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Prostaglandins & other Lipid Mediators 88 (2009) 51–61

Contents lists available at ScienceDirect

Prostaglandins and Other Lipid Mediators

TP-induced apoptosis involves a Ca2+-independent phospholipase A2 and-lipoxygenase in macrophages

elio Miranda Costa-Juniora, Anderson Nogueira Mendesa,ustavo Henrique Nolasco Grimmer Davisa, Cristiane Monteiro da Cruza,na Lúcia Marques Venturab, Carlos Henrique Serezanic, Lucia Helena Facciolid,uro Nomizod, Célio G. Freire-de-Limae, Rodrigo da Cunha Bisaggioa, Pedro Muanis Persechinia,∗

Laboratório de Imunobiofísica, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, 21941-902 Rio de Janeiro, RJ, BrazilLaboratório de Neuroquímica, Instituto de Biologia, Universidade Federal Fluminense, 24030-210 Niterói, RJ, BrazilInternal Medicine, Pulmonary and Critical Care Medicine, University of Michigan, 1150 West Medical Center Drive, Ann Arbor, MI 46109-0642, United StatesDepartamento de Análises Clínicas Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto,niversidade de São Paulo, 14040-903 Ribeirão Preto, SP, BrazilLaboratório de Biologia Imunitária, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, 21941-902 Rio de Janeiro, RJ, Brazil

r t i c l e i n f o

rticle history:eceived 6 July 2008eceived in revised form6 September 2008ccepted 29 September 2008vailable online 15 October 2008

eywords:TP2X7

hospholipase A2

acrophagepoptosis

a b s t r a c t

Macrophages express P2X7 and other nucleotide (P2) receptors, and display the phenomena of extracel-lular ATP (ATPe)-induced P2X7-dependent membrane permeabilization and cell death by apoptosis andnecrosis. P2X7 receptors also cooperate with toll-like receptors (TLRs) to induce inflammasome activa-tion and IL-1� secretion. We investigated signaling pathways involved in the induction of cell death byATPe in intraperitoneal murine macrophages. Apoptosis (hypodiploid nuclei) and necrosis (LDH release)were detected 6 h after an induction period of 20 min in the presence of ATP. Apoptosis was blockedby caspase 3 and caspase 9 inhibitors and by cyclosporin A. The MAPK inhibitors PD-98059, SB-203580and SB-202190 provoked no significant effect on apoptosis, but SB-203580 blocked LDH release. Neitherapoptosis nor necrosis was inhibited when both intra- and extracellular Ca2+ were chelated during theinduction period. Mepacrine, a generic PLA2 inhibitor and BEL, an inhibitor of Ca2+-independent PLA2

(iPLA2) blocked apoptosis, while pBPB and AACOOPF3, inhibitors of secretory and Ca2+-dependent PLA2

ecrosis-Lipoxygenase

PLA2

respectively, had no significant effect. Cycloxygenase inhibitors had no effect on apoptosis, while theinhibitors of lipoxygenase (LOX) and leukotriene biosynthesis nordihydroguaiaretic acid (NDGA), zileu-ton, AA-861, and MK-886 significantly decreased apoptosis. Neither NDGA nor MK-886 blocked apoptosisof 5-LOX−/− macrophages. CP-105696 and MK-571, antagonists of leukotriene receptors, had no signifi-cant effect on apoptosis. None of the inhibitors of PLA2 and LOX/leukotriene pathway had a significantinhibitory effect on LDH release5-LOX are involved in the trigge

1

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Abbreviations: AAS, acetyl salicylic acid; ATPe, extracellular ATP; BBG, BrilhantBlue G; BEL, 2-bromo-enol-lactone; BzATP, 2′-3′-O-(4-benzoylbenzoyl)-ATP; CsA,cyclosporin A; cPLA2, Ca2+-dependent cytosolic PLA2; COX, cycloxygenase; FLAP,5-lipoxygenase-activating protein; iPLA2, Ca2+-independent cytosolic PLA2; LDH,lactate dehydrogenase; LOX, lipoxygenase; NDGA, nordihydroguaiaretic acid; oxATP,periodate-oxidized ATP; pBPB, 4-bromophenyl bromide; PD-98059, 2′-amino-

′

-methoxyflavone; PLA2, phospholipase A2; PTP, mitochondrial permeabilityransition pore; sPLA2, secretory PLA2.∗ Corresponding author at: Laboratório de Imunobiofísica, Instituto de Biofísicaarlos Chagas Filho, UFRJ, Bloco G do CCS, Ilha do Fundão, 21941-590 Rio de Janeiro,J, Brazil. Tel.: +55 21 2562 6560.

E-mail address: pedromp@biof.ufrj.br (P.M. Persechini).

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098-8823/$ – see front matter © 2008 Elsevier Inc. All rights reserved.oi:10.1016/j.prostaglandins.2008.09.004

. Our results indicate that a Ca2+-independent step involving an iPLA2 andring of apoptosis but not necrosis by P2X7 in macrophages.

© 2008 Elsevier Inc. All rights reserved.

. Introduction

Modulation of macrophage differentiation and viability in dif-erent microenvironments is a key element of innate and acquiredmmunity [1,2]. Among the major immunomodulators produceduring inflammation and immune response, the extracellularucleotides and adenosine [3–5] can be secreted by several means6] and their activity are mediated in great part by the nucleotideP2) and adenosine (P1) receptors [7,8]. Those receptors are modu-

ated by ecto-nucleotidases such as CD39 and CD73 and expressedn macrophages and endothelial cells that participate in themmune response [9].

P2X7 receptors are unique among the members of the P2X sub-amily of P2 receptors that are ATP-gated channels (P2X1–7) since

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hey open a small ion channel selective for Na+, K+ and Ca2+. Theseeceptors are also associated with the activation of a non-selectiveransport mechanism that allows the uptake of molecules up to00 Da, a phenomenon called ATPe-induced permeabilization andsually ascribed to the opening of a non-selective pore [8,10–12].hey are also associated with many immune effects such as apopto-is and necrosis, cytokine release, killing of intracellular pathogens,nd membrane blebbing [3,4,13]. In addition, P2X7 receptors arepregulated by IFN-� plus LPS [4] and cooperate with toll-likeeceptors (TLRs) and NOD-like receptors (NLRs) in the process orathogen recognition, activation of inflammasome, and maturationnd secretion of IL-1� and IL-18 [14].

