Limits...
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.

Show MeSH
Modulation of IL-1β  release by apyrase and hexokinase  in N13 microglial cells. (A) Where  indicated, cells were incubated in  the presence of apyrase (apy, 0.4  U/ml) or hexokinase (hex, 100  μg/ml) throughout LPS treatment  (10 μg for 24 h). As a control, the  enzymes were boiled for 30 min (b  apy and b hex) before being added  to the cell monolayers. Pretreatment with oATP (300 μM) was  peformed for 2 h; then the monolayers were rinsed and challenged  with the different stimuli. (B) Cells  were first stimulated for 2 h with  LPS (10 μg/ml), and then stimulated with either 1 mM ATP or  ADP for 30 min. Other experimental conditions were as shown  in Fig. 1.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2196027&req=5

Figure 2: Modulation of IL-1β release by apyrase and hexokinase in N13 microglial cells. (A) Where indicated, cells were incubated in the presence of apyrase (apy, 0.4 U/ml) or hexokinase (hex, 100 μg/ml) throughout LPS treatment (10 μg for 24 h). As a control, the enzymes were boiled for 30 min (b apy and b hex) before being added to the cell monolayers. Pretreatment with oATP (300 μM) was peformed for 2 h; then the monolayers were rinsed and challenged with the different stimuli. (B) Cells were first stimulated for 2 h with LPS (10 μg/ml), and then stimulated with either 1 mM ATP or ADP for 30 min. Other experimental conditions were as shown in Fig. 1.

Mentions: Autocrine/paracrine stimulation of purinergic receptors can also in principle be prevented by exogenously added ATP-consuming enzymes such as apyrase or hexokinase. Fig. 2 A shows that apyrase completely inhibits LPS-dependent IL-1β release (the inactivated enzyme has no such effect). Surprisingly, hexokinase does not inhibit but rather stimulates IL-1β release. The main difference between apyrase and hexokinase is that the first hydrolyzes ATP and ADP, thus generating AMP, whereas hexokinase uses ATP as phosphorus donor to phosphorylate glucose, thus generating glucose 6 phosphate and ADP. It is known that ADP is an agonist at P2Z/P2X7 receptor, though less potent than ATP (12). Thus we checked whether the potentiating effect of hexokinase is mediated by stimulation of the P2Z/ P2X7 receptor by accumulated ADP. This seems to be the case because pretreatment with oATP blocks IL-1β secretion due to the combined addition of LPS and hexokinase (Fig. 2 A), and more importantly, exogenous ADP (ADPe) is a much more potent stimulus than ATP (Fig. 2 B). These experiments suggest that IL-1β release could be modulated by ATPe and ADPe, probably released by the inflammatory cells themselves under LPS stimulation.


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)

Modulation of IL-1β  release by apyrase and hexokinase  in N13 microglial cells. (A) Where  indicated, cells were incubated in  the presence of apyrase (apy, 0.4  U/ml) or hexokinase (hex, 100  μg/ml) throughout LPS treatment  (10 μg for 24 h). As a control, the  enzymes were boiled for 30 min (b  apy and b hex) before being added  to the cell monolayers. Pretreatment with oATP (300 μM) was  peformed for 2 h; then the monolayers were rinsed and challenged  with the different stimuli. (B) Cells  were first stimulated for 2 h with  LPS (10 μg/ml), and then stimulated with either 1 mM ATP or  ADP for 30 min. Other experimental conditions were as shown  in Fig. 1.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Modulation of IL-1β release by apyrase and hexokinase in N13 microglial cells. (A) Where indicated, cells were incubated in the presence of apyrase (apy, 0.4 U/ml) or hexokinase (hex, 100 μg/ml) throughout LPS treatment (10 μg for 24 h). As a control, the enzymes were boiled for 30 min (b apy and b hex) before being added to the cell monolayers. Pretreatment with oATP (300 μM) was peformed for 2 h; then the monolayers were rinsed and challenged with the different stimuli. (B) Cells were first stimulated for 2 h with LPS (10 μg/ml), and then stimulated with either 1 mM ATP or ADP for 30 min. Other experimental conditions were as shown in Fig. 1.
Mentions: Autocrine/paracrine stimulation of purinergic receptors can also in principle be prevented by exogenously added ATP-consuming enzymes such as apyrase or hexokinase. Fig. 2 A shows that apyrase completely inhibits LPS-dependent IL-1β release (the inactivated enzyme has no such effect). Surprisingly, hexokinase does not inhibit but rather stimulates IL-1β release. The main difference between apyrase and hexokinase is that the first hydrolyzes ATP and ADP, thus generating AMP, whereas hexokinase uses ATP as phosphorus donor to phosphorylate glucose, thus generating glucose 6 phosphate and ADP. It is known that ADP is an agonist at P2Z/P2X7 receptor, though less potent than ATP (12). Thus we checked whether the potentiating effect of hexokinase is mediated by stimulation of the P2Z/ P2X7 receptor by accumulated ADP. This seems to be the case because pretreatment with oATP blocks IL-1β secretion due to the combined addition of LPS and hexokinase (Fig. 2 A), and more importantly, exogenous ADP (ADPe) is a much more potent stimulus than ATP (Fig. 2 B). These experiments suggest that IL-1β release could be modulated by ATPe and ADPe, probably released by the inflammatory cells themselves under LPS stimulation.

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