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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: Clin.Invest. 95:1207- 1216).These observations support our previous hypothesis that the P2Z/P2X7 receptor is involved in macrophage fusion.

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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Double staining of multinucleated giant cells with lucifer yellow and Texas red. Two batches of P2Zhyper cells were separately allowed to pinocytose lucifer yellow (10 mg/ml) or Texas red (1 mg/ml) for 1 h at 37°C and then rinsed several times with complete RPMI medium, mixed together, and layered in 24-well dishes at the concentration of 5 × 105/well. After 3 d, the cultures were examined for formation of MGCs and photographed with a fluorescence microscope. (A) Phase contrast; (B) rhodamine filter; (C)  fluorescein filter. Bars, 25 μm.
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Figure 7: Double staining of multinucleated giant cells with lucifer yellow and Texas red. Two batches of P2Zhyper cells were separately allowed to pinocytose lucifer yellow (10 mg/ml) or Texas red (1 mg/ml) for 1 h at 37°C and then rinsed several times with complete RPMI medium, mixed together, and layered in 24-well dishes at the concentration of 5 × 105/well. After 3 d, the cultures were examined for formation of MGCs and photographed with a fluorescence microscope. (A) Phase contrast; (B) rhodamine filter; (C) fluorescein filter. Bars, 25 μm.

Mentions: P2Zhyper cells were very fragile and easily died upon reaching confluence. Nonetheless, in many cultures we observed that shortly after reaching confluence (usually 3 d after plating), macrophages formed dense aggregates in which MGCs were easily detected (Fig. 5). During the last two years we have selected >20 P2Zhyper cell clones, and invariably, although to a different extent, we have observed formation of MGCs in all these cell populations. MGCs had different shapes: round, with short pseudopods (Fig. 5 A); star-like, with dendritic-like elongations (Fig. 5 B); polygonal, with a regular cellular contour (Fig. 5 C). MGCs contained from 2 to >10 large nuclei usually located in the center and were also readily observed in monolayers of HEK293 cells stably transfected with the P2X7 receptor cDNA (Fig. 5 D); on the contrary, fused cells were never observed in monolayers of P2Zhypo or P2Zwt macrophages (Fig. 6). In principle, MGCs could originate either from a real membrane fusion event or from kariokinesis without cell division. To solve this issue, we pooled two batches of P2Zhyper cells that had been previously labeled with different endosome–lysosome markers, lucifer yellow, and Texas red. Real fusion events were detected by observing both yellow/green and red vesicles within the same MGC. Two examples of such fusion are shown by arrowheads, which identify MGCs of different shapes in Fig. 7. On the contrary, individual macrophages are labeled by either the yellow/green or red tracer (Fig. 7, closed triangles). Obviously, cell fusion can also occur between macrophages labeled with the same tracer, such as the spindle-shaped MGC visible in the left-hand side of Fig. 7 A (closed diamond), that is stained only by Texas red.


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)

Double staining of multinucleated giant cells with lucifer yellow and Texas red. Two batches of P2Zhyper cells were separately allowed to pinocytose lucifer yellow (10 mg/ml) or Texas red (1 mg/ml) for 1 h at 37°C and then rinsed several times with complete RPMI medium, mixed together, and layered in 24-well dishes at the concentration of 5 × 105/well. After 3 d, the cultures were examined for formation of MGCs and photographed with a fluorescence microscope. (A) Phase contrast; (B) rhodamine filter; (C)  fluorescein filter. Bars, 25 μm.
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Related In: Results  -  Collection

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Figure 7: Double staining of multinucleated giant cells with lucifer yellow and Texas red. Two batches of P2Zhyper cells were separately allowed to pinocytose lucifer yellow (10 mg/ml) or Texas red (1 mg/ml) for 1 h at 37°C and then rinsed several times with complete RPMI medium, mixed together, and layered in 24-well dishes at the concentration of 5 × 105/well. After 3 d, the cultures were examined for formation of MGCs and photographed with a fluorescence microscope. (A) Phase contrast; (B) rhodamine filter; (C) fluorescein filter. Bars, 25 μm.
Mentions: P2Zhyper cells were very fragile and easily died upon reaching confluence. Nonetheless, in many cultures we observed that shortly after reaching confluence (usually 3 d after plating), macrophages formed dense aggregates in which MGCs were easily detected (Fig. 5). During the last two years we have selected >20 P2Zhyper cell clones, and invariably, although to a different extent, we have observed formation of MGCs in all these cell populations. MGCs had different shapes: round, with short pseudopods (Fig. 5 A); star-like, with dendritic-like elongations (Fig. 5 B); polygonal, with a regular cellular contour (Fig. 5 C). MGCs contained from 2 to >10 large nuclei usually located in the center and were also readily observed in monolayers of HEK293 cells stably transfected with the P2X7 receptor cDNA (Fig. 5 D); on the contrary, fused cells were never observed in monolayers of P2Zhypo or P2Zwt macrophages (Fig. 6). In principle, MGCs could originate either from a real membrane fusion event or from kariokinesis without cell division. To solve this issue, we pooled two batches of P2Zhyper cells that had been previously labeled with different endosome–lysosome markers, lucifer yellow, and Texas red. Real fusion events were detected by observing both yellow/green and red vesicles within the same MGC. Two examples of such fusion are shown by arrowheads, which identify MGCs of different shapes in Fig. 7. On the contrary, individual macrophages are labeled by either the yellow/green or red tracer (Fig. 7, closed triangles). Obviously, cell fusion can also occur between macrophages labeled with the same tracer, such as the spindle-shaped MGC visible in the left-hand side of Fig. 7 A (closed diamond), that is stained only by Texas red.

Bottom Line: Clin.Invest. 95:1207- 1216).These observations support our previous hypothesis that the P2Z/P2X7 receptor is involved in macrophage fusion.

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