Although different signaling pathways are known to be triggeredy P2X7 [8,10,11,15–18] the specific pathways that lead to each ofhe P2X7-dependent responses are still poorly understood [10,15].n particular, the elucidation of pathways that link ATPe and P2X7 toell death still await further clarification [10,19,20] and the possiblearticipation of other P2 receptor subtypes has not been discarded.

Cell death is a complex phenomena that may involve necro-is, apoptosis, and autophagy [21–24]. ATPe-induced macrophageeath has been characterized as apoptosis and necrosis by differentpproaches [25,26]. It involves the activation of caspase 3, 8 and 93,25,27] and the generation of ceramide [28].

The biochemical pathway(s) that triggers apoptosis-relatedvents are not known. Phospholipase A2 (PLA2) comprises a setf intracellular and extracellular enzymes – secretory, calcium-ependent (sPLA2); cytoplasmic, calcium-dependent (cPLA2); andytoplasmic, calcium-independent (iPLA2) – that can release aatty acid such as arachidonic acid (AA) [29–31] and may benvolved in apoptosis in different experimental models [32–35].

acrophages express different PLA2 isoforms, including iPLA229,32] that can be activated by P2X7 receptors. Both cPLA2 andPLA2 have been involved in P2X7-mediated calichrein secretiony rat submandibular glands [36] and 2-bromo-enol-lactone (BEL),selective antagonist of iPLA2 inhibits P2X7-induced IL-1� secre-

ion in murine macrophages [37]. In addition, P2X7 activationnduces leukotriene secretion in rat astrocytes [38] indicating that-lipoxygenase (5-LOX) is also activated by ATPe. Since other P2eceptors also activate PLA2-mediated signaling (reviewed in Ref.10]), and macrophages express several P2X and P2Y receptors3,8,39] we raised the hypothesis that one or more PLA2 and 5-LOXould be required in ATPe-induced apoptosis of macrophages.

In this work, we investigate the putative intracellular sig-aling events that could be associated with ATPe-induced2X7-dependent cell death in thioglycollate-elicited intraperi-oneal murine macrophages. Our data indicates that differentathways link purinergic receptors to apoptosis and necrosis andhat an iPLA2 and 5-LOX, but not the overload of intracellular Ca2+,

AP kinases (MAPKs) or cyclooxygenase (COX), are involved inriggering apoptosis. By showing that ATPe-induced apoptosis andecrosis follow different pathways, our data open new possibilitieso design P2-based strategies of immune modulation.

. Methods

.1. Materials

DMEM, fetal bovine serum, penicillin and streptomycinere obtained from Gibco/BRL (São Paulo, RJ, Brazil); 1-acyl-2-

ydroxy-sn-glycero-3-phosphate (LPA) was obtained from AvantiAlabaster, AL, USA); 3-[1-(para-chlorobenzyl)-5-(isopropyl)--t-butylthioindol-2-yl]-2,2-dimethylpropanoic acid (MK-886),ordihydroguaiaretic acid (NDGA), arachidonyltrifluoromethyletone (AACOCF3), palmitoyl trifluoromethyl ketone (PACOCF3),

ci2ba

ther Lipid Mediators 88 (2009) 51–61

-acetyl-Tyr-Val-Ala-Asp-chloromethyl ketone (Z-DEVD-FMK),B-202190, SB-203580, and PD-98059 were from CalbiochemSan Diego, CA, USA); thioglycollate medium was from DifcoDetroit, MI, USA). NaCl, MgCl2, CaCl2, KCl were from Reagen (Rioe Janeiro, RJ, Brazil); acetyl-Leu-Glu-His-Asp-aldehyde (Ac-LEHD-HO) was from Peptide Institute, Inc. (Osaka, Japan); cyclosporin

(CsA) from Amersham Pharmacia Biotech (São Paulo, SP,razil); 4-bromophenyl bromide (pBPB), mepacrine, adenosine--triphosphate (ATP), acetyl salicylic acid (AAS), Triton-X 100,rachidonic acid, lyso-phosphatydilcholine (LPC), 2-bromo-nol-lactone (BEL), EGTA, ATP, 2′-3′-O-(4-benzoylbenzoyl)-ATPBzATP), periodate-oxidized ATP (oxATP), ethidium bromide,EPES, dimethyl sulfoxide (DMSO), Brilliant Blue G (BBG), Triton-

100, and phosphate buffered saline (PBS) were purchasedrom Sigma–Aldrich (St. Louis, MO, USA); BAPTA-AM were from

olecular Probes (Eugene, OR, USA). Zileuton, 2,3,5-trimethyl--(12-hydroxy-5,10-dodecadiynyl)-1, 4-benzoquinone (AA-861),nd N-(2-cyclohexyloxy-4-nitrophenyl)-methanesulfonamideNS-398) was from Cayman Chemical (Ann Arbor, MI, USA). (E)--[[[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl]-[[(3-dimethyl-mino)-3-oxopropyl]thio]methyl]thio]-propanoic acid (MK-571).P-105696 was a kind gift of Pfizer, Groton, CT, USA.

.2. Animals

Swiss-Webster and C57/Bl6 mice were obtained from the animalacilities of the Instituto de Microbiologia Paulo de Goes and fromhe Transgenic Animal Facility of the Instituto de Biofísica Carloshagas Filho of the Federal University of Rio de Janeiro. Male mice

acking the 5-LOX enzyme gene (5-LO−/−) were obtained from Theackson Laboratory, and age-matched male wild type (WT) micebackground, strain 129) were used as controls. All animals were–12-week old, weighed approximately 16–30 g and were handledccording to the guidelines for animal use in scientific experimentsf the Instituto de Biofísica Carlos Chagas Filho of the Federal Uni-ersity of Rio de Janeiro and the Animal Care Committee of theniversidade Estadual de Campinas.

.3. Cell isolation and culture

Experiments were performed with thioglycollate-elicitedacrophages obtained from the intraperitoneal cavity of mice, col-

ected 4 days after thioglycollate injection, as described [40]. Inrief, cells were washed three times in PBS and kept on ice at a con-entration of 106 cells/mL until used in the flow cytometry-basedembrane-permeabilization assays or in cell death assays (freshly

solated macrophages), or plated in 35 mm culture dishes in ordero obtain adherent macrophages, described as follows. To obtaindherent macrophages, cells were first added to the culture dishest a concentration of 2 × 106 cells/dish in 2 mL DMEM medium sup-lemented with 10% fetal bovine serum, 2 g/L sodium bicarbonate,.3 mg/L l-glutamine, 100 U/mL penicillin, and 100 �g/mL strep-omycin (complete medium) at 37 ◦C in a humidified atmosphereontaining 5% CO2. Non-adherent cells were then removed after 2 hnd the macrophages were kept for 4 days under the same cultureonditions.

