The anti apoptotic effect of leukotriene B4 in neutrophils

The anti-apoptotic effect of leukotriene B 4in neutrophils:A role for phosphatidylinositol 3-kinase,extracellularsignal-regulated kinase and Mcl-1Darlaine Pe ´trin,Sylvie Turcotte,Annie-Kim Gilbert,Marek Rola-Pleszczynski,Jana Stankova *Immunology Division,Department of Pediatrics,Faculty of Medicine,Universite ´de Sherbrooke,3001,North 12th Avenue,Sherbrooke,Que ´bec,Canada J1H 5N4Received 25April 2005;accepted 24May 2005Available online 20June 2005AbstractThe constitutive commitment of neutrophils to apoptosis is a key process for the control and resolution of inflammation and it can be delayed by various inflammatory mediators including leukotriene B 4(LTB 4).The mechanisms by which LTB 4contributes to neutrophil survival are still unclear and the present work aims at identifying intracellular pathways underlying this effect.Inhibition of human neutrophil apoptosis by LTB 4was abrogated by the phosphatidylinositol 3-kinase (PI3-K)inhibitor wortmannin and by the specific MEK inhibitor PD98059.In contrast,inhibitors of p38MAPK,Jak2/3and Src did not hinder the anti-apoptotic effect of LTB 4.We also investigated the effects of members of the Bcl-2family as they play a crucial role in the regulation of programmed cell death.When neutrophils were incubated with LTB 4for 1to 6h,the mRNA levels of the anti-apoptotic protein Mcl-1were upregulated approximately 2-fold,while those of the pro-apoptotic protein Bax were downregulated 3-to 4-fold,as determined by real-time PCR.Accordingly,Western blot analysis revealed that the expression of Mcl-1was upregulated in presence of LTB 4,while flow cytometric analysis revealed that Bax protein was downregulated.Furthermore,the modulatory effects of LTB 4on Mcl-1and Bax proteins were abolished in the presence of either wortmannin or PD98059.Taken together,these results demonstrate the participation of PI3-K and MEK/ERK kinases,as well as regulatory apoptotic proteins such as Mcl-1and Bax,in the anti-apoptotic effects of LTB 4in human neutrophils.D 2005Elsevier Inc.All rights reserved.Keywords:Human neutrophils;Lipid mediators;Apoptosis;Signal transduction;Leukotriens;Mcl-11.IntroductionNeutrophils,also known as polymorphonuclear leuko-cytes (PMN)3,represent a first line of defense againstinvading microorganisms.These phagocytic cells are key players of inflammation,rapidly responding to chemotactic agents and producing additional inflammatory mediators [1,2].Neutrophils have a very short half-life (8–20h)as they undergo constitutive apoptosis,a process assuring the elimination of dead cells without the release of cytotoxic granules into the surrounding tissues.Among the different inflammatory mediators,leuko-triene B 4(LTB 4)is well recognized for its ability to delay neutrophil apoptosis [3–5].However,the mechanisms underlying this anti-apoptotic effect are not clear.LTB 4is a lipid mediator derived from nuclear membrane phospholipids via the action of 5-lipoxygenase [6].LTB 4is rapidly synthesized by neutrophils and alveolar macro-0898-6568/$-see front matter D 2005Elsevier Inc.All rights reserved.doi:10.1016/j.cellsig.2005.05.021Abbreviations:PMN,Polymorphonuclear leukocyte;MAPK,Mitogen-activated protein kinase;ERK,Extracellular signal-regulated kinase;MEK,MAPK/ERK kinase;PI3-K,Phosphatidylinositol 3-kinase;PI,Propidium iodide;COPD,Chronic obstructive pulmonary disease;Jak,Janus kinase;STAT,Signal transducer and activator of transcription;GPCR,G protein coupled receptor;LTB 4,Leukotriene B 4;PKC,Protein kinase C;LPA,Lysophosphatidic acid.