paola ricciardi - castagnoli   ( university of milano , italy ) and were grown in rpmi 1640 medium ( paa , linz , austria ) supplemented with 2 mm glutamine   and 10% ( heat - inactivated ) fcs ( life technologies ltd . ,
paisley ,   scotland ) , 100 u / ml penicillin , and 100 g / ml streptomycin as   described previously ( 2 ) .
human monocytes were isolated from   buffy coats by one - step gradient ( percoll ; pharmacia biotech spa ,   cologno monzese , italy ) or by adherence on plastic petri dishes .  
after isolation , cells were kept in culture for 5 d in rpmi medium containing 2 mm glutamine , 5% human serum , 100 u / ml   penicillin , and 100 g / ml streptomycin . il-1 and il-6 in the   supernatant of lps ( sigma chemical co. ,
st . louis , mo ) treated   cells were measured with the intertext-1x mouse il-1 elisa   kit and intertext-6x mouse il-6 elisa kit , respectively ( genzyme   srl , cinisello balsamo , italy ) .
all reagents used were dissolved in   endotoxin - free water ( sigma ) and checked for endotoxin contamination .
microglial cells ( 25  10/ well ) were plated in microtiter plastic wells in culture medium   and incubated in a co2 incubator at 37c in the absence or presence of lps for 24 h. at the end of this incubation , the monolayers were thoroughly rinsed with saline solution and supplemented   with 100 l of a special diluent buffer ( firezyme ltd .
, san diego , ca ) to stabilize extracellular atp and directly placed in the   test chamber of a luminometer ( firezyme ) .
then , 100 l of luciferin - luciferase solution ( firezyme ) was added , and light emission was recorded . as a control ,
paola ricciardi - castagnoli   ( university of milano , italy ) and were grown in rpmi 1640 medium ( paa , linz , austria ) supplemented with 2 mm glutamine   and 10% ( heat - inactivated ) fcs ( life technologies ltd . ,
paisley ,   scotland ) , 100 u / ml penicillin , and 100 g / ml streptomycin as   described previously ( 2 ) .
human monocytes were isolated from   buffy coats by one - step gradient ( percoll ; pharmacia biotech spa ,   cologno monzese , italy ) or by adherence on plastic petri dishes .  
after isolation , cells were kept in culture for 5 d in rpmi medium containing 2 mm glutamine , 5% human serum , 100 u / ml   penicillin , and 100 g / ml streptomycin . il-1 and il-6 in the   supernatant of lps ( sigma chemical co. ,
st . louis , mo ) treated   cells were measured with the intertext-1x mouse il-1 elisa   kit and intertext-6x mouse il-6 elisa kit , respectively ( genzyme   srl , cinisello balsamo , italy ) .
all reagents used were dissolved in   endotoxin - free water ( sigma ) and checked for endotoxin contamination .
microglial cells ( 25  10/ well ) were plated in microtiter plastic wells in culture medium   and incubated in a co2 incubator at 37c in the absence or presence of lps for 24 h. at the end of this incubation , the monolayers were thoroughly rinsed with saline solution and supplemented   with 100 l of a special diluent buffer ( firezyme ltd . , san diego , ca ) to stabilize extracellular atp and directly placed in the   test chamber of a luminometer ( firezyme ) . then
, 100 l of luciferin - luciferase solution ( firezyme ) was added , and light emission was recorded . as a control ,
1 shows that a 24-h incubation in the presence of 10   g / ml lps triggers release of il-1 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-1 release is restored in lps - treated , oatpinhibited cells by the k ionophore nigericin , an agent   known to cause il-1 release through a receptor - independent pathway ( 4 , 10 ) .
autocrine / paracrine stimulation of purinergic receptors   can also in principle be prevented by exogenously added   atp - consuming enzymes such as apyrase or hexokinase .  
2 a shows that apyrase completely inhibits lps - dependent il-1 release ( the inactivated enzyme has no such effect ) .
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-1 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 .
these   experiments suggest that il-1 release could be modulated   by atpe and adpe , probably released by the inflammatory   cells themselves under lps stimulation .
an obvious sine qua non of this hypothesis is that microglial cells must release atp in response to lps .
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 ) .  
the lps dose response curve for atp release closely matched that for il-1   release , as shown in fig .