.4. Induction and analysis of cell death

Suspensions of freshly isolated intraperitoneal cells (2 × 105

ells/200 �L) or adherent macrophages in culture dishes were pre-ncubated at 37 ◦C with/without each drug or vehicle (controls) for0 min in DMEM medium containing 2 g/L sodium bicarbonate anduffered with 10 mM Na-HEPES, pH 7.4 and then 5 mM ATP wasdded to the same medium for an additional 20 min (induction

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eriod). In the experiments with high extracellular K+ concentra-ion, a salt solution (composition in mM: NaCl 5; KCl 140; CaCl2 1;

gCl2 1, K-HEPES 10, pH 7.4) was substituted for culture mediumuring the induction period. After this period, cells were gentlyashed twice with complete medium to remove ATP and further

ncubated for 6 h at 37 ◦C (effector period). Unless otherwise speci-ed, the drugs were kept in the medium during the effector period.he whole process was carried out in complete DMEM mediumn the standard culture conditions described above. The cells werehen centrifuged at 5600 × g for 1 min, the supernatant was col-ected for measurement of LDH content (see below) and the pelletas used for the determination of apoptosis as described [41]. Inrief, the cells were gently suspended in 250 �L of a lysing bufferhat preserved the nuclei for the determination of DNA content50 �g/mL ethidium bromide, 0.1% sodium citrate, 0.1% Triton-X00). The nuclei were then analyzed by flow cytometry (FACSCal-bur, Becton Dickinson, Mountain View, CA, USA) and the degree ofpoptosis was quantified by counting the number of hypodiploidvents. At least 5000 events were collected per sample and nor-alized values were calculated by subtracting the spontaneous

poptosis obtained in the controls (absence of ATP or any otherrugs) from all experimental values and taking the % of numberhe hypodiploid events induced by 5 mM ATP as 100%.

Cell lysis (necrosis) was determined by measuring lactate dehy-rogenase (LDH) enzymatic activity in the supernatants collected asbove, using a commercially available colorimetric assay kit accord-ng to the maker’s instructions (CytoTox, Promega, Madison, WI,SA) [42]. Control lyses were obtained by using supernatants ofntreated cells (0%) and cells treated with 0.1% Triton-X 100 (100%).ach reading was subtracted from the reading obtained in mediumontaining drugs without cells in order to avoid any interference ofhe drugs with the reagents in the LDH detection kit. Normalizedalues of LDH release were calculated by subtracting the sponta-eous values obtained in the absence of ATP or any other drug fromll experimental values and taking the OD value obtained for 5 mMTP as 100%.

In some experiments the 20 min induction period was per-ormed in an extracellular solution containing, in mM: 135 NaCl,

KCl, 1 MgCl2, 1 CaCl2, and 10 Na-HEPES, pH 7.4. In calcium-freexperiments, 1 mM EGTA was substituted for CaCl2 and in high K+

xperiments, KCl was substituted for NaCl.

.5. Intracellular calcium buffering

In experiments aimed to avoid intracellular calcium signaling,acrophages were incubated with 1 �g/mL BAPTA-AM for 30 min

t room temperature in HEPES-buffered culture medium contain-ng 2.5 mM probenicide, washed twice in DMEM medium andxposed to ATPe as described above either in the presence or notf 1 mM EGTA. Under these experimental conditions we did notbserve any calcium signals induced by ATPe [15].

.6. Permeabilization assays

ATPe-induced membrane permeabilization was measured byetecting ethidium uptake using a flow cytometer (FACSCaliburytometer, Becton, Dickinson and Co., Franklin Lakes, NJ, USA). Inrief, freshly isolated macrophages (106 per mL) were removedrom ice, pre-warmed at 37 ◦C for 5 min, treated with the indicatedrugs for 20 min at 37 ◦C, and then incubated with ATP or BzATP

or 10 min in the presence of the drugs. Ethidium bromide (2 �Mnal concentration) was added during the last 5 min of incubationnd the intensity of dye uptake was immediately determined byow cytometry using an excitation wavelength of 488 nm and anmission wavelength of either 590 or 670 nm. At least 5000 data

celvo

ther Lipid Mediators 88 (2009) 51–61 53

oints were collected for each sample. The results were analyzedsing the WinMDI program (Multiple Document Interface Flowytometry Application, version 2.8, by Joseph Trotter, The Scrippsesearch Institute, La Jolla, CA, USA). In some experiments, adherentacrophages of 3–5-day-old cultures were used for the perme-

bilization assays. In this case, dye uptake was determined usingfluorescence microscope (Axiovert 100, Karl Zeiss, Oberkochen,ermany) equipped with a HBO lamp and appropriate filters.

.7. Statistical analysis

Differences between experimental groups were evaluated byhe two-tailed unpaired Student’s t-test. A *p value < 0.05, **palue < 0.01 and ***p < 0.001 were considered statistically signifi-ant.

. Results

.1. ATPe induces P2X7-dependent apoptosis and necrosis inacrophages after a 20 min induction period

In order to investigate signaling pathways triggered by ATPe

n macrophages, we used an experimental protocol that alloweds to better distinguish between the initial steps and the latertages of ATPe-induced P2X7-dependent cell death. Cells were firstncubated in the presence of ATP for 20 min (induction period),ashed carefully and kept in culture medium supplemented with

erum, in the absence of ATPe for 6 h (effector period). Apop-osis was quantified by measuring the percent of hypodiploidvents by flow cytometry and necrosis was quantified by LDHelease at the end of the effector period, as described in Sec-ion 2. Under these experimental conditions, 5 mM ATPe lead to30–60% apoptosis and 30–90% necrosis (Fig. 1a) and a sponta-

eous cell death of no more than 15%. The induction of apoptosisas also ascertained in selected experiments by showing that ATPe

reatment induced bleb formation (detected by light and electronicroscopy), nuclear fragmentation (observed by acridin-orange

taining and electron microscopy), DNA fragmentation (observedn agarose gel) and FITC-ANEXIN binding after 1 h (detected byow cytometry) (data not shown). The involvement of P2X7 recep-ors in ATPe-induced macrophage death was firmly established inhe literature since it was shown that both apoptosis and necrosisre absent in macrophages derived from P2X7