*Corresponding author.Tel.:+18195645268;fax:+18195645215.E-mail address:Jana.Stankova@USherbrooke.ca (J.Stankova).Cellular Signalling 18(2006)479–487/locate/cellsigphages,and it stimulates neutrophil chemotaxis and degranulation among other effects[7–10].Elevated con-centrations of LTB4have been found in various inflam-matory conditions including bronchial asthma,cystic fibrosis,COPD,psoriasis,and inflammatory bowel disease [11–15].Two types of plasma membrane receptors for LTB4 have been identified in human neutrophils,named BLT1 and BLT2[16,17].Both receptors are members of the heptahelical,G protein-coupled receptor(GPCR)family. Regarding neutrophil survival,we have previously shown that BLT antagonists prevented the delay in neutrophil apoptosis induced by LTB4[5].In leukocytes,most responses to LTB4are pertussis toxin(PTX)-sensitive, suggesting the involvement of G a i/o subunits[18,19]. Subsequent to the activation of PTX-sensitive G proteins, phosphatidylinositol3-kinase(PI3-K),a heterodimeric enzyme that phosphorylates phosphatidylinositol-4,5-biphosphate(PtdIns(4,5)P2),may be activated[20].For example,it has been shown that LTB4,via its BLT1 receptor,has the ability to activate PI3-K through PTX-sensitive G proteins[21].PI3-K and its best-characterized downstream target,the serine/threonine kinase Akt, occupy a central position in modulating neutrophil activation,chemotaxis and apoptosis[22].Besides PI3-K,mitogen-activated protein kinases (MAPKs)are also involved in many facets of cellular regulation and the mechanisms by which GPCRs activate MAPK cascades have been studied extensively.Previous reports have indicated that G i protein-coupled receptors, such as the one for lysophosphatidic acid(LPA),induce activation of MEK through a PI3-K g-dependent pathway, in a manner that can be both Ras-dependent and independent[23,24].The Ras-dependent pathway is known to be activated by Src-family kinases,themselves activated by G h g subunits or PI3-K[23,25–27].Among the different MAPK cascades,the extracellular signal-regulated kinase(ERK)pathway is often associated with cell survival[28].Neutrophil exposure to GM-CSF or IL-8delays apoptosis by stimulating PI3-K and ERK-dependent pathways[29].Although p38MAPK mediates primarily pro-apoptotic and growth inhibitory signals,it may also induce antiapoptotic signals,depending on the tissues and isoforms involved[30].p38MAPK resides among the mechanisms involved in interleukin-15-induced suppresssion of human neutrophil apoptosis[31].Neutrophil apoptosis is a tightly regulated process important in the control and resolution of inflammation. The Bcl-2family comprises a series of pro-apoptotic(e.g. Bax,Bad,Bid)and anti-apoptotic(e.g.Mcl-1,A1,Bcl-X L)members,essential for the regulation of neutrophil apoptosis.Recent studies have demonstrated that Mcl-1 plays a vital role for PMN survival[32,33].Mcl-1 expression may be regulated by signal transduction through ERK and transcriptional activation through Signal Transducer and Activator of Transcription(STAT)mole-cules[34,35].In contrast,Bax promotes neutrophil death, given that antisense Bax oligonucleotides delay PMN apoptosis[36].It has also been demonstrated that human neutrophils exposed to diverse apoptosis-delaying agents significantly diminished Bax protein expression.The role of Mcl-1and Bax in LTB4-treated neutrophils is presently unknown.The present series of experiments were aimed at identifying the intracellular pathways activated by LTB4 in order to understand the mechanisms underlying the inhibition of spontaneous apoptosis by LTB4.Pertussis toxin was used to assess the