3 b. it has been shown previously   that atp is a powerful stimulus for il-1 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 ,
4 shows that   atp is released by human macrophages isolated from three   different subjects after stimulation with lps .
the mechanism of il-1 processing and release is a key   issue in immunology ( 59 , 15 ) .
rather surprisingly , recent   evidence points to a decrease in cytoplasmic k as a pivotal   stimulus for ice activation and il-1 maturation ( 4 , 10 ) .  
however , lps itself does not directly activate plasma membrane k channels , and mouse microglial cells express inwardly but not outwardly rectifying k channels ( 16 ) , thus   raising the issue of the mechanism responsible for lowering   the cytoplasmic k concentration .
it has been suggested   that this might be achieved by a lps - dependent increase in   the number of voltage - dependent k channels in the macrophage plasma membrane ( 4 ) , but typical k channel inhibitors blocked il-1 release only at concentrations far   above those necessary to inhibit these channels ( 4 ) .
the   p2z / p2x7 receptor is a good candidate to mediate cytoplasmic k depletion .
this receptor is typically expressed   in macrophages and macrophage - like cells ( 2 , 17 , 18 ) , and   it is modulated by inflammatory cytokines ( 17 , 19 ) .
a brief   stimulation with atpe triggers massive k efflux ( 12 ) and   release of processed il-1 ( 2 , 10 , 11 ) , whereas a sustained   activation causes cell death ( 1 , 17 , 20 ) . our data suggest   that il-1 release from microglial cells requires a double   stimulation : first , lps - dependent transcription of the il-1   gene and cytoplasmic accumulation of proil-1 ; second ,   paracrine / autocrine activation of the p2z / p2x7 receptor   that causes release of the mature cytokine .
adenine nucleotides can originate from many different sources : ( a ) the microglial cells themselves can release atpe , either spontaneously or under lps stimulation ; ( b ) injured or damaged   cells certainly release significant amounts of this nucleotide ,   a process likely to occur in vivo at sites of inflammation ; ( c )  in   the central nervous system , atpe can be released by neurons that establish close contact with the microglial cells .
it might seem paradoxical that adp is a better il-1 releasing agent than atp , although notoriously it is a less   potent stimulus at the p2z / p2x7 receptor .
however , this   is not unexpected because adp , in contrast with atp , is   devoid of cytotoxic activity , and data from our laboratory   show that release of il-1 is optimal in response to a submaximal , noncytotoxic stimulation of the p2z / p2x7 receptor , such as that due to adpe ( d. ferrari and f. di virgilio , manuscript in preparation ) .
involvement of the p2z / p2x7 purinergic receptor in   lps - dependent il-1 release may allow the development   of new pharmacological antagonists ( i.e. , oatp and derivatives ) to modulate the in vivo production of this cytokine   in pathological conditions such as septic shock or chronic   inflammatory diseases .
oxidized atp inhibits lps - dependent release of il-1.   n13 microglial cells were incubated in 24-well plates in rpmi medium   supplemented with 10% fcs at a concentration of 2  10 and incubated   24 h in the presence or absence ( controls ) of 10 g / ml lps . in the experiments with oatp , cells were treated with this inhibitor ( 300 m ) for 2 h   and then rinsed before addition of lps . stimulation with nigericin ( 20 m )   was performed for 30 min after removal of oatp .
data are averages of duplicate determinations from a single experiment repeated on three separate occasions .
( a ) where   indicated , cells were incubated in   the presence of apyrase ( apy , 0.4   u / ml ) or hexokinase ( hex , 100   g / ml ) throughout lps treatment   ( 10 g 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 m ) 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 g / ml ) , and then stimulated with either 1 mm atp or   adp for 30 min .
microglial   cells were plated in 24-well plates as described in fig . 1 for il-1 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   37c .
for measurement of atp , release samples were processed as follows : monolayers were rinsed and 100 l of diluent buffer ( firezyme )   were added ( see materials and methods ) .
data for il-1 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 .
macrophages were isolated from three different donors ( a  c  )   as described in materials and methods and plated in microtiter plastic   wells at a concentration of 50  10/well . after plating ,
cells were stimulated for 24 h with lps and atp release measured as detailed in fig .
 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 . 
 16:524528 ) . the physiological role of this newly cloned ( surprenant , a. , f. rassendren , e. kawashima , r.a . 
 north   and g. buell . 
 1996 . 
 science ( wash . 
 dc ) . 
 272:735737 ) 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. in the present report we investigated the   role of microglial p2z / p2x7 receptor in il-1 release triggered by lps . 
 our data suggest that   lps - dependent il-1 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 secretion . 