−/− animals exposedo ATPe [20,43]. We confirmed that both apoptosis and necro-is are dependent on fully functional P2X7 receptors under ourxperimental conditions since formation of hypodiploid nuclei andDH release are blocked by Mg2+, and oxATP (data not shown).TPe-induced apoptosis and LDH release was also blocked by BBGt a concentration that is also required to block P2X7-associatednward current and membrane permeabilization (ethidium uptake)n murine macrophages (unpublished observations). In addition,

e also found that the inhibitors of caspase 3 (Z-DEVD-FMK) andaspase 9 (Ac-LEHD-CHO) inhibited apoptosis (data not shown) asreviously reported by others [25,27].

An important issue to be considered in our cell-death inductionrotocol is membrane integrity during the effector phase. Duringhe induction period, cell membranes are permeabilized due to thepening of the P2X7-associated pores and the continuous main-enance of the pore at the open state would eventually lead to

oloidosmotic electrolyte imbalance, making it more difficult tovaluate the physiological significance of the intracellular signalingeading to cell death. These questions are even more important iniew of the fact that P2X7 receptors do not desensitize [8]. Washingut ATPe after the 20 min induction period avoids this problem by

54 H.M. Costa-Junior et al. / Prostaglandins & other Lipid Mediators 88 (2009) 51–61

Fig. 1. ATPe-induced macrophages death after a 20 min induction period. Macrophages were exposed to zero or 5 mM ATP for 20 min and cell death (a) was accessed 6 hlater. Alternatively, membrane permeabilization (ethidium uptake) was measured either at the end of the initial 20 min period or 15 min after ATP withdrawing (b). (a) Celldeath induced by ATP. Absolute values of apoptosis (% hypodiploid nucleus) and necrosis (LDH release) were evaluated in adherent and freshly isolated macrophages asi olatedb in aft compv

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ndicated. (b) Closure of the permeabilization pore after ATP withdrawing. Freshly isromide was added either 5 min before the end of this period (closed bars) or 15 mhree experiments performed in triplicates. Statistical significance was obtained byalues obtained by the exposure to ATP alone. **p value < 0.01 and ***p < 0.001.

losing the pores and preserving membrane integrity as can be ver-fied by the absence of ethidium uptake 15 min after the initiationf the effector period (Fig. 1b). These data indicate that neither theore nor the cation channel are required during the effector periodf apoptosis and that P2X7-associated signaling is important onlyuring the induction period.

.2. Intracellular K+ depletion and Ca2+ overflow are not requiredo trigger ATPe-induced macrophage death

One possible scenario for the triggering of apoptosis by ATPe

n macrophages is that the opening of the P2X7-associated cationhannels and permeabilization pores would induce the depletionf K+ and an overload of Ca2+ in the cytosol; two phenomena that

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ig. 2. Intracellular K+ depletion and Ca2+ overflow are not required to trigger ATPe-inducedxposed to 0–10 mM (a) or 5 mM (b and c) ATP for an additional 20 min under the sameontaining or not the same drugs, as indicated below. Cell death was accessed 6 h lateralt solution during the induction period and in complete medium without ATP during tacrophages pre-incubated or not with 1 or 10 �M CsA for 20 min and maintained in the p

c) Apoptosis and LDH release of freshly isolated macrophages pre-incubated or not with 1GTA and maintained in the same medium during the induction period. The cells were kepresent the mean % ± S.E.M. of at least three experiments performed in triplicates. Statrug with the values obtained for ATP alone. **p value < 0.01 and ***p < 0.001.

macrophages were incubated for 20 min in the presence of ATP and 2 �M ethidiumter ATP withdrawing (dashed bars). Data represent the mean % ± S.E.M. of at leastaring cell death or ethidium uptake induced by ATP plus drug with the respective

ould lead to the activation of caspases and the opening of per-eability transition pores (PTPs), mitochondrial rupture, as well

s the release of cytochrome c and other pro-apototic molecules20,24,44].

In order to investigate whether K+ depletion is involved on thenduction of apoptosis by P2X7, we kept the cells in a high K+ saltolution during the 20 min induction period in the presence of ATPe.nder these experimental conditions we observed no significantifferences in the degree of apoptosis (Fig. 2a). We next showed

hat cyclosporin A, an inhibitor of apoptosis mediated by PTP, pre-ented ATPe-induced apoptosis but not LDH release (Fig. 2b). Inrder to investigate whether the cyclosporin A-sensitive step isependent on the Ca2+ overflow that occurs during the induc-ion phase we pre-loaded the cells with BAPTA-AM and exposed

macrophage death. Macrophages were pre-incubated or not in different conditions,conditions (induction period), and transferred to complete medium without ATP,(effector period). (a) Apoptosis of freshly isolated macrophages kept in a high K+

he effector period. (b) Normalized apoptosis and LDH release induced in adherentresence of the same concentration of CsA during the induction and effector periods.�g/mL BAPTA-AM for 30 min in complete culture medium containing or not 1 mM

ept in complete medium without EGTA and ATP during the induction period. Dataistical significance was obtained by comparing the cell death induced by ATP plus

H.M. Costa-Junior et al. / Prostaglandins & other Lipid Mediators 88 (2009) 51–61 55

Fig. 3. MAPKs are not required to trigger ATPe-induced macrophage death. Adherent macrophages were pre-incubated or not in the presence of MAPK inhibitors for 20 min,e ductid ssed( expert alue <

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xposed to zero or 5 mM ATP for an additional 20 min under the same conditions (inrugs. Normalized apoptosis (closed bars) and LDH release (dashed bars) were accec) SB-202190; 0.1, 1 and 10 �M. Data represent the mean % ± S.E.M. of at least threehe cell death induced by ATP plus drug with the values obtained for ATP alone. *p v

hem to ATPe for 20 min in culture medium containing EGTA, aondition that completely abrogates any Ca2+ signaling withoutnhibiting P2X7-associated cation channels and permeabilizationores [15,45]. Under these conditions, we observed no significantifferences either in apoptosis or LDH release (Fig. 2c). These results

ndicate that K+ depletion and the increase of intracellular free cal-ium concentration, two phenomena associated with the activation

f P2X7 receptor under physiological conditions, are not required torigger cell death signaling cascade. These data also suggest that yetnrecognized calcium-independent events trigger apoptosis dur-

ng the initial 20 min in the presence of ATPe. We could not testhe effect of maintaining the high K+ concentration or completely

