Rapid, single-step isolation of equine neutrophils on a discontinuous Percoll density gradient

Pycock, Jonathan F.; Allen, W. Edward; Morris, Tim

doi:10.1016/s0034-5288(18)30727-6

Cited by 56 publications

(32 citation statements)

References 8 publications

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“…Neutrophils were isolated from EDTA (1.6 mg/ml) anticoagulated blood drawn from the jugular vein of healthy horses that were fed and bred in identical conditions and were not subjected to any medical treatment (Faculty of Veterinary Medicine, University of Liege, Belgium). The neutrophils were isolated at room temperature (18-22˚C) by centrifugation (400 x g, 45 min, 20˚C) on a discontinuous Percoll density gradient according to the method of Pycock et al (22). The cells were gently collected and washed with 2 volumes of physiological saline solution.…”

Section: Methodsmentioning

confidence: 99%

Modulatory activities of Agelanthus dodoneifolius (Loranthaceae) extracts on stimulated equine neutrophils and myeloperoxidase activity

Boly¹,

Dessy²,

Kohnen³

et al. 2011

Int J Mol Med

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Abstract. Agelanthus dodoneifolius DC Danser (Loranthaceae)is used for the treatment of various diseases including asthma. The aqueous and hydroalcoholic extracts have been reported to have anti-inflammatory, spasmolytic and bronchorelaxant activities. The present study investigates the effects of the aqueous decoction and the diethyl ether, ethyl acetate and butanolic fractions of Agelanthus dodoneifolius DC Danser (Loranthaceae) on reactive oxygen species (ROS) production and myeloperoxidase (MPO) release by phorbol 12-myristate 13-acetate (PMA)-stimulated equine neutrophils and on purified equine MPO activity. ROS production and MPO release by the PMA-stimulated neutrophils were measured by the lucigenin-enhanced chemiluminescence and ELISA assays, respectively. Specific immunological extraction followed by enzymatic detection (SIEFED) was used to specifically measure the equine MPO activity. Identification and quantification of the individual and total phenolic and flavonoid compounds were performed using UPLC-MS/MS equipment and colorimetric methods involving Folin-Ciocalteu and AlCl 3 , respectively. All the tested extracts displayed dose-dependent inhibitory effects on the oxidant activities of neutrophils; a stronger effect was observed with the organic fractions than the aqueous decoction. These findings could be correlated with a high content of phenolic and flavonoid compounds. The results confirm the previously shown anti-inflammatory effect of Agelanthus dodoneifolius and its potential use for the treatment of neutrophil-dependent inflammatory diseases. IntroductionPolymorphonuclear neutrophils (PMNs) are the primary effector cells in host responses to injury and infection (1-4). The antimicrobial function of stimulated PMNs is based on their phagocytic capacity and their ability to release oxidant and proteolytic enzymes as well as reactive oxygen species (ROS) involved in the destruction of microorganisms. ROS play important roles in the pathogenesis of various serious diseases, such as neurodegenerative disorders, aging, cancer, cataracts, inflammation and cardiovascular diseases including atherosclerosis (5,6).Myeloperoxidase (MPO), a heme peroxidase enzyme stored in the azurophilic granules of neutrophils, plays a crucial role in the destruction of bacteria inside PMNs. The bacterial killing process mainly uses hydrogen peroxide (H 2 O 2 ) and the chloride anion (Cl -) to produce hypochlorous

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Section: Methodsmentioning

confidence: 99%

Modulatory activities of Agelanthus dodoneifolius (Loranthaceae) extracts on stimulated equine neutrophils and myeloperoxidase activity

Boly¹,

Dessy²,

Kohnen³

et al. 2011

Int J Mol Med

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“…Equine neutrophils were isolated by density centrifugation of heparinized blood from healthy horses on a discontinuous Percoll gradient as described previously (15). After washing, cells were resuspended in MEM at 1 ϫ 10 5 cells/ml and used immediately for further experimentation.…”

