Limits...
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.

Show MeSH

Related in: MedlinePlus

Enhancement of  ATP-dependent lucifer yellow uptake by P2Zhyper  cells. Macrophages were incubated in complete RPMI  medium at 37°C and stimulated with 3 mM ATP in the  presence of 1 mg/ml of lucifer yellow for 15 min. After  this incubation time, they  were rinsed several times  with complete RPMI medium and photographed  with a fluorescence microscope (40× objective). (A  and D) P2Zhyper; (B and E)  P2Zwt; (C and F) P2Zhypo.  Bars, 25 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2141639&req=5

Figure 1: Enhancement of ATP-dependent lucifer yellow uptake by P2Zhyper cells. Macrophages were incubated in complete RPMI medium at 37°C and stimulated with 3 mM ATP in the presence of 1 mg/ml of lucifer yellow for 15 min. After this incubation time, they were rinsed several times with complete RPMI medium and photographed with a fluorescence microscope (40× objective). (A and D) P2Zhyper; (B and E) P2Zwt; (C and F) P2Zhypo. Bars, 25 μm.

Mentions: P2Z/P2X7 receptor function can be demonstrated by the ATP-mediated uptake of extracellular hydrophilic fluorescent markers such as lucifer yellow or YO-PRO. Fig. 1 shows that incubation in the presence of 3 mM ATP triggered a massive uptake of lucifer yellow by P2Zhyper cells (Fig. 1, A and D), while on the contrary almost no uptake by P2Zhypo cells was detectable (Fig. 1, C and F). P2Zwt showed an intermediate behavior (Fig. 1, B and E). Nearly all P2Zhyper cells were positive for lucifer yellow uptake to varying levels. Because the sustained activation of the P2Z/P2X7 receptor leads to cell death, we tested for the release of the cytoplasmic enzyme lactic dehydrogenase in response to ATP. Whereas P2Zhypo were unaffected, P2Zhyper rapidly released lactic dehydrogenase, and P2Zwt again showed an intermediate behavior (Fig. 2).


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)

Enhancement of  ATP-dependent lucifer yellow uptake by P2Zhyper  cells. Macrophages were incubated in complete RPMI  medium at 37°C and stimulated with 3 mM ATP in the  presence of 1 mg/ml of lucifer yellow for 15 min. After  this incubation time, they  were rinsed several times  with complete RPMI medium and photographed  with a fluorescence microscope (40× objective). (A  and D) P2Zhyper; (B and E)  P2Zwt; (C and F) P2Zhypo.  Bars, 25 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Enhancement of ATP-dependent lucifer yellow uptake by P2Zhyper cells. Macrophages were incubated in complete RPMI medium at 37°C and stimulated with 3 mM ATP in the presence of 1 mg/ml of lucifer yellow for 15 min. After this incubation time, they were rinsed several times with complete RPMI medium and photographed with a fluorescence microscope (40× objective). (A and D) P2Zhyper; (B and E) P2Zwt; (C and F) P2Zhypo. Bars, 25 μm.
Mentions: P2Z/P2X7 receptor function can be demonstrated by the ATP-mediated uptake of extracellular hydrophilic fluorescent markers such as lucifer yellow or YO-PRO. Fig. 1 shows that incubation in the presence of 3 mM ATP triggered a massive uptake of lucifer yellow by P2Zhyper cells (Fig. 1, A and D), while on the contrary almost no uptake by P2Zhypo cells was detectable (Fig. 1, C and F). P2Zwt showed an intermediate behavior (Fig. 1, B and E). Nearly all P2Zhyper cells were positive for lucifer yellow uptake to varying levels. Because the sustained activation of the P2Z/P2X7 receptor leads to cell death, we tested for the release of the cytoplasmic enzyme lactic dehydrogenase in response to ATP. Whereas P2Zhypo were unaffected, P2Zhyper rapidly released lactic dehydrogenase, and P2Zwt again showed an intermediate behavior (Fig. 2).

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