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ig. 4. Involvement of iPLA2 in ATPe-induced apoptosis. Freshly isolated or adherent mac0 min, exposed to zero or 5 mM ATP for an additional 20 min under the same conditionshe same drugs. Normalized apoptosis and LDH release were accessed 6 h later (effector pelease (b) of freshly isolated (closed bars) and adherent (dashed bars) macrophages. (c) Efars) of freshly isolated macrophages. (d) Effect of AACOCF3 10 and 100 �M on the apoptond f) Effect of BEL 1 and 10 �M on the apoptosis (e) and LDH release (f) of freshly isolate± S.E.M. of at least three experiments performed in triplicates. Statistical significance wbtained for ATP alone. *p value < 0.05, **p value < 0.01 and ***p < 0.001.

on period), and transferred to complete medium without ATP, containing the same6 h later (effector period). (a) PD-98059; 25 �M. (b) SB-203580; 1, 10 and 100 �M.iments performed in triplicates. Statistical significance was obtained by comparing0.05 and **p value < 0.01.

emoving Ca2+ ions during the whole 6 h period because of exten-ive cell death observed under our experimental conditions evenn the absence of ATPe (data not shown).

.3. MAPKs are not required to trigger ATPe-induced macrophagepoptosis

We have recently shown that MAPKs are not involved in theormation of the P2X7-associated permeabilization pore in murinentraperitoneal macrophages [15]. However, p38 has been impliedn P2X7-associated cell death [46–48]. Based in these evidences,

e decided to investigate the effect of inhibitors of MAPKs in

rophages were pre-incubated or not in the presence of different PLA2 inhibitors for(induction period), and transferred to complete medium without ATP, containingeriod). (a and b) Effect of mepacrine 10 and 100 �M on the apoptosis (a) and LDHfect of pBPB 10 and 100 �M on the apoptosis (closed bars) and LDH release (dashedsis (closed bars) and LDH release (dashed bars) of freshly isolated macrophages. (ed (closed bars) and adherent (dashed bars) macrophages. Data represent the meanas obtained by comparing the cell death induced by ATP plus drug with the values

56 H.M. Costa-Junior et al. / Prostaglandins & other Lipid Mediators 88 (2009) 51–61

F macroo for anc as acce aring

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ig. 5. Cycloxygenases are not involved in ATPe-induced cell death. Freshly isolatedf indicated doses of AAS (a) or NS-398 (b) for 20 min, exposed to zero or 5 mM ATPomplete medium without ATP, containing the same drugs. Normalized apoptosis wxperiments performed in triplicates. Statistical significance was obtained by comp

TPe-induced cell death. None of the drugs used (PD-98059 annhibitor of ERK 1/2 phosphorylation; SB-203580 and SB-202190,wo inhibitors of p38) displayed any significant decrease in ATPe-nduced apoptosis in doses up to 100 �M (Fig. 3). However, 10 �MB-203580 (Fig. 3b), but neither PD-98059 (Fig. 3a) nor SB-202190Fig. 3c), blocked approximately 50% of ATPe-induced LDH release,uggesting that ATPe-induced necrosis of macrophages requires thectivation of p38.

.4. iPLA2 inhibitors decrease ATPe-induced apoptosis inacrophages

We next investigated the effects of several inhibitors of PLA2 onTPe-induced macrophage death (Fig. 4). Mepacrine, a generic PLA2

nhibitor, inhibited ATPe-induced apoptosis in both adherent andreshly isolated macrophages, the latter being more sensitive to therug (Fig. 4a). LDH release was significantly enhanced by mepacrinend an increase of more than twofold was observed in adherentacrophages (Fig. 4b). The inhibitor of sPLA2, pBPB, had no signif-

cant effect either on apoptosis or on LDH release (Fig. 4c). Theseata suggest that one or more cytoplasmic PLA2 could be involved

n the induction of apoptosis. In order to further clarify this possibil-ty, we showed that AACOCF3, a specific inhibitor of Ca2+-dependenthospholipase A2 (cPLA2), had no significant effect either on apop-osis or on LDH release (Fig. 4d), while BEL, a selective blocker ofPLA2 [49] completely abrogated ATPe-induced apoptosis in bothdherent and freshly isolated macrophages (Fig. 4e) without signif-cantly affecting LDH release (Fig. 4f). In none of these experimentsid we observe significant induction of apoptosis or LDH release byhe drugs or by the DMSO-containing buffer alone (Fig. 4). Theseesults indicate that an iPLA2 is involved in P2-associated signalingascade that leads to apoptosis in macrophages and are consistentith the existence of a Ca2+-independent step triggered by ATPe,

s shown in the previous section. We also conclude that a signalpstream to this iPLA2 remains activated and can still lead to celleath by necrosis.

.5. Apoptosis induced by ATPe involves 5-lipoxygenase but notycloxygenases

The effect of iPLA2 inhibitors suggest that lipid derivatives

eleased by these enzymes from plasma or organelle mem-ranes are downstream mediators of ATPe-induced apoptosis inacrophages. Macrophages can express both cycloxygenases (COX-and COX-2) and also 5- and 12/15-lipoxygenases (5-LOX and

2/15-LOX); two enzymes widely known to produce eicosanoids

3A

d

phages (a) or adherent macrophages (b) were pre-incubated or not in the presenceadditional 20 min under the same conditions (induction period), and transferred toessed 6 h later (effector period). Data represent the mean % ± S.E.M. of at least threethe cell death induced by ATP plus drug with the values obtained for ATP alone.

rom arachidonic acid derived from PLA2 activity [29,50,51]. There-ore, we next investigated the effects of inhibitors of COX and LOXathways on ATPe-induced apoptosis in macrophages. AAS, a COX-1nd COX-2 inhibitor, and NS-398, a COX-2 inhibitor, had no signif-cant effect either in apoptosis (Fig. 5a and b) or LDH release (dataot shown).