involvement of PTX-sensitive G proteins in this response.Pharmacological inhibitors such as wortmannin,PD98059,SB203580,PP1,and AG490allowed us to examine the participation of PI3K, MEK,p38MAPK,Src and Jak2/3kinases,respectively,in the anti-apoptotic effect of LTB4.The modulation of Mcl-1 and Bax in the prolonged PMN survival induced by LTB4 was examined at the mRNA level by real-time PCR,and at the protein level by western blot or flow cytometric analyses.2.Materials and methods2.1.MaterialsRPMI1640medium was purchased from Invitrogen Life Technologies(Burlington,Ontario,Canada).LTB4 was from Cayman Chemicals(Ann Arbor,MI,USA). PD98059wortmannin,PP1,PP2,and SB203580were from Biomol(Plymouth Meeting,PA,USA).AG490was from Calbiochem(San Diego,California,USA).GM-CSF was from Peprotech Canada(Ottawa,Ontario,Canada). The polyclonal anti-Mcl-1(S-19)and anti-Bax(N20) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz,CA,USA).HRP-conjugated sheep anti-mouse IgG,or HRP-conjugated donkey anti-rabbit Ig were from Amersham Biosciences(Baie d’Urfe´,Que´bec, Canada).Fetal bovine serum(FBS),the monoclonal vinculin antibody,pertussis toxin(PTX),streptomycin, HEPES,aprotinin,leupeptin,soybean trypsin inhibitor,4-(2-Aminoethyl-benzene sulfanyl fluoride)(AEBSF),phe-nylmethyl-sulfonyl fluoride(PMSF),and sodium orthova-nadate were purchased from Sigma-Aldrich(Oakville, Ontario,Canada).Sodium fluoride and sodium pyrophos-phate were from Fisher Scientific(Nepean,Ontario, Canada).2.2.Isolation of neutrophils and culture conditionsVenous blood was collected from healthy medication-free volunteers,after informed consent,in accordance with an Internal Review Board-approved protocol.Peripheral blood neutrophils were isolated as previously described [5].Briefly,following a first centrifugation for15min,D.Pe´trin et al./Cellular Signalling18(2006)479–487 480dextran sedimentation and Ficoll–Hypaque density gra-dient were used to isolate peripheral blood leukocytes. Subsequent to a second centrifugation at400Âg for20 min,neutrophils were obtained from the pellet,and contaminating erythrocytes were eliminated by hypotonic lysis.Neutrophils were resuspended to2.5Â106cells/ml (or as indicated)in RPMI1640medium containing5% heat-inactivated FBS,100U/ml penicillin(Novopharm), 100A g/ml streptomycin,and HEPES0.01M.Cells were pre-incubated in a humidified atmosphere with5%carbon dioxide at37-C for1h in polypropylene conical tubes in presence of pertussis toxin(0.1,1,or10ng/ ml),wortmannin(0.1or1A M),PD98059(0.1,1,or10 A M),SB203580(10A M),PP1(10A M),PP2(10A M), AG490(10A M)or their respective vehicles(controls). Following this pre-incubation,neutrophils were cultured with LTB4(100nM)or its vehicle(ethanol)for an additional19h,or as indicated.2.3.Assessment of apoptosisCell apoptosis was assayed by cell surface binding of phosphatidylserine by annexin V.Briefly,neutrophils were stained with annexin V-FITC(BD PharMingen,Missis-sauga,Ontario,Canada)for15min at room temperature, in conjunction with a vital dye,propidium iodide,to distinguish apoptotic cells from necrotic cells.Stained cells were examined with a FACScan flow cytometer(BD Biosciences)and analyzed with the CellQuest Pro soft-ware.Results illustrated represent the percentage of cells stained for annexin V,but not for propidium iodide.2.4.RNA extraction and reverse transcriptionCells were incubated at a concentration of1Â107cells/ml RPMI medium for indicated times,in presence of LTB4(100 nM)or its vehicle(ethanol).Total RNAwas isolated using the TriPure isolation reagent(Roche Applied Science,Laval, Que´bec,Canada)according to the