Section: Cellsmentioning

confidence: 99%

Herpesvirus Chemokine-Binding Glycoprotein G (gG) Efficiently Inhibits Neutrophil Chemotaxis In Vitro and In Vivo

Walle

May

Sukhumavasi

et al. 2007

The Journal of Immunology

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Glycoprotein G (gG) of alphaherpesviruses has been described to function as a viral chemokine-binding protein (vCKBP). More recently, mutant viruses devoid of gG have been shown to result in increased virulence, but it remained unclear whether the potential of gG to serve as a vCKBP is responsible for this observation. In this study, we used equine herpesvirus type 1 (EHV-1) as a model to study the pathophysiological importance of vCKBP activity. First, in vitro chemotaxis assays studying migration of immune cells, an important function of chemokines, were established. In such assays, supernatants of EHV-1-infected cells significantly inhibited IL-8-induced chemotaxis of equine neutrophils. Identification of gG as the responsible vCKBP was achieved by repeating similar experiments with supernatants from cells infected with a gG-negative mutant, which were unable to alter IL-8-induced equine neutrophil migration. Furthermore, rEHV-1 gG was able to significantly reduce neutrophil migration, establishing gG as a bona fide vCKBP. Second, and importantly, in vivo analyses in a murine model of EHV-1 infection showed that neutrophil migration in the target organ lung was significantly reduced in the presence of gG. In summary, we demonstrate for the first time that EHV-1 gG not only binds to chemokines but is also capable of inhibiting their chemotactic function both in vitro and in vivo, thereby contributing to viral pathogenesis and virulence.

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“…Thereafter three different methods [8,26,27] for isolating equine neutrophils were compared. The methods of Jain et al [26] and Pycock et al [27] both employed a single-step isolation technique with discontinuous isotonic Percoll gradients (59 ϩ 75 and 70 ϩ 85% Percoll in HBSS, respectively) and were used as originally detailed. The third technique was a modification of a method originally described for isolating human neutrophils which uses discontinuous hypotonic plasma/Percoll gradients [8].…”

Section: Preparation Of Equine Neutrophilsmentioning

confidence: 99%

Priming induces functional coupling of N-formyl-methionyl-leucyl-phenylalanine receptors in equine neutrophils

Brazil

Rossi

Haslett

et al. 1998

Journal of Leukocyte Biology

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The synthetic formylpeptide fMLP is widely used as a model chemoattractant and secretagogue for mammalian neutrophils. Despite possessing fMLP receptors, equine neutrophils do not produce superoxide anions in response to fMLP and there is no inflammatory reaction in the horse when fMLP is injected intradermally. The functional capability of these receptors was investigated after pretreatment with recognized priming agents. Purified neutrophils were pretreated with lipopolysaccharide (LPS), platelet-activating factor (PAF), or tumor necrosis factor ␣ (TNF-␣) and superoxide anion generation and shape change quantified by lucigenin-dependent chemiluminescence (LDCL) and flow cytometry, respectively. LPS, TNF-␣, and PAF pretreatment induced significant LDCL in response to fMLP; similarly LPS pretreatment was a prerequisite for fMLP-stimulated neutrophil polarization in response to fMLP. However, LPS failed to induce fMLP-mediated chemotaxis of equine neutrophils. These data indicate that equine neutrophil fMLP receptors are not vestigial as previously thought but can trigger both respiratory burst activity and cell polarization responses after priming.

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Rapid, single-step isolation of equine neutrophils on a discontinuous Percoll density gradient

Cited by 56 publications

References 8 publications

Modulatory activities of Agelanthus dodoneifolius (Loranthaceae) extracts on stimulated equine neutrophils and myeloperoxidase activity

Modulatory activities of Agelanthus dodoneifolius (Loranthaceae) extracts on stimulated equine neutrophils and myeloperoxidase activity

Herpesvirus Chemokine-Binding Glycoprotein G (gG) Efficiently Inhibits Neutrophil Chemotaxis In Vitro and In Vivo

Priming induces functional coupling of N-formyl-methionyl-leucyl-phenylalanine receptors in equine neutrophils

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