ATP-Evoked Intracellular Ca2+ Responses in M-CSF Differentiated Human Monocyte-Derived Macrophage are Mediated by P2X4 and P2Y11 Receptor Activation.

Janice A. Layhadi,Samuel J. Fountain

doi:10.3390/ijms20205113

Janice A. Layhadi, Samuel J. Fountain

Open Access

https://doi.org/10.3390/ijms20205113

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Abstract

Tissues differentially secrete multiple colony stimulating factors under conditions of homeostasis and inflammation, orientating recruited circulating monocytes to differentiate to macrophage with differing functional phenotypes. Here, we investigated ATP-evoked intracellular Ca2+ responses in human macrophage differentiated with macrophage colony-stimulating factor (M-CSF). Extracellular ATP evoked (EC50 13.3 ± 1.4 μM) robust biphasic intracellular Ca2+ responses that showed a dependency on both metabotropic (P2Y) and ionotropic (P2X) receptors. qRT-PCR and immunocytochemistry revealed the expression of P2Y1, P2Y2, P2Y6, P2Y11, P2Y13, P2X1, P2X4, P2X5, and P2X7. Pharmacological analysis revealed contribution of only P2X4 and P2Y11 to the Ca2+ response evoked by maximal ATP concentrations (100 µM). This study reveals the contribution of P2X4 and P2Y11 receptor activation to ATP-evoked intracellular Ca2+ responses, and makes comparison with macrophage differentiated using granulocyte colony-stimulating factor (GM-CSF).

Highlights

The generation of monocyte-derived macrophage can be achieved by exposure to colony-stimulating factors (CSFs)
Human macrophage produced by macrophage colony-stimulating factor (M-CSF) differentiation of monocytes displayed phenotypically elongated2.c1h
Sci. 2019, 20, x responses evoked by ATP at different concentrations. (E) Comparison of the total Ca2+ response evoked by 100 μM ATP in granulocyte macrophage colony-stimulating factor (GM-CSF) (10 ng/mL, 6 days) versus M-CSF differentiated macrophage (3 donors each). ** p < 0.01

Summary

Introduction

The generation of monocyte-derived macrophage can be achieved by exposure to colony-stimulating factors (CSFs). CSF are secreted glycoproteins which can recognize and bind to receptors expressed on the surface of haemopoietic stem cells and stimulate proliferation and differentiation. M-CSF and GM-CSF activate different receptors, but both are capable of promoting macrophage survival, proliferation, differentiation, and activation [4,5]. M-CSF orientated human blood monocytes are widely used to generate monocyte-derived macrophage and model tissue macrophage. Macrophage differentiated using M-CSF are often referred to as M2-macrophage (alternatively activated) with an anti-inflammatory cytokine profile [6]. Macrophage differentiated using GM-CSF have a pro-inflammatory cytokine profile, referred to as M1-macrophage (classically activated), and resemble tissue macrophage in lung alveoli [9]. Circulating monocytes that extravasate into tissues expressing different

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