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Spontaneous cell fusion in macrophage cultures expressing high levels of the P2Z/P2X7 receptor.

Chiozzi P, Sanz JM, Ferrari D, Falzoni S, Aleotti A, Buell GN, Collo G, Di Virgilio F - J. Cell Biol. (1997)

Bottom Line: Spisani, S.Clin.Invest. 95:1207- 1216).

View Article: PubMed Central - PubMed

Affiliation: Institute of General Pathology, University of Ferrara, I-44100 Ferrara, Italy.

ABSTRACT
Mouse and human macrophages express a plasma membrane receptor for extracellular ATP named P2Z/P2X7. This molecule, recently cloned, is endowed with the intriguing property of forming an aqueous pore that allows transmembrane fluxes of hydrophylic molecules of molecular weight below 900. The physiological function of this receptor is unknown. In a previous study we reported experiments suggesting that the P2Z/P2X7 receptor is involved in the formation of macrophage-derived multinucleated giant cells (MGCs; Falzoni, S., M. Munerati, D. Ferrari, S. Spisani, S. Moretti, and F. Di Virgilio. 1995. J. Clin. Invest. 95:1207- 1216). We have selected several clones of mouse J774 macrophages that are characterized by either high or low expression of the P2Z/P2X7 receptor and named these clones P2Zhyper or P2Zhypo, respectively. P2Zhyper, but not P2Zhypo, cells grown to confluence in culture spontaneously fuse to form MGCs. As previously shown for human macrophages, fusion is inhibited by the P2Z/P2X7 blocker oxidized ATP. MGCs die shortly after fusion through a dramatic process of cytoplasmic sepimentation followed by fragmentation. These observations support our previous hypothesis that the P2Z/P2X7 receptor is involved in macrophage fusion.

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P2X7 RNA expression in J774 mouse macrophages,  P2Zhyper, and P2Zhypo clones. P2Zhyper and P2Zhypo clones,  wild-type J774 macrophages, and HEK293 cells stably transfected with P2X7 cDNA were incubated with digoxigenin antisense riboprobe (A–D, respectively). Bars, 10 μm.
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Figure 3: P2X7 RNA expression in J774 mouse macrophages, P2Zhyper, and P2Zhypo clones. P2Zhyper and P2Zhypo clones, wild-type J774 macrophages, and HEK293 cells stably transfected with P2X7 cDNA were incubated with digoxigenin antisense riboprobe (A–D, respectively). Bars, 10 μm.

Mentions: P2Zhyper cells overexpress the P2Z/P2X7 mRNA and protein as seen by in situ hybridization (Fig. 3) and western (Fig. 4) blot analysis. P2Zhyper macrophages (Fig. 3, A) show a very strong reactivity to the specific P2X7 anti-sense riboprobe, while P2Zhypo cells (Fig. 3 B) have little, if any, reactivity with the notable exception of one highly reactive cell. J774 wild-type cells (Fig. 3 C) show an intermediate reactivity. A stable transfectant (HEK293 cells) for rat P2X7 is shown for comparison (Fig. 3 D). On average, we found that in the P2Zhyper population ∼83 ± 5% of the cells were strongly positive, as compared to ∼65 ± 8% of positive cells in the J774 wild-type population and only 2–3% of positive cells in the P2Zhypo macrophages. No hybridization was detected with the sense riboprobe (not shown). Fig. 4 shows that the specific polyclonal Ab raised against the carboxy-terminal tail of the receptor recognized in J774 wild-type cells a protein of the expected molecular weight of ∼72. This band was stronger in P2Zhyper cells and absent in P2Zhypo and control cells.


Spontaneous cell fusion in macrophage cultures expressing high levels of the P2Z/P2X7 receptor.

Chiozzi P, Sanz JM, Ferrari D, Falzoni S, Aleotti A, Buell GN, Collo G, Di Virgilio F - J. Cell Biol. (1997)

P2X7 RNA expression in J774 mouse macrophages,  P2Zhyper, and P2Zhypo clones. P2Zhyper and P2Zhypo clones,  wild-type J774 macrophages, and HEK293 cells stably transfected with P2X7 cDNA were incubated with digoxigenin antisense riboprobe (A–D, respectively). Bars, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2141639&req=5

Figure 3: P2X7 RNA expression in J774 mouse macrophages, P2Zhyper, and P2Zhypo clones. P2Zhyper and P2Zhypo clones, wild-type J774 macrophages, and HEK293 cells stably transfected with P2X7 cDNA were incubated with digoxigenin antisense riboprobe (A–D, respectively). Bars, 10 μm.
Mentions: P2Zhyper cells overexpress the P2Z/P2X7 mRNA and protein as seen by in situ hybridization (Fig. 3) and western (Fig. 4) blot analysis. P2Zhyper macrophages (Fig. 3, A) show a very strong reactivity to the specific P2X7 anti-sense riboprobe, while P2Zhypo cells (Fig. 3 B) have little, if any, reactivity with the notable exception of one highly reactive cell. J774 wild-type cells (Fig. 3 C) show an intermediate reactivity. A stable transfectant (HEK293 cells) for rat P2X7 is shown for comparison (Fig. 3 D). On average, we found that in the P2Zhyper population ∼83 ± 5% of the cells were strongly positive, as compared to ∼65 ± 8% of positive cells in the J774 wild-type population and only 2–3% of positive cells in the P2Zhypo macrophages. No hybridization was detected with the sense riboprobe (not shown). Fig. 4 shows that the specific polyclonal Ab raised against the carboxy-terminal tail of the receptor recognized in J774 wild-type cells a protein of the expected molecular weight of ∼72. This band was stronger in P2Zhyper cells and absent in P2Zhypo and control cells.

Bottom Line: Spisani, S.Clin.Invest. 95:1207- 1216).

View Article: PubMed Central - PubMed

Affiliation: Institute of General Pathology, University of Ferrara, I-44100 Ferrara, Italy.

ABSTRACT
Mouse and human macrophages express a plasma membrane receptor for extracellular ATP named P2Z/P2X7. This molecule, recently cloned, is endowed with the intriguing property of forming an aqueous pore that allows transmembrane fluxes of hydrophylic molecules of molecular weight below 900. The physiological function of this receptor is unknown. In a previous study we reported experiments suggesting that the P2Z/P2X7 receptor is involved in the formation of macrophage-derived multinucleated giant cells (MGCs; Falzoni, S., M. Munerati, D. Ferrari, S. Spisani, S. Moretti, and F. Di Virgilio. 1995. J. Clin. Invest. 95:1207- 1216). We have selected several clones of mouse J774 macrophages that are characterized by either high or low expression of the P2Z/P2X7 receptor and named these clones P2Zhyper or P2Zhypo, respectively. P2Zhyper, but not P2Zhypo, cells grown to confluence in culture spontaneously fuse to form MGCs. As previously shown for human macrophages, fusion is inhibited by the P2Z/P2X7 blocker oxidized ATP. MGCs die shortly after fusion through a dramatic process of cytoplasmic sepimentation followed by fragmentation. These observations support our previous hypothesis that the P2Z/P2X7 receptor is involved in macrophage fusion.

Show MeSH
Related in: MedlinePlus