NDGA, a LOX inhibitor, MK-886, an inhibitor of the 5-LOX cou-ling protein FLAP [52], and, zileuton and AA-861, two selective-LOX inhibitors, inhibited ATPe-induced apoptosis (Fig. 6a, c, end f) without significant effects in LDH release (Fig. 6b and d,nd data not shown). Some of these inhibitors, such as NDGA andK-886, have been described as inducing cell death through a

OX/FLAP-independent mechanism [53]. However, most of theseeports involve incubation with the drugs for a much longer periodf time and under our experimental conditions they did not induceither apoptosis or LDH release (Fig. 6).

.6. Arachidonic acid- and ATPe-induced macrophage deathnvolves different pathways

The effects of the inhibitors of iPLA2 and 5-LOX described aboveead to the suggestion that these enzymes cooperate during the pro-ess of ATPe-induced macrophage apoptosis. This is an unexpectedossibility since, while iPLA2 has been involved in the releasef oleic acid and other fatty acids, organelle and plasma mem-rane remodeling, bleb formation, phosphatidyl serine turnover,nd the release of pro-apoptotic molecules from mitochondria,PLA2 is considered to be the main cytoplasmic enzyme thateleases arachidonic acid for the biosynthesis of eicosanoids by-LOX [29–31,50,51,54–58].

To further clarify these possibilities we asked whether ATPe

nd exogenous arachidonic acid could induce similar effects inacrophage (Fig. 7). We observed that, although arachidonic acid

an also induce both apoptosis (Fig. 7a) and LDH release (Fig. 7b) inacrophages, the pathway used by this fatty acid is different from

he one used by ATPe since neither NDGA nor CsA, two inhibitorsf ATPe-induced apoptosis, decreased arachidonic acid-inducedacrophage death (Fig. 7a and b). In addition, consistent with its

ole as a selective inhibitor of iPLA2, BEL did not block apoptosisnduced by exogenous arachidonic acid (Fig. 7a).

.7. 5-LOX activity but not leukotriene secretion is required forTPe-induced cell death

Collectively, our data indicate that ATPe-induced and arachi-onic acid-induced apoptosis follow different intracellular path-

H.M. Costa-Junior et al. / Prostaglandins & other Lipid Mediators 88 (2009) 51–61 57

Fig. 6. Involvement of 5-lipoxygenase in ATPe-induced apoptosis. Freshly isolated (closed bars) or adherent (dashed bars) macrophages were pre-incubated or not in thepresence of different 5-LOX inhibitors for 20 min, exposed to zero or 5 mM ATP for an additional 20 min under the same conditions (induction period), and transferred toc a, c, eN f) AA-p l deatha

wppipnas

sm

Fdtrd

omplete medium without ATP, containing the same drugs. Normalized apoptosis (DGA; 10 and 50 �M. (c and d) MK-886; 0.1 and 1.0 �M. (e) Zileuton; 1 and 10 �M. (erformed in triplicates. Statistical significance was obtained by comparing the celnd ***p < 0.001.

ays in macrophages. However, these results do not exclude theossibility that macrophages could secrete leukotrienes only in theresence of ATPe or that apoptosis could be triggered by the bind-

ng of these eicosanoids with their receptors. In order to discard thisossibility, we showed that neither CP-105696 nor MK-571, antago-ists of the leukotriene receptors BLT1 and CysLT, respectively, hadny significant effect either on ATPe-induced macrophage apopto-is (Fig. 8a) or on LDH release (Fig. 8b).

muUdb

ig. 7. Arachidonic acid- and ATPe-induced macrophage death involves different pathwaysrugs, exposed to zero, 5 mM ATP (closed bars), or 20 �M arachidonic acid (AA) (dashedransferred to complete medium without ATP, containing the same drugs. Normalizedepresent the mean % ± S.E.M. of at least three experiments performed in triplicates. Statrug with the values obtained for ATP alone. **p value < 0.01 and ***p < 0.001.

and f) and LDH release (b and d) were accessed 6 h later (effector period). (a and b)861; 1 and 10 �M. Data represent the mean % ± S.E.M. of at least three experiments

induced by ATP plus drug with the values obtained for ATP alone. **p value < 0.01

These results suggested that 5-LOX-derivatives other thanecreted leukotrienes are necessary to induce ATPe-inducedacrophage death. In order to further demonstrate the involve-

ent of 5-LOX, we performed ATPe-induced cell death experiments

sing macrophages derived from 5-LOX−/− mice [59,60] (Fig. 9).nexpectedly, 5-LOX−/− macrophages were as sensitive to celleath by 5 mM ATPe as their wild type counterpart (Fig. 9a and). These data raised the possibility that either all the inhibitors of

. Adherent macrophages were pre-incubated or not in the presence of the indicatedbars) for an additional 20 min under the same conditions (induction period), andapoptosis (a) and LDH release (b) were accessed 6 h later (effector period). Dataistical significance was obtained by comparing the cell death induced by ATP plus

58 H.M. Costa-Junior et al. / Prostaglandins & other Lipid Mediators 88 (2009) 51–61

Fig. 8. Leukotriene receptors are not involved in ATPe-induced cell death. Adherent macrophages were pre-incubated or not in the presence of 1 or 10 �M CP-10596 (closedbars), or MK-571 (dashed bars), exposed to zero or 5 mM ATP for an additional 20 min under the same conditions (induction period), and transferred to complete mediumwithout ATP, containing the same drugs. Normalized apoptosis (a) and LDH release (b) were accessed 6 h later (effector period). Data represent the mean % ± S.E.M. of atleast three experiments performed in triplicates. Statistical significance was obtained by comparing the cell death induced by ATP plus drug with the values obtained for ATPalone.

Fig. 9. ATPe-induced apoptosis in macrophages derived from 5-LOX−/− mice. (a and b) Freshly isolated macrophages were obtained from wild type (WP, closed bars) or 5-LOX−/− (dashed bars) mice were exposed or not to 0.1, 1 and 5 mM ATP for 20 min and apoptosis (a) and LDH release (b) were determined 6 h later. (c) Adherent macrophageso d or nf red tow rimenc e < 0.0

tappnfiwatbum

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btained from WT (closed bars) or 5-LOX−/− (dashed bars) mice were pre-incubateor an additional 20 min under the same conditions (induction period), and transferas determined 6 h later. Data represent the mean % ± S.E.M. of at least three expe

ell death induced by ATP plus drug with the values obtained for ATP alone. *p valu

he LOX–leukotriene pathway used above to inhibit apoptosis havedifferent target or that 5-LOX-deficient mice develop alternativeathways to ATPe-induced cell death. Consistent with the latterossibility we showed that apoptosis of 5-LOX−/− macrophages areot sensitive to NDGA or MK-886 (Fig. 9c), a result that also con-rms the specific action of these drugs in the 5-LOX pathway in theild type macrophages. Therefore, we conclude that ATPe-induced

poptosis in wild type macrophages involves 5-LOX activity but nothe secretion of leukotrienes. These results do not exclude the possi-le involvement of other pathways that are silent in wild type micender our experimental conditions but are activated in 5-LOX−/−

ice.

