◽  
Edwin W. Ades
Keyword(s):  
Single Dose ◽  
Combination Therapy ◽  
Polymorphonuclear Leukocytes ◽  
Low Dose ◽  
Control Level ◽  
Pneumococcal Infection ◽  
Amino Acid Peptide ◽  
Opsonophagocytic Killing

ABSTRACT P4, a 28-amino-acid peptide, is a eukaryotic cellular activator that enhances specific in vitro opsonophagocytic killing of multiple bacterial pathogens. In a previous study, we successfully recreated this phenomenon in mice in vivo by using a two-dose regimen of P4 and pathogen-specific antibodies, which significantly reduced moribundity in mice. For the present study, we hypothesized that the inclusion of a low-dose antibiotic would make it possible to treat the infected mice with a single dose containing a mixture of P4 and a pathogen-specific antibody. A single dose consisting of P4, intravenous immunoglobulin (IVIG), and ceftriaxone effectively reduced moribundity compared to that of untreated controls (n = 10) by 75% (P < 0.05) and rescued all (10 of 10) infected animals (P < 0.05). If rescued animals were reinfected with Streptococcus pneumoniae and treated with a single dose containing P4, IVIG, and ceftriaxone, they could be rerescued. This observation of the repeated successful use of P4 combination therapy demonstrates a low risk of tolerance development. Additionally, we examined the polymorphonuclear leukocytes (PMN) derived from infected mice and observed that P4 enhanced in vitro opsonophagocytic killing (by >80% over the control level; P < 0.05). This finding supports our hypothesis that PMN are activated by P4 during opsonophagocytosis and the recovery of mice from pneumococcal infection. P4 peptide-based combination therapy may offer an alternative and rapid immunotherapy to treat fulminant pneumococcal infection.

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