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Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation
Published on: April 29, 2015
Secretion of IL-1β triggered by dynasore in murine peritoneal macrophages
Michèle Seil1, Malika El Ouaaliti, Jean-Paul Dehaye
1Laboratoire de Chimie Biologique et Médicale et de Microbiologie Pharmaceutique, Institut de Pharmacie C.P. 205/3, Université Libre de Bruxelles, Belgium.
Abstract:
The interaction of lipopolysaccharide-primed murine peritoneal macrophages with ivermectin, an antiparasite drug which potentiates P2X(4) receptors and dynasore which inhibits the GTPase activity of dynamin, a protein contributing to the internalization of plasma membrane proteins, was tested. Murine peritoneal macrophages express P2X(4) receptors which are mostly intracellular. In cells from P2X(7)-knockout mice (KO mice), 10 µm adenosine triphosphate (ATP) provoked a transient increase of the intracellular concentration of calcium. Ivermectin had no effect by itself but potentiated the increase of the intracellular concentration of calcium by ATP. The combination of ATP plus ivermectin also decreased the intracellular concentration of potassium and promoted the secretion of IL-1β. Concentrations of dynasore above 50 µm affected the integrity of mitochondria (MTT test) and of the plasma membrane (release of lactate dehydrogenase, LDH). At a 10 µm concentration, dynasore had no effect on the responses to ATP and on the internalization of P2X(4) receptors. By itself dynasore promoted the release of potassium and the secretion of IL-1β after activation of caspase-1. In conclusion, our results confirm that ivermectin potentiates the responses coupled to P2X(4) receptors probably by interaction with an allosteric site. We also show that this potentiation triggers the release of IL-1β by macrophages. As opposed to ivermectin, dynasore has no effect on P2X(4) receptors. This drug triggers a potassium efflux via a mechanism which does not involve purinergic receptors and generates, in consequence, the activation of caspase-1 and the secretion of IL-1β.
Insights
Ivermectin enhances responses to ATP in macrophages by interacting with P2X(4) receptors, promoting IL-1β secretion. Dynasore, however, triggers potassium efflux and IL-1β release independently of purinergic receptors.
Area of Science:
- Immunology
- Pharmacology
Background:
- Murine peritoneal macrophages express P2X(4) receptors, primarily intracellular.
- P2X(7)-knockout mice models were used to study calcium signaling.
- Adenosine triphosphate (ATP) is a known activator of purinergic receptors.
Purpose of the Study:
- To investigate the effects of ivermectin and dynasore on macrophage responses.
- To elucidate the role of P2X(4) receptors in macrophage activation.
- To determine the mechanisms of IL-1β secretion induced by these drugs.
Main Methods:
- Lipopolysaccharide-primed murine peritoneal macrophages were treated with ivermectin and dynasore.
- Intracellular calcium and potassium concentrations were measured.
- Interleukin-1β (IL-1β) secretion was quantified.
- Mitochondrial integrity and plasma membrane permeability were assessed.
Main Results:
- Ivermectin potentiated ATP-induced calcium increases and promoted IL-1β secretion.
- Dynasore, at higher concentrations, compromised cell integrity but not P2X(4) receptor internalization at 10 µm.
- Dynasore alone induced potassium efflux and IL-1β release via caspase-1 activation, independent of purinergic receptors.
Conclusions:
- Ivermectin potentiates P2X(4) receptor-mediated responses and triggers IL-1β release.
- Dynasore activates caspase-1 and IL-1β secretion through a non-purinergic mechanism.
- Ivermectin and dynasore exhibit distinct mechanisms of action on macrophages.
