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Purinergic modulation of interleukin-1 beta release from microglial cells stimulated with bacterial endotoxin.

Ferrari D, Chiozzi P, Falzoni S, Hanau S, Di Virgilio F - J. Exp. Med. (1997)

Bottom Line: Immunol.LPS-dependent release of ATP is also observed in monocyte-derived human macrophages.It is suggested that bacterial endotoxin activates an autocrine/paracrine loop that drives ATP-dependent IL-1 beta secretion.

View Article: PubMed Central - PubMed

Affiliation: Institute of General Pathology, University of Ferrara, Italy.

ABSTRACT
Microglial cells express a peculiar plasma membrane receptor for extracellular ATP, named P2Z/P2X7 purinergic receptor, that triggers massive transmembrane ion fluxes and a reversible permeabilization of the plasma membrane to hydrophylic molecules of up to 900 dalton molecule weight and eventual cell death (Di Virgilio, F. 1995. Immunol. Today, 16:524-528). The physiological role of this newly cloned (Surprenant, A., F. Rassendren, E. Kawashima, R. A. North and G. Buell, 1996. Science (Wash. DC). 272:735-737) cytolytic receptor is unknown. In vitro and in vivo activation of the macrophage and microglial cell P2Z/P2X7 receptor by exogenous ATP causes a large and rapid release of mature IL-1 beta. In the present report we investigated the role of microglial P2Z/P2X7 receptor in IL-1 beta release triggered by LPS. Our data suggest that LPS-dependent IL-1 beta release involves activation of this purinergic receptor as it is inhibited by the selective P2Z/P2X7 blocker oxidized ATP and modulated by ATP-hydrolyzing enzymes such as apyrase or hexokinase. Furthermore, microglial cells release ATP when stimulated with LPS. LPS-dependent release of ATP is also observed in monocyte-derived human macrophages. It is suggested that bacterial endotoxin activates an autocrine/paracrine loop that drives ATP-dependent IL-1 beta secretion.

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Oxidized ATP inhibits LPS-dependent release of IL-1β.  N13 microglial cells were incubated in 24-well plates in RPMI medium  supplemented with 10% FCS at a concentration of 2 × 106 and incubated  24 h in the presence or absence (controls) of 10 μg/ml LPS. In the experiments with oATP, cells were treated with this inhibitor (300 μM) for 2 h  and then rinsed before addition of LPS. Stimulation with nigericin (20 μM)  was performed for 30 min after removal of oATP. Closed bars, IL-1β; open  bars, IL-6. Data are averages of duplicate determinations from a single experiment repeated on three separate occasions. Similar results were obtained with the N9 cell line.
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Figure 1: Oxidized ATP inhibits LPS-dependent release of IL-1β. N13 microglial cells were incubated in 24-well plates in RPMI medium supplemented with 10% FCS at a concentration of 2 × 106 and incubated 24 h in the presence or absence (controls) of 10 μg/ml LPS. In the experiments with oATP, cells were treated with this inhibitor (300 μM) for 2 h and then rinsed before addition of LPS. Stimulation with nigericin (20 μM) was performed for 30 min after removal of oATP. Closed bars, IL-1β; open bars, IL-6. Data are averages of duplicate determinations from a single experiment repeated on three separate occasions. Similar results were obtained with the N9 cell line.

Mentions: Fig. 1 shows that a 24-h incubation in the presence of 10 μg/ml LPS triggers release of IL-1β and that this is blocked by pretreatment with the selective P2Z/P2X7 inhibitor (13) oATP. To show that the effect of oATP is not due to a nonspecific inhibition of cell responses, we have also monitored IL-6 release, which is much less affected. As further proof that oATP does not have nonspecific effects, we show that IL-1β release is restored in LPS-treated, oATPinhibited cells by the K+ ionophore nigericin, an agent known to cause IL-1β release through a receptor-independent pathway (4, 10).


