Activation of Macrophages by P2X7-Induced Microvesicles from Myeloid Cells Is Mediated by Phospholipids and Is Partially Dependent on TLR4

Thomas, L. Michael; Salter, Russell D.

doi:10.4049/jimmunol.1001231

Cited by 71 publications

(67 citation statements)

References 68 publications

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“…Combined this data highlights the importance of ensuring that antagonists used in intracellular signalling studies downstream of P2X7 activation do not directly affect P2X7 itself. In this regard, CAY10593 has been used at 50 μM to support a role for PLD in the generation of P2X7-induced microvesicles capable of [27]. However, our data suggests that CAY10593 may have also acted on P2X7 itself in this previous study.…”

Section: Discussioncontrasting

confidence: 53%

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CAY10593 inhibits the human P2X7 receptor independently of phospholipase D1 stimulation

Pupovac

Stokes

Sluyter

2013

Purinergic Signalling

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The P2X7 receptor is a trimeric ATP-gated cation channel important in health and disease. We have observed that the specific phospholipase D (PLD)1 antagonist, CAY10593 impairs P2X7-induced shedding of the 'low affinity' IgE receptor, CD23. The current study investigated the mode of action of this compound on P2X7 activation. Measurements of ATP-induced ethidium + uptake revealed that CAY10593 impaired P2X7-induced pore formation in human RPMI 8226 B cells, P2X7-transfected HEK-293 cells and peripheral blood mononuclear cells. Concentration response curves demonstrated that CAY10593 impaired P2X7-induced pore formation in RPMI 8226 cells more potently than the PLD2 antagonist CAY10594 and the non-specific PLD antagonist halopemide. Electrophysiology measurements demonstrated that CAY10593 also inhibited P2X7-induced inward currents. Notably, RT-PCR demonstrated that PLD1 was absent in RPMI 8226 cells, while choline-Cl medium or 1-butanol, which block PLD stimulation and signalling respectively did not impair P2X7 activation in these cells. This data indicates that CAY10593 impairs human P2X7 independently of PLD1 stimulation and highlights the importance of ensuring that compounds used in signalling studies downstream of P2X7 activation do not affect the receptor itself.

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Section: Discussioncontrasting

confidence: 53%

“…P2X7 activation can stimulate PLD in B cells [21,22] and macrophages [23,24]. P2X7-induced PLD stimulation in macrophages plays a role in the killing of intracellular mycobacteria [25,26] and the generation of microvesicles capable of further macrophage activation [27]. In contrast, the role of P2X7-induced PLD stimulation in B cells remains unknown.…”

Section: +mentioning

confidence: 99%

CAY10593 inhibits the human P2X7 receptor independently of phospholipase D1 stimulation

Pupovac

Stokes

Sluyter

2013

Purinergic Signalling

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“…Inflammation, coagulation Cell lipase D, which does not affect the release of microvesicles but inhibits hydrolysis of phosphatidylcholine to phosphatidic acid, this further supports the lipid nature of the active component (Thomas and Salter, 2010). In addition, exosomes from DCs and macrophages, and microvesicles from human plasma, contain functional enzymes for synthesis of leukotrienes, which are potent lipid inflammatory mediators (Esser et al, 2010).…”

Section: Cellmentioning

confidence: 79%

Classification, Functions, and Clinical Relevance of Extracellular Vesicles

et al. 2012

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“…M059J cells were pre-treated with the selective P2X7R antagonist A740003 (10 μM) [13][14][15][16][17][18] or with the two non-selective P2 receptor antagonists suramin (30 μM) or RB2 (100 μM), and after 20 min with ATP (5 mM) for 24 h. At the end of this period, the medium was removed, the cells were washed with calcium magnesium-free medium (CMF) and 100 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide solution (MTT) (MTT 5 mg/ml in PBS in 90 % DMEM/10 % FBS) was added to the cells and incubated for 3 h. The formazan crystals were dissolved with 100 μl of dimethyl sulfoxide (DMSO). The absorbance was quantified in 96-well plates (Spectra Max M2e, Molecular Devices) at 595 nm.…”

Section: Cell Viabilitymentioning

confidence: 99%

P2X7 receptor activation leads to increased cell death in a radiosensitive human glioma cell line

Gehring

Pereira

Zanin

et al. 2012

Purinergic Signalling

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Gliomas are the most lethal tumors of central nervous system. ATP is an important signaling molecule in CNS and it is a selective P2X7 purinergic receptor ligand at high concentrations. Herein, we investigated whether the activation of P2X7R might be implicated in death of a radiosensitive human glioma lineage. The effects of P2X7R agonists (ATP and BzATP) and irradiation (2 Gy) on glioma cells were analyzed by MTT assay and annexin-V/PI determination, whereas mRNA and protein P2X7R expression was assessed by qRT-PCR and flow cytometry, respectively. P2X7R pore formation was functionality examined by analyzing ethidium bromide uptake. The human glioma cells U-138 MG and U-251 MG were resistant to death when treated with either ATP (5 mM) or BzATP (100 μM), but the radiosensitive M059J glioma cells displayed a significant decrease of cell viability (32.4±4.1 % and 25.6 ± 3.3 %, respectively). The M059J lineage expresses significantly higher mRNA P2X7R levels when compared to the U-138 MG and U-251 cell lines (0.40± 0.00; 0.28±0.01, and 0.31±0.01, respectively), and irradiation upregulated P2X7R expression (0.55 ±0.08) in this lineage. Noteworthy, P2X7R protein doubled after irradiation on M059J lineage, and increased in 50 % and 42.6 % when comparing M059J-irradiated to irradiated U-138 MG and U-251 MG cells, respectively. Ethidium bromide uptake was significantly increased in 104 % and 77.8 % when comparing M059J to U-138 MG and U-251MG, respectively. Finally, the selective P2X7R antagonist A740003 significantly decreased the cell death caused by irradiation. We provide novel evidence indicating that M059J human glioma cell line is ATP-P2X7R sensitive, pointing out the relevance of the purinergic P2X7R on glioma radiosensitivity.

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Activation of Macrophages by P2X7-Induced Microvesicles from Myeloid Cells Is Mediated by Phospholipids and Is Partially Dependent on TLR4

Abstract: Material

Cited by 71 publications

References 68 publications

CAY10593 inhibits the human P2X7 receptor independently of phospholipase D1 stimulation

CAY10593 inhibits the human P2X7 receptor independently of phospholipase D1 stimulation

Classification, Functions, and Clinical Relevance of Extracellular Vesicles

P2X7 receptor activation leads to increased cell death in a radiosensitive human glioma cell line

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