Optimal conditions for simultaneous purification of mononuclear and polymorphonuclear leucocytes from human blood by the hypaque-ficoll method

Fèrrante, Antonio; Thong, Y.H.

doi:10.1016/0022-1759(80)90036-8

Cited by 543 publications

(239 citation statements)

References 9 publications

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“…Neutrophils from heparinized (10 U/ml) peripheral venous blood were isolated by density-gradient centrifugation on Mono-Poly resolving medium (ICN Biomedical, Costa Mesa, CA) at 300 ϫ g for 30 min at room temperature (30). The fraction containing neutrophils was harvested and washed three times with PBS at 4°C and suspended in RPMI 1640 medium.…”

Section: Cells and Cell Culturesmentioning

confidence: 99%

Activation of Human Oral Epithelial Cells by Neutrophil Proteinase 3 Through Protease-Activated Receptor-2

Uehara¹,

Sugawara²,

Muramoto

et al. 2002

The Journal of Immunology

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Proteinase 3 (PR3), a 29-kDa serine proteinase secreted from activated neutrophils, also exists in a membrane-bound form, and is suggested to actively contribute to inflammatory processes. The present study focused on the mechanism by which PR3 activates human oral epithelial cells. PR3 activated the epithelial cells in culture to produce IL-8 and monocyte chemoattractant protein-1 and to express ICAM-1 in a dose- and time-dependent manner. Incubation of the epithelial cells for 24 h with PR3 resulted in a significant increase in the adhesion to neutrophils, which was reduced to baseline levels in the presence of anti-ICAM-1 mAb. Activation of the epithelial cells by PR3 was inhibited by serine proteinase inhibitors and serum. The epithelial cells strongly express protease-activated receptor (PAR)-1 and PAR-2 mRNA and weakly express PAR-3 mRNA. The expression of PAR-2 on the cell surface was promoted by PR3, and inhibited by cytochalasin B, but not by cycloheximide. PR3 cleaved the peptide corresponding to the N terminus of PAR-2 with exposure of its tethered ligand. Treatment with trypsin, an agonist for PAR-2, and a synthetic PAR-2 agonist peptide induced intracellular Ca2+ mobilization, and rendered cells refractory to subsequent stimulation with PR3 and vice versa. The production of cytokine induced by PR3 and the PAR-2 agonist peptide was completely abolished by a phospholipase C inhibitor. These findings suggest that neutrophil PR3 activates oral epithelial cells through G protein-coupled PAR-2 and actively participates in the process of inflammation such as periodontitis.

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Section: Cells and Cell Culturesmentioning

confidence: 99%

Activation of Human Oral Epithelial Cells by Neutrophil Proteinase 3 Through Protease-Activated Receptor-2

Uehara¹,

Sugawara²,

Muramoto

et al. 2002

The Journal of Immunology

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“…The neutrophils were prepared essentially as pioneered by Böyum (Böyum, 1968), a methodology that was later optimized by others (Ferrante and Thong, 1980). A density …”

Section: Neutrophilsmentioning

confidence: 99%

Role of the actin cytoskeleton during respiratory burst in chemoattractant‐stimulated neutrophils

Bengtsson

Orselius

Wetterö

2006

Cell Biology International

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Running Title: The actin cytoskeleton and the respiratory burstThe actin cytoskeleton and the respiratory burst -Bengtsson et al. 2 ABSTRACTThe aim of this study was to clarify the role of the actin cytoskeleton during chemotactic peptide fMet-Leu-Phe (fMLF)-stimulated respiratory burst in human neutrophil granulocytes. Reactive oxygen species (ROS) was measured as luminol-amplified chemiluminescence (CL) and F-actin content as bodipy phallacidin fluorescence in neutrophils treated with latrunculin B or jasplakinolide, an inhibitor and activator of actin polymerization, respectively. Latrunculin B markedly decreased, whereas jasplakinolide increased, the F-actin content in neutrophils, unstimulated or stimulated with fMLF.Latrunculin B enhanced the fMLF-triggered ROS production more than tenfold.Jasplakinolide initially inhibited the fMLF-induced CL-response, however, caused a potent second sustained phase (>400% of control). Both actin-drugs triggered a substantial CL-response when added 5-25 min after fMLF. This was also valid for chemotactic doses of fMLF, where latrunculin B and jasplakinolide amplified the ROSproduction 5-10 times. By using specific signal transduction inhibitors, we found that the NADPH-oxidase activation triggered by destabilization of the actin cytoskeleton occurs downstream of phospholipase C and protein kinase C but is mediated by Rho GTPases and tyrosine phosphorylation. In conclusion, this study indicates that rearrangements of the actin cytoskeleton are a prerequisite in connecting ligand/receptor activation, generation of second messengers and assembly of the NADPH oxidase in neutrophil granulocytes.The actin cytoskeleton and the respiratory burst -Bengtsson et al. 3

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“…The neutrophils were prepared essentially according to the methodology pioneered by Böyum [18] and later optimized by others [19]. A density separation medium was prepared by careful layering of one part of Lymphoprep over four parts of Polymorphprep (Axis-Shield PoC AS, Oslo, Norway).…”

Section: Isolation Of Neutrophils and Plateletsmentioning

confidence: 99%

Platelets stimulated by IgG-coated surfaces bind and activate neutrophils through a selectin-dependent pathway

2003

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Blood platelets bind rapidly to foreign surfaces and interact with adsorbed proteins and neutrophil granulocytes. We demonstrate by use of luminol-amplified chemiluminescence under stirred and nonstirred conditions that platelets at IgG-coated surfaces amplify the neutrophil extracellular release of reactive oxygen species (ROS). The neutrophil response involved tyrosine phosphorylation, but was only in part induced by neutrophil F c -receptor stimulation. The platelet mediated effects were contact-dependent since the respiratory burst was inhibited when the IgG-stimulated platelets were removed by filtration, but not when they were fixed in paraformaldehyde. Bodipyphallacidin-staining of filamentous actin (F-actin) revealed that an actin-dependent platelet adhesion supported the subsequent adhesion and spreading of neutrophils. The neutrophil ROS-response was lowered when the interaction between platelet P-selectin (CD62P) and neutrophil P-selectin glycoprotein ligand-l (PSGL-1 or CD162) was inhibited. The blocking of L-selectin (CD62L) or blocking of the interaction between platelet glycoprotein (Gp) IIb/IIIa and neutrophil complement receptor 3 (CR3) showed no effect. We conclude that platelet activation on immobilized IgG trigger a contact-dependent "frustrated" phagocytosis by neutrophils, associated with a release of toxic ROS. IgG-stimulated platelets activate neutrophils -Wetterö et al.3

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Optimal conditions for simultaneous purification of mononuclear and polymorphonuclear leucocytes from human blood by the hypaque-ficoll method

Cited by 543 publications

References 9 publications

Activation of Human Oral Epithelial Cells by Neutrophil Proteinase 3 Through Protease-Activated Receptor-2

Activation of Human Oral Epithelial Cells by Neutrophil Proteinase 3 Through Protease-Activated Receptor-2

Role of the actin cytoskeleton during respiratory burst in chemoattractant‐stimulated neutrophils

Platelets stimulated by IgG-coated surfaces bind and activate neutrophils through a selectin-dependent pathway

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