. Discussion

The role of P2 receptors and extracellular nucleotides in theodulation of the immune system has gained increasing attention

uring the last decade [3,14,18,43]. The expression of P2 receptorsre modulated during the differentiation of haematopoietic cells61] and a group of 5 G protein-coupled P2 receptors were foundo be associated with M2 polarization (induced by IL-4), while theene of the ATP-gated ion channel P2X7 was transiently upregu-

mtiai

ot with 100 �M NDGA or 1 �M MK-886 for 20 min, exposed to zero or 5 mM ATPcomplete medium without ATP, containing the same drugs. Normalized apoptosis

ts performed in triplicates. Statistical significance was obtained by comparing the5, and ***p < 0.001.

ated during the maturation from monocyte to macrophage [62].ATPe-induced macrophage death is dependent on the presence

f P2X7 receptors and involves the activation of caspases 3, 8,nd 9 [3,25,27] and the generation of ceramide [28], but little isnown regarding the signaling cascades that couple P2X7 to celleath programs. Because of the presence of multiple P2 receptors inacrophages, the opening of different ion channels, the presence

f a permeabilization pore, and the activation of multiple signal-ng cascades [10], several candidates should be considered. Thesenclude the overload of Ca2+ and the efflux of K+ [11,20,45], MAPKs15,16,63,64], and phospholipase A2 (PLA2) [36,37,65,66], amongthers. Interestingly, although Ca2+ overload does occur after pore-pening under physiological conditions [11], pore formation andRK 1/2 activation are Ca2+-independent. [15,45,64]. These datauggest that a Ca2+-independent phenomena may be involved inTPe-induced apoptosis and other P2X7-associated phenomena.

In this paper we studied cell death induced by ATPe in

acrophages using a protocol that allowed us to better inves-

igate the triggering events that occur during the initial 20 minnduction period in the presence of ATPe. We first found thatmong the quickly activated signals induced by ATPe, the risen the intracellular free Ca2+ concentration and the phosphoryla-

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ion of ERK 1/2 are not involved in the triggering of macrophagepoptosis and LDH release (Figs. 2c and 3). Interestingly, we haveecently demonstrated that these same signals are not requiredor the opening of the permeabilization pores [15]. In addition,he inhibitors of p38, SB-203580 and SB-202190, two reagentshat also do not block the opening of the permeabilization porender our experimental conditions [15], were also unable to blockTPe-induced apoptosis at doses up to 100 �M (data not shown).owever 10 �M SB-203580, but not SB-202190, blocked approx-

mately 50% of ATPe-induced LDH release (Fig. 3c). These resultsuggest that the necrosis induced by ATPe in macrophages can beegulated independently of apoptosis and requires the activationf p38, as already described in myocardium ischemic necrotic zoneormation [67]. The outcome of an immune response may be modu-ated by the type of macrophage death and further characterizationf these pathways may help the development of new pharmacolog-cal tools for immune-intervention.

The persistence of death in cells exposed to ATPe in conditionshat completely abrogated intracellular Ca2+ signaling was an unex-ected result since it has been well documented that ATPe can

nduce a marked increase in the free intracellular Ca2+ concen-ration in macrophages through the activation of P2X7, P2Y2, andther receptors [7,10,11]. Also, the opening of the permeabilizationore can cause an overflow of Ca2+ ions, a condition that has beeneported to induce apoptosis through the opening of permeabilityransition pores of mitochondria and the decrease of intracellular+ concentration in other cell types such as neurons stimulated

hrough NMDA receptors [24,44,68]. In these cells, apoptosis cane prevented by exposing the cells to the agonist either in thebsence of extracellular Ca2+ or in a medium containing high K+

oncentration. Our results obtained using EGTA and BAPTA-AMtrongly support the possibility that ATPe-induced cell death inacrophages can be triggered by a different pathway involving a

a2+-independent mechanism. In accordance with this possibility,xchanging K+ for Na+ during the 20 min induction period to avoid+ efflux through P2X channels and/or the permeabilization poreid not prevent ATPe-induced apoptosis (Fig. 2a). It is important toote that our results are valid only for the induction period. Apop-osis is known to require several Ca2+-dependent events [24] anduring the effector period, after the withdrawing of ATPe, othera2+-dependent mechanisms may occur. In fact, CsA, a drug thatan avoid the collapse of mitochondria potential in ATPe-stimulatedacrophages [69] effectively blocked ATPe-induced apoptosis, sug-

esting that a Ca2+-dependent step requiring either the opening ofermeability transition pores of mitochondria or the activation ofalcineurin is required (Fig. 2b).

The search for a Ca2+-independent step that could link P2 recep-ors to cell death apoptosis prompted us to investigate the role ofLA2. Our data indicate that an iPLA2 but not a sPLA2 or a cPLA2 ismplicated in ATPe-induced macrophage apoptosis (Fig. 4).

