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Published on: May 31, 2018
N-Alkyl-Substituted Isatins Enhance P2X7 Receptor-Induced Interleukin-1β Release from Murine Macrophages
1School of Biological Sciences, University of Wollongong, Wollongong, NSW 2522, Australia; Centre for Medical and Molecular Bioscience, University of Wollongong, Wollongong, NSW 2522, Australia; Illawarra Health and Medical Research Institute, Wollongong, NSW 2522, Australia.
Abstract:
Extracellular adenosine 5'-triphosphate (ATP) activates the P2X7 receptor channel to induce the rapid release of the proinflammatory cytokine, interleukin- (IL-) 1β, from macrophages. Microtubule rearrangements are thought to be involved in this process. Some isatin derivatives alter microtubules and display anticancer activities. The current study investigated the effect of isatin and seven structurally diverse isatin derivatives on P2X7-mediated IL-1β release from murine J774 macrophages. ATP-induced IL-1β and lactate dehydrogenase (LDH) release were assessed by specific colorimetric assays. P2X7 activity was determined by flow cytometric measurements of ATP-induced cation dye uptake. Cytotoxicity of isatin derivatives was determined using a tetrazolium-based colorimetric assay. ATP caused rapid IL-1β release in a concentration-dependent manner, and this process was completely impaired by the P2X7 antagonist, AZ10606120. In contrast, 5,7-dibromo-N-(p-methoxybenzyl)isatin (NAI) and 3-{4-[5,7-dibromo-1-(4-methoxybenzyl)-2-oxoindolin-3-ylidenamino]phenyl}propanoic acid (NAI-imine) enhanced P2X7-induced IL-1β release by twofold compared to that of isatin and the parent molecule, 5,7-dibromoisatin. NAI and NAI-imine had minimal effect on P2X7-induced dye uptake and LDH release. In contrast, 24-hour incubation with NAI and NAI-imine (in the absence of exogenous ATP) induced macrophage death in a concentration-dependent manner. In conclusion, this study demonstrates that N-alkyl-substituted isatins enhance P2X7 receptor-induced IL-1β release from murine macrophages. Thus, in addition to direct anticancer effects, these compounds may also impact inflammatory and immune cells within the tumor microenvironment.
Insights
Certain isatin derivatives, like NAI and NAI-imine, boost the release of interleukin-1β (IL-1β) from macrophages by activating P2X7 receptors. These compounds may influence immune cells in the tumor microenvironment.
Area of Science:
- Immunology
- Pharmacology
- Cell Biology
Background:
- Extracellular adenosine 5'-triphosphate (ATP) activates P2X7 receptors on macrophages, triggering the release of the pro-inflammatory cytokine interleukin-1β (IL-1β).
- Microtubule dynamics are implicated in P2X7 receptor-mediated IL-1β secretion.
- Isatin derivatives are known to modulate microtubules and possess anticancer properties.
Purpose of the Study:
- To investigate the impact of isatin and its derivatives on P2X7 receptor-mediated IL-1β release from murine macrophages.
- To assess the effects of these compounds on P2X7 receptor activity and macrophage cytotoxicity.
Main Methods:
- Murine J774 macrophages were treated with ATP and various isatin derivatives.
- IL-1β and lactate dehydrogenase (LDH) release were quantified using colorimetric assays.
- P2X7 receptor activity was measured by flow cytometry assessing cation dye uptake.
- Cytotoxicity was evaluated using a tetrazolium-based colorimetric assay.
Main Results:
- ATP induced a concentration-dependent release of IL-1β, which was blocked by the P2X7 antagonist AZ10606120.
- Two specific isatin derivatives, 5,7-dibromo-N-(p-methoxybenzyl)isatin (NAI) and NAI-imine, significantly enhanced ATP-induced IL-1β release.
- NAI and NAI-imine showed minimal impact on P2X7-mediated dye uptake and LDH release but induced concentration-dependent macrophage death upon prolonged incubation without exogenous ATP.
Conclusions:
- N-alkyl-substituted isatins, specifically NAI and NAI-imine, potentiate P2X7 receptor-induced IL-1β release from macrophages.
- These findings suggest that isatin derivatives can modulate inflammatory responses in immune cells.
- Beyond direct anticancer effects, isatin derivatives may influence the tumor microenvironment by affecting immune cell function.