manufacturer’s instruc-tions.Total RNA was treated with10U DNase I(Amersham Biosciences)and20U Rnasin(Fisher Scientific)for15min at 37-C and inactivated at70-C for10min.Afterwards,1A g of total RNAwas used for the synthesis of cDNA,along with0.5 A g pd(T)(Amersham Biosciences),100mM dithiothreitol (DTT)(Fisher Scientific),250A M2V-deoxynucleoside5V-triphosphate(dNTP)(Amersham Biosciences)and200U/A l M-MLV reverse transcriptase(BioCan scientific,Missis-sauga,Ontario,Canada).2.5.Quantitative real-time PCRThe real-time PCR reactions were carried out using the Platinum Quantitative PCR SuperMix-UDG(Invitrogen Life Technologies)combined with SyBR Green,and analyzed with the RotorGene apparatus(Corbett Research, Montreal Biotech,Kirkland,Quebec,Canada).All samples were performed in triplicates.The means of the triplicate for the gene of interest were normalized over GAPDH,and controls(samples stimulated with ethanol)were set to1. The primer sequences were designed as follows:Mcl-1 forward,5V-TTCCAAGGCATGCTTCGGAAAC-3V,and reverse,5V-TCTGCTAATGGTTCGATGCAGC-3,Bax for-ward,5V-TCAGGATGCGTCCACCAAGAAGC-3V,and reverse,5V-CCTTGAGCACCAGTTTGCTGGCA-3V.The primers for GAPDH were designed based on published sequences[37]:forward,5V-GATGACATCAAGAAG-GTGGTGAA-3V,and reverse,5V-GTCTTACTCCTT-GGAGGCCATGT-3V.The predicted sizes of the PCR-amplified products were193bp for Mcl-1,216bp for Bax,and246bp for GAPDH.2.6.Western blot analysisAfter isolation and culture of neutrophils for24h,cells were incubated with a series of protease inhibitors(aprotinin 2A g/ml,leupeptin5A g/ml,PMSF1mM,AEBSF100A g/ml, and soybean trypsin inhibitor10A g/ml)and phosphatase inhibitors(sodium orthovanadate2mM,sodium fluoride10 mM,sodium pyrophosphate2mM).Whole cell extracts were prepared by heating cell preparations directly in Laemmli buffer for5min at100-C.50A g of proteins,as determined by the Bradford assay,were separated by SDS-PAGE on a10% acrylamide gel.Proteins were transferred onto an Immun-Blot i PVDF membrane(Bio-Rad Laboratories,Missis-sauga,Ontario,Canada).The membranes were incubated overnight with polyclonal anti-Mcl-1or monoclonal anti-vinculin antibodies.After three-15min washes with Tris-bufferred saline containing Tween20(0.1%)(TBST), membranes were incubated with HRP-conjugated anti-rabbit or anti-mouse antibodies,and revealed using ECL detection reagents(Amersham Biosciences).2.7.Flow cytometryNeutrophils were washed with PBS1X and fixed in2% paraformaldehyde for15min at room temperature.Cells were washed and resuspended in presence of human Ig to block non-specific sites,then resuspended in PBS2% BSA,and labelled for30min with anti-Bax antibody or isotypic control(Inter Sciences)at room temperature. Neutrophils were then washed and incubated in presence of FITC-conjugated goat anti-rabbit IgG(BioCan Scien-tific)diluted in PBS2%BSA.Cells were washed again and resuspended in PBS before immunofluorescence analysis.Ten thousand events were analyzed on a FACScan flow cytometer.2.8.Statistical analysesData are expressed as mean+SEM.For all experiments, paired Student’s t-tests were performed to compare treat-ments,except for experiments involving the use of pharma-D.Pe´trin et al./Cellular Signalling18(2006)479–487481cological inhibitors.For the latter,two-way analysis ofvariance (ANOVA)were performed (GraphPad Prism software,San Diego,CA).For real-time PCR experiments,data were first transformed into log units and then compared using ratio paired t -tests.P values smaller than 0.05were considered significant.3.Results3.1.Kinetic of LTB 4-induced delay in neutrophil apoptosis The assessment of