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The purinergic P2Z receptor of human macrophage cells. Characterization and possible physiological role
S Falzoni1, M Munerati, D Ferrari
1Institute of General Pathology, University of Ferrara, Italy.
Abstract:
We have investigated responses of human monocyte/macrophage cells to extracellular ATP (ATPe). Freshly isolated peripheral blood monocytes showed responses linked to P2Y but not P2Z purinergic receptors; however, during in vitro macrophage differentiation, these cells also exhibited responses suggestive of the presence of the membrane-permeabilizing P2Z receptor. In fact, in human macrophages a brief (15-min) exposure to ATPe, but not other nucleotides, caused (1) a rapid and long-lasting plasma membrane depolarization; (2) a large increase in intracellular Ca2+ concentration followed by efflux of the Ca2+ indicator; (3) uptake of low molecular weight hydrophilic molecules such as Lucifer yellow and ethidium bromide; and (4) cell rounding, swelling, and eventual release of the cytoplasmic enzyme lactate dehydrogenase. rIFN-gamma enhanced both membrane-permeabilizing and cytotoxic ATPe effects. Membrane permeabilization and cytotoxicity were fully blocked by pretreatment of the cells with oxidized ATP, a compound recently shown to block P2Z receptors covalently in macrophages. Blocking of the P2Z receptor by oxidized ATP also inhibited multinucleated giant cell generation stimulated by concanavalin A or rIFN-gamma without decreasing monocyte migration or membrane adhesion molecule expression. These data suggest that human macrophages express rIFN-gamma-modulated purinergic P2Z receptors in vitro and hint at a role for these plasma membrane molecules in the generation of macrophage polykarions.
Insights
Human macrophages develop P2Z purinergic receptors during differentiation, responding to extracellular ATP (ATPe) with membrane permeabilization and cytotoxicity, modulated by rIFN-gamma.
Area of Science:
- Immunology
- Cell Biology
- Molecular Biology
Background:
- Monocytes differentiate into macrophages in vitro.
- Purinergic receptors, including P2Y and P2Z, play roles in cellular signaling.
- Extracellular ATP (ATPe) can trigger cellular responses via purinergic receptors.
Purpose of the Study:
- To investigate human monocyte/macrophage responses to extracellular ATP (ATPe).
- To characterize the purinergic receptors involved in these responses.
- To determine the role of rIFN-gamma and P2Z receptors in macrophage function.
Main Methods:
- Isolated human peripheral blood monocytes were differentiated into macrophages in vitro.
- Cells were exposed to extracellular ATP (ATPe) and other nucleotides.
- Responses measured included plasma membrane depolarization, intracellular Ca2+ concentration, molecule uptake, cell morphology changes, lactate dehydrogenase release, and multinucleated giant cell formation.
- Effects of rIFN-gamma and oxidized ATP (a P2Z receptor blocker) were assessed.
Main Results:
- Differentiating macrophages, but not fresh monocytes, responded to ATPe with membrane permeabilization and cytotoxicity, indicative of P2Z receptor activity.
- ATPe exposure caused rapid membrane depolarization, increased intracellular Ca2+, uptake of hydrophilic molecules, and cell damage.
- rIFN-gamma enhanced ATPe-induced effects, while oxidized ATP blocked ATPe responses and inhibited concanavalin A/rIFN-gamma-stimulated multinucleated giant cell generation.
Conclusions:
- Human macrophages express functional, rIFN-gamma-modulated P2Z purinergic receptors in vitro.
- P2Z receptor activation by ATPe leads to membrane permeabilization and cytotoxicity.
- P2Z receptors are implicated in multinucleated giant cell formation, suggesting a role in macrophage polykaryon generation.
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