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

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.

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.

Figure 2

Differential sensitivity to ATP-mediated cytotoxicity of P2Zhyper, P2Zwt, and P2Zhypo macrophages. Macrophages were incubated in serum-free RPMI medium at 37°C at a concentration of 5 × 10⁵/ml and stimulated with 3 mM ATP for 6 h. Releases of lactic dehydrogenase (LDH) from two clones of P2Zhyper cells (□ □, ♦ ♦), two clones of P2Zhypo cells (⋄ ⋄, ▴ ▴), and P2Zwt (▪  ▪) are shown.

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.

Figure 4

Western blot analysis with a specific anti-P2X₇ antiserum. Proteins (30 μg/lane) extracted from J774 mouse macrophages, P2Zhyper, and P2Zhypo clones were separated by electrophoresis and transferred to nitrocellulose. P2X₇ was visualized by incubation with the P2X₇ specific antibody, followed by peroxidase-conjugated secondary antibody and chemiluminescent substratum. Stable P2X₇ refers to proteins extracted from HEK293 cells stably transfected with P2X₇ (positive control). Negative control refers to proteins extracted from HEK293 cells stably transfected with P2X₂.

Figure 5

Spontaneous formation of multinucleated giant cells in monolayers of P2Zhyper cells and HEK293 cells transfected with the P2X₇ receptor. Cells were plated at the concentration of 5 × 10⁵ cells/ml and grown for 3 d. (A–C) P2Zhyper macrophages; (D) HEK293 cells stably transfected with the P2X₇ receptor. Photographs were taken with a 20× objective in A and a 40× objective in B–D. Bars: (A) 50 μm; (B–D) 25 μm.

Figure 6

Lack of formation of multinucleated giant cells in monolayers of P2Zwt and P2Zhypo macrophages. P2Zhypo (A) and P2Zwt (B) macrophages were plated as described in Fig. 5 and photographed with a 20× objective. Bar, 50 μm.

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.

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.

Figure 8

Early cellular interactions in cultures of P2Zhyper macrophages. Experimental conditions as in Fig. 5.

Figure 9

Detail of sites of close cellular interaction in cultures of P2Zhyper macrophages. Macrophage monolayers were incubated as described in Fig. 5, fixed, and processed for electron microscopy, as described in Materials and Methods. Bar, 0.17 μm.

Figure 10

Inhibition of multinucleated giant cell formation by oxidized ATP. Macrophages from the same batch of P2Zhyper cells were plated in 24-well dishes at a concentration of 5 × 10⁵/ ml. 6 h after the plating, cells shown in B were treated with 300 μM oATP for 2 h and then rinsed thoroughly and supplemented with fresh, complete RPMI medium.

Figure 11

Multinucleated giant cells die after fusion. P2Zhyper macrophages were plated as described in Fig. 5. Sequential pictures were taken from the same field at ∼12-h interval.