apoptosis can be performed using various techniques.Since the appearance of phosphatidyl-serine on the outer surface is a general feature of apoptotic neutrophils,we used annexin V ,which binds phosphati-dylserine,to quantify apoptosis.LTB 4was able to reduce the number of annexin-positive cells,and was be seen within 10h of incubation with stimulus (control 14T 7%vs LTB 46T 3%apoptosis)(Fig.1).A significant difference was observed following 20h of incubation (control 67T 2%vs LTB 443T 2%apoptosis),and this time was selected for the following series of experiments.3.2.Involvement of PTX-sensitive G proteins in the prolonged neutrophil survival induced by LTB 4Leukocytes express abundantly G i proteins and responses to LTB 4in PMN leukocytes are mainly PTX-sensitive [19,38].However,it has been shown that BLT1could couple to the PTX-resistant G a 16subunit,indicating that different signaling pathways can be triggered by LTB 4[39].Consequently,we studied the involvement of the PTX-sensitive G proteins in the anti-apoptotic effect of LTB 4.As shown in Fig.2,even low concentrations of pertussis toxin (0.1to 10ng/ml)inhibited the anti-apoptotic effect of LTB 4by 76%to 100%.These results,indicating the involvement of PTX-sensitive G proteins,are in agreement with previously published data [4].3.3.Involvement of PI3-K and MEK/ERK pathways in the anti-apoptotic effect of LTB 4In vivo and in vitro studies have shown that PI3-K participates in neutrophil activation,chemotaxis and apop-tosis [22].Moreover,it is part of the mechanism underlying the anti-apoptotic effect of GM-CSF [29].Therefore,we investigated the role of PI3-K activation in LTB 4-treated neutrophils.The use of wortmannin (0.1and 1A M),a potent but not isoform-specific PI3-K inhibitor,reversed the anti-apoptotic effect of LTB 4(in absence of LTB 4:DMSO 60T 3%,0.1A M wortmannin 68T 5%,and 1A M wortman-nin 65T 4%apoptosis;in presence of LTB 4:DMSO 39T 3%,0.1A M wortmannin 55T 6%,1A M wortmannin 57T 5%apoptosis)(Fig.3A).Similarly,we studied the effects of PD98059,a potent and selective MEK inhibitor.Preincubation of human neutrophils with PD98059(1and 10A M)completely inhibited the anti-apoptotic effect of LTB 4(in absence of LTB 4:DMSO 57T 6%,10A M PD9805957T 6%apoptosis;in presence of LTB 4:DMSO 31T 4%,0.1A M PD9805965%BC o u n t s 204060Annexin V-FITC Control41%204060LTB4C o u n t s10110210310410010102104101103Annexin V-FITCA*Control LTB4% n e u t r o p h i l a p o p t o s i sHours in cultureFig.1.Kinetics of LTB 4-mediated delay of neutrophil apoptosis.(A)PMN were incubated in presence of LTB 4(100nM)or its vehicle (ethanol)for indicated times.Cells were analyzed for annexin V-FITC binding in conjunction with propidium iodide.Results are illustrated as annexin V-positive,propidium iodide-negative cells,and represent the mean T SEM of at least three independent experiments.Statistical significance was assessed using paired Student’s t -tests (*p <0.01).(B)Representative histograms of annexin V-FITC binding in PMN incubated for 20h in presence of LTB 4(100nM)or ethanol (control).Numbers represent the percent annexin V-positive and propidium iodide-negative cells.Fig.2.Involvement of PTX-sensitive G proteins in the anti-apoptotic effect of LTB 4.Neutrophils were exposed to graded concentrations of pertussis toxin,or medium alone,for 1h prior to an incubation of 19h in presence of LTB 4(100nM)or its vehicle (ethanol).Cells were analyzed for annexin V-FITC binding in conjunction with propidium iodide.Results are illustrated as annexin V-positive,propidium iodide-negative cells,and represent the mean T SEM