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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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LPS dose response for IL-1β and ATP release. Microglial  cells were plated in 24-well plates as described in Fig. 1 for IL-1β secretion or microtiter plastic wells as described in Materials and Methods for  ATP release and stimulated with LPS for 24 h in a CO2 incubator at  37°C. For measurement of ATP, release samples were processed as follows: monolayers were rinsed and 100 μl of diluent buffer (Firezyme)  were added (see Materials and Methods). Accumulation of extracellular  ATP was measured by the luciferin/luciferase assay. Data for IL-1β release are duplicates from a single experiment repeated with similar results  with three different batches of microglial cells. Data for ATP release are  means of quadruplicate determinations ± SD from a single experiment  repeated in three different occasions.
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Figure 3: LPS dose response for IL-1β and ATP release. Microglial cells were plated in 24-well plates as described in Fig. 1 for IL-1β secretion or microtiter plastic wells as described in Materials and Methods for ATP release and stimulated with LPS for 24 h in a CO2 incubator at 37°C. For measurement of ATP, release samples were processed as follows: monolayers were rinsed and 100 μl of diluent buffer (Firezyme) were added (see Materials and Methods). Accumulation of extracellular ATP was measured by the luciferin/luciferase assay. Data for IL-1β release are duplicates from a single experiment repeated with similar results with three different batches of microglial cells. Data for ATP release are means of quadruplicate determinations ± SD from a single experiment repeated in three different occasions.

Mentions: An obvious sine qua non of this hypothesis is that microglial cells must release ATP in response to LPS. Fig. 3 A shows that microglial cells chronically stimulated with LPS release ATP. Because the incubation medium is changed right before ATP determination, extracellular ATP measured in this experiment is very likely not accumulated in the bulk phase but continuously generated by the microglial cells. In support of this interpretation, we consistently found very little extracellular ATP in the cell-free supernatant (not shown). This observation is consistent with that previously reported by Filippini et al. (14) in T lymphocytes. The LPS dose– response curve for ATP release closely matched that for IL-1β release, as shown in Fig. 3 B. It has been shown previously that ATP is a powerful stimulus for IL-1β secretion from macrophages (10, 11), thus suggesting that this nucleotide might also have a role in autocrine/paracrine stimulation of these cells. In support of this hypothesis, Fig. 4 shows that ATP is released by human macrophages isolated from three different subjects after stimulation with LPS.


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)

LPS dose response for IL-1β and ATP release. Microglial  cells were plated in 24-well plates as described in Fig. 1 for IL-1β secretion or microtiter plastic wells as described in Materials and Methods for  ATP release and stimulated with LPS for 24 h in a CO2 incubator at  37°C. For measurement of ATP, release samples were processed as follows: monolayers were rinsed and 100 μl of diluent buffer (Firezyme)  were added (see Materials and Methods). Accumulation of extracellular  ATP was measured by the luciferin/luciferase assay. Data for IL-1β release are duplicates from a single experiment repeated with similar results  with three different batches of microglial cells. Data for ATP release are  means of quadruplicate determinations ± SD from a single experiment  repeated in three different occasions.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: LPS dose response for IL-1β and ATP release. Microglial cells were plated in 24-well plates as described in Fig. 1 for IL-1β secretion or microtiter plastic wells as described in Materials and Methods for ATP release and stimulated with LPS for 24 h in a CO2 incubator at 37°C. For measurement of ATP, release samples were processed as follows: monolayers were rinsed and 100 μl of diluent buffer (Firezyme) were added (see Materials and Methods). Accumulation of extracellular ATP was measured by the luciferin/luciferase assay. Data for IL-1β release are duplicates from a single experiment repeated with similar results with three different batches of microglial cells. Data for ATP release are means of quadruplicate determinations ± SD from a single experiment repeated in three different occasions.
Mentions: An obvious sine qua non of this hypothesis is that microglial cells must release ATP in response to LPS. Fig. 3 A shows that microglial cells chronically stimulated with LPS release ATP. Because the incubation medium is changed right before ATP determination, extracellular ATP measured in this experiment is very likely not accumulated in the bulk phase but continuously generated by the microglial cells. In support of this interpretation, we consistently found very little extracellular ATP in the cell-free supernatant (not shown). This observation is consistent with that previously reported by Filippini et al. (14) in T lymphocytes. The LPS dose– response curve for ATP release closely matched that for IL-1β release, as shown in Fig. 3 B. It has been shown previously that ATP is a powerful stimulus for IL-1β secretion from macrophages (10, 11), thus suggesting that this nucleotide might also have a role in autocrine/paracrine stimulation of these cells. In support of this hypothesis, Fig. 4 shows that ATP is released by human macrophages isolated from three different subjects after stimulation with LPS.

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