Purinergic modulation of interleukin-1 beta release from microglial cells stimulated with bacterial endotoxin.

Ferrari D, Chiozzi P, Falzoni S, Hanau S, Di Virgilio F - J. Exp. Med. (1997)

Oxidized ATP inhibits LPS-dependent release of IL-1β.  N13 microglial cells were incubated in 24-well plates in RPMI medium  supplemented with 10% FCS at a concentration of 2 × 106 and incubated  24 h in the presence or absence (controls) of 10 μg/ml LPS. In the experiments with oATP, cells were treated with this inhibitor (300 μM) for 2 h  and then rinsed before addition of LPS. Stimulation with nigericin (20 μM)  was performed for 30 min after removal of oATP. Closed bars, IL-1β; open  bars, IL-6. Data are averages of duplicate determinations from a single experiment repeated on three separate occasions. Similar results were obtained with the N9 cell line.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196027&req=5

Figure 1: Oxidized ATP inhibits LPS-dependent release of IL-1β. N13 microglial cells were incubated in 24-well plates in RPMI medium supplemented with 10% FCS at a concentration of 2 × 106 and incubated 24 h in the presence or absence (controls) of 10 μg/ml LPS. In the experiments with oATP, cells were treated with this inhibitor (300 μM) for 2 h and then rinsed before addition of LPS. Stimulation with nigericin (20 μM) was performed for 30 min after removal of oATP. Closed bars, IL-1β; open bars, IL-6. Data are averages of duplicate determinations from a single experiment repeated on three separate occasions. Similar results were obtained with the N9 cell line.
Mentions: Fig. 1 shows that a 24-h incubation in the presence of 10 μg/ml LPS triggers release of IL-1β and that this is blocked by pretreatment with the selective P2Z/P2X7 inhibitor (13) oATP. To show that the effect of oATP is not due to a nonspecific inhibition of cell responses, we have also monitored IL-6 release, which is much less affected. As further proof that oATP does not have nonspecific effects, we show that IL-1β release is restored in LPS-treated, oATPinhibited cells by the K+ ionophore nigericin, an agent known to cause IL-1β release through a receptor-independent pathway (4, 10).

Bottom Line: Immunol.LPS-dependent release of ATP is also observed in monocyte-derived human macrophages.It is suggested that bacterial endotoxin activates an autocrine/paracrine loop that drives ATP-dependent IL-1 beta secretion.

View Article: PubMed Central - PubMed

Affiliation: Institute of General Pathology, University of Ferrara, Italy.

ABSTRACT
Microglial cells express a peculiar plasma membrane receptor for extracellular ATP, named P2Z/P2X7 purinergic receptor, that triggers massive transmembrane ion fluxes and a reversible permeabilization of the plasma membrane to hydrophylic molecules of up to 900 dalton molecule weight and eventual cell death (Di Virgilio, F. 1995. Immunol. Today, 16:524-528). The physiological role of this newly cloned (Surprenant, A., F. Rassendren, E. Kawashima, R. A. North and G. Buell, 1996. Science (Wash. DC). 272:735-737) cytolytic receptor is unknown. In vitro and in vivo activation of the macrophage and microglial cell P2Z/P2X7 receptor by exogenous ATP causes a large and rapid release of mature IL-1 beta. In the present report we investigated the role of microglial P2Z/P2X7 receptor in IL-1 beta release triggered by LPS. Our data suggest that LPS-dependent IL-1 beta release involves activation of this purinergic receptor as it is inhibited by the selective P2Z/P2X7 blocker oxidized ATP and modulated by ATP-hydrolyzing enzymes such as apyrase or hexokinase. Furthermore, microglial cells release ATP when stimulated with LPS. LPS-dependent release of ATP is also observed in monocyte-derived human macrophages. It is suggested that bacterial endotoxin activates an autocrine/paracrine loop that drives ATP-dependent IL-1 beta secretion.

Show MeSH
Related in: MedlinePlus