Related Experiment Video
Updated: Jul 25, 2025
Macrophage Differentiation and Polarization into an M2-Like Phenotype using a Human Monocyte-Like THP-1 Leukemia Cell Line
Published on: August 2, 2021
Prevention of M2 polarization and temporal limitation of differentiation in monocytes by extracellular ATP
Benedikt F Scherr1,2, Martin F Reiner3, Flavia Baumann4
1Department of Pneumology, Medical Center, Faculty of Medicine, University of Freiburg, Engesserstr. 4 5thFloor, 79106 79108, Freiburg, Germany.
Background:
Elevated levels of extracellular adenosine triphosphate (ATP) modulate immunologic pathways and are considered to be a danger signal in inflammation, lung fibrosis and cancer. Macrophages can be classified into two main types: M1 macrophages are classically activated, pro-inflammatory macrophages, whereas M2 macrophages are alternatively activated, pro-fibrotic macrophages. In this study, we examined the effect of ATP on differentiation of native human monocytes into these macrophage subtypes. We characterized M1 and M2 like macrophages by their release of Interleukin-1beta (IL-1β) and Chemokine (C-C motif) ligand 18 (CCL18), respectively.
Results:
Monocytes were stimulated with ATP or the P2X7 receptor agonist Benzoylbenzoyl-ATP (Bz-ATP), and the production of various cytokines was analyzed, with a particular focus on CCL18 and IL-1β, along with the expression of different purinergic receptors. Over a 72 h period of cell culture, monocytes spontaneously differentiated to M2 like macrophages, as indicated by an increased release of CCL18. Immediate stimulation of monocytes with ATP resulted in a dose-dependent reduction in CCL18 release, but had no effect on the concentration of IL-1β. In contrast, delayed stimulation with ATP had no effect on either CCL18 or IL-1β release. Similar results were observed in a model of inflammation using lipopolysaccharide-stimulated human monocytes. Stimulation with the P2X7 receptor agonist Bz-ATP mimicked the effect of ATP on M2-macrophage differentiation, indicating that P2X7 is involved in ATP-induced inhibition of CCL18 release. Indeed, P2X7 was downregulated during spontaneous M2 differentiation, which may partially explain the ineffectiveness of late ATP stimulation of monocytes. However, pre-incubation of monocytes with PPADS, Suramin (unselective P2X- and P2Y-receptor blockers) and KN62 (P2X7-antagonist) failed to reverse the reduction of CCL18 by ATP.
Conclusions:
ATP prevents spontaneous differentiation of monocytes into M2-like macrophages in a dose- and time-dependent manner. These effects were not mediated by P2X and P2Y receptors.
Insights
Extracellular adenosine triphosphate (ATP) inhibits the differentiation of monocytes into M2-like macrophages. This effect is dose- and time-dependent and does not involve P2X or P2Y receptors, offering new insights into immune regulation.
Area of Science:
- Immunology
- Cell Biology
- Molecular Biology
Background:
- Extracellular adenosine triphosphate (ATP) acts as a danger signal in inflammation, fibrosis, and cancer by modulating immune pathways.
- Macrophages are key immune cells, with M1 (pro-inflammatory) and M2 (pro-fibrotic) subtypes playing distinct roles.
- Understanding how ATP influences monocyte differentiation into these macrophage subtypes is crucial for immune response modulation.
Purpose of the Study:
- To investigate the effect of extracellular adenosine triphosphate (ATP) on the differentiation of human monocytes into M1 and M2 macrophage subtypes.
- To characterize M1 and M2 macrophage phenotypes by analyzing the release of Interleukin-1beta (IL-1β) and Chemokine (C-C motif) ligand 18 (CCL18).
Main Methods:
- Human monocytes were stimulated with ATP or the P2X7 receptor agonist Benzoylbenzoyl-ATP (Bz-ATP).
- Cytokine production (CCL18, IL-1β) and purinergic receptor expression were analyzed over 72 hours.
- Inhibition studies were performed using P2X/P2Y receptor blockers (PPADS, Suramin) and a P2X7 antagonist (KN62).
Main Results:
- Monocytes spontaneously differentiated into M2-like macrophages, indicated by increased CCL18 release.
- ATP significantly reduced CCL18 release in a dose- and time-dependent manner, without affecting IL-1β levels.
- P2X7 receptor activation mimicked ATP's effect, but receptor blockers failed to reverse ATP-induced inhibition, suggesting a non-canonical pathway.
Conclusions:
- Extracellular adenosine triphosphate (ATP) effectively inhibits the spontaneous differentiation of monocytes into M2-like macrophages.
- This inhibitory effect is dose- and time-dependent and appears to be independent of P2X and P2Y receptor signaling.
- These findings highlight a novel mechanism by which ATP regulates macrophage polarization, with potential implications for inflammatory and fibrotic diseases.
More Related Videos
08:37Polarization and Characterization of M1 and M2 Human Monocyte-Derived Macrophages on Implant Surfaces
Published on: December 6, 2024
10:43Polarization of M1 and M2 Human Monocyte-Derived Cells and Analysis with Flow Cytometry upon Mycobacterium tuberculosis Infection
Published on: September 18, 2020