The possible mechanism used by iPLA2 to induce apoptosis inTPe-activated macrophages is not clear. Arachidonic acid is anbvious candidate since this fatty acid can be liberated by groupI iPLA2 from macrophages and other cells [54], and it can induce

he death of several cell types, including neutrophils, monocytic cellines and macrophages [70–72]. However, we showed that exoge-ously added arachidonic acid and ATPe use different pathwayso induce macrophage apoptosis (Fig. 7). Several authors report airect participation of iPLA2 in mitochondrial-mediated pathwaysf apoptosis [56,57,73] and an elegant mechanism whereby cas-

ase 3 cleavage activates the pro-apoptotic iPLA2 while inhibitingPLA2, an enzyme involved in the production of pro-inflammatoryicosanoids, has been proposed by Atsumi et al. [74]. In insulin-ecreting cells, caspase 3-processed iPLA2 translocates to theerinuclear region and plays a functional role in the develop-

fpoIv

ther Lipid Mediators 88 (2009) 51–61 59

ent of apoptosis induced by endoplasmic reticulum stress [33].urther experiments are needed to investigate whether similarechanisms could be involved in P2X7-dependent macrophage

poptosis.Apoptosis is a complex phenomena that can be triggered by

number of different extrinsic or intrinsic stimuli. It is gener-lly agreed that apoptosis requires the activation of caspase 3y two alternative pathways. The intrinsic pathways involves theelease of cytochrome c by mitochondria, apoptosome formationnd caspase 9 activation, while extrinsic stimuli are usually asso-iated with death receptors and leads to the activation of caspase[24,75,76]. A link between the two pathways established by the

ctivation of the BH3-only pro-apoptotic molecule BID by caspase. Apoptosis triggered by P2X7 receptors seem to involve bothathways since it is blocked by inhibitors of caspase 3, 8, and 93,25,27] (and data not shown). P2X7 activation could trigger theseathways by a number of signals. These include the overload ofa2+ and the efflux of K+ through the ATP-gated P2X7 channelsnd the large permeabilization pore, a direct activation of cas-ases through a putative C-terminal death domain [77] (reviewed

n Ref. [10]). A link has been established between intracellularepletion of K+, iPLA2 activity, caspase 1 activation, and in IL-1�rocessing and secretion [78,79] and the involvement of P2X7 as aathway for K+ efflux in inflammasome activation and IL-1� pro-essing triggered by ATPe has been firmly established [78,80,81].owever, the involvement of this mechanism in the activation of

he caspases that initiate apoptosis was not studied. Our resultshows that although an iPLA2 seems to be also involved in apop-osis, neither the K+ depletion nor the Ca2+ overload that occurfter P2X7 activation are required to trigger apoptosis. Interest-ngly, K+ depletion by pore-forming toxins can activate caspase-1nd engage the cell in survival-associated pathways [82], sug-esting that the P2X7-associated pore could also activate thisurvival pathway and that live-or-death decision would dependn other factors such as dose and extent of exposure to ATP,nd the expression of nucleotidases and other P2 receptors onacrophages.A more precise identification of the iPLA2 associated with

acrophage apoptosis and its mechanism of activation by P2X7emains to be elucidated. It would be also interesting to furthernvestigate the similarities and differences between the pathwayshat links P2X7 to inflammasome activation and apoptosis. Inter-stingly, in the same way that our results indicate that K+ depletionay not be a necessary requirement during the triggering phase

f apoptosis, it has recently been shown that extracellular bacteriahat signals through TLRs, but not intracellular bacteria, requireso-stimulation by ATPe and depletion of cytoplasmic K+ through2X7 and pannexin [83,84], indicating that intracellular bacteriaan activate the inflammasome directly via cryopyrin.

One possible scenario to better understand ATPe-induced apop-osis in macrophages is considering that ATPe induces the depletionf intracellular Ca2+ stores in macrophages [85] and causes endo-lasmic reticulum stress, a phenomenon that can lead apoptosis

nvolving iPLA2 and caspase 8 [22,33,55,86].Using 5-LOX and FLAP inhibitors and 5-LOX−/− macrophages we

lso showed that, in addition to an iPLA2 these two proteins areequired for apoptosis, which lead us to the unexpected sugges-ion that iPLA2, 5-LOX, and FLAP cooperate to form a downstreamro-apoptotic signal in macrophages exposed to ATPe. It is well doc-mented that 5-LOX translocates to the nuclear membrane and

orms a complex with cPLA2 and the integral nuclear membranerotein FLAP, initiating leukotriene production [87,88]. However, tour knowledge, no evidence for a similar complex with iPLA2 exists.t is therefore important to further identify the isoform of iPLA2 acti-ated by ATPe in macrophages, the mechanism of cooperation with

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-LOX and FLAP, their exact location, and the pro-apoptotic signalshey generate.

We have also shown that macrophages derived from 5-LOX−/−

ice undergo NDGA- and MK-886-insensitive ATPe-induced apop-osis (Fig. 9). We have not investigated whether the upstream

echanisms of induction (e.g. involvement of P2X7 and iPLA2nd non-involvement of intracellular Ca2+) are the same as inheir wild type counterparts. However, these results make it clearhat there are alternative pathways for ATPe-induced apoptosisn macrophages and that these pathways could be modulatedn special circumstances. 5-LOX-deficient mice should representn interesting model to reveal these pathways. In addition, welso speculate that oxidants derived from 5-LOX activity mighte an important pathway involved in the effects of ATP-inducedell death. Since the pharmacological approaches partially inhibit-LOX activity and LT biosynthesis, we believe that the residualctivity might be effective in the protection of cell death. Thus,ur results also suggest that a possible LT-independent effect ofoth FLAP and 5-LOX inhibitors which could account for the effectsf ATP. In keeping with this possibility we notice that the 5-LOXnhibitors zileuton [89] and NDGA [90] have anti-oxidant activity.he precise role of 5-LOX and FLAP inhibitor remains to be deter-ined and the possible involvement of intracellular receptors of

ipid mediators should also be considered.Apoptosis of cells of the immune system is an impor-

ant phenomenon in immune-modulation causing immune-uppression/tolerance by depleting immune-competent cells, bynducing macrophages to shift the immune system to a Th2 pat-ern or causing anergy in macrophages and dendritic cells thatngest apoptotic cells [91–93]. Macrophages are considered rel-tively resistant to apoptosis and the control of its cell deathrograms can be important for immune modulation in rheumatoidrthritis [94] and atherosclerosis [95]. The modulation of P2 recep-ors and their signaling pathways in macrophages and other cells ofhe immune system may be important to elucidate and control thene tuning of the immune system in several immune-pathologicalituations.

cknowledgements

The authors wish to thank Drs. Bruno Diaz, Cláudio Canetti andobson Coutinho-Silva for helpful discussions, Marc Peters-Goldenor donating inhibitor of leukotriene synthesis, and Vandir da Costa,ercules Antonio da Silva Souza, and Carlos Arterio Sorgi for tech-ical assistance.

This work was supported by Conselho Nacional de Desenvolvi-ento Científico e Tecnológico (CNPq), Fundacão Carlos Chagas

ilho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ),undacão de Amparo à Pesquisa do Estado de São Paulo (FAPESP)nd American Lung Association.

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