of at least three independent experiments.Statistical signifi-cance was assessed using two-way ANOV A (*p <0.05).D.Pe ´trin et al./Cellular Signalling 18(2006)479–48748249T 6%,1A M PD9805958T 4%,and 10A M PD9805961T 6%apoptosis)(Fig.3B).Following these observations,and based on previous studies suggesting the induction of MEK/ERK activation by PI3-K [23,24],we assessed whether PI3-K and MEK/ERK could be part of the same signaling pathway.Simultaneous inhibition of PI3-K and MEK did not cause an additional increase in apoptosis when compared to either inhibitor alone,in the presence of LTB 4(Fig.3C),suggesting that indeed they could.In addition to MEK/ERK pathway,p38MAPK is also activated in human neutrophils stimulated by inflammatory cytokines [40].As illustrated in Table 1,the inhibition of this kinase by SB 203580(10A M)did not reverse the anti-apoptotic effect of LTB 4.Recently,Src family tyrosine kinases has been shown to participate in GPCR signaling [41],and this link betweenSrc kinases and GPCRs was also found to be associatedwith the regulation of apoptosis [42].However,the use of PP1(10A M)(Table 1),a Src-family kinase inhibitor,did not reverse the anti-apoptotic effect of LTB 4.Various other downstream effectors may also be acti-vated following GPCR activation.For example,our group has shown that platelet-activating factor receptor signals through the Janus kinase (Jak)/STAT pathway [43].We examined the effect of AG490(10A M),a potent inhibitor of the Jak2/3tyrosine kinase,on the delay in neutrophil apoptosis induced by LTB 4.As shown in Table 1,this inhibitor did not hinder the anti-apoptotic response induced by LTB 4.3.4.Mcl-1mRNA and protein expression in LTB 4-treated neutrophilsBecause of the importance of Bcl-2family for the survival of numerous cell types,we investigated the effect of LTB 4on Mcl-1expression,an anti-apoptotic protein expressed in neutrophils and previously shown to be inducible by GM-CSF,IL-1h ,and LPS [32].We first examined the level of mRNA of Mcl-1by real-time PCR (Fig.4A).Cell exposure to LTB 4(100nM)caused a transient increase in Mcl-1mRNA when compared to control.Depending on blood donors,an approximately 2.5-fold induction was observed after 1and 3h incubations.A Western blot was then performed in order to determine whether this induction at the mRNA level was associated with an increase at the protein level.Fig.4b is a representative experiment demonstrating that Mcl-1protein expression was greatly diminished after 24h of incubation,but treatments with GM-CSF (10ng/ml)or LTB 4(100nM)prevented the loss of Mcl-1protein expression.Densitometric analysis of Mcl-1over vinculin revealed that there was a 3-fold increase in Mcl-1expression in LTB 4-treated cells when compared to Mcl-1expression in ethanol-treated cells (data not shown).This is comparable to Mcl-1expression in GM-CSF-treated cells,where a 2.5-fold increase was observed.Table 1Effect of inhibition of p38kinase,Jak2/3,and Src kinase on the anti-apoptotic effect of LTB 4Control (%apoptosis)LTB 4(%apoptosis)DMSO 60T 441T 4SB20358055T 1133T 10PP158T 640T 6AG49061T 744T 5Freshly isolated neutrophils were pretreated for 1h with SB203580(10A M),AG490(10A M),PP1(10A M),or their respective controls (DMSO)prior to an incubation for 19h in presence of LTB 4(100nM)or its vehicle ethanol (control).Cells were analyzed for annexin V-FITC binding in conjunction with propidium iodide.Results are illustrated as annexin V-positive,propidium iodide-negative cells,and represent the mean T SEM of at least three independentexperiments.Fig.3.Role of PI3-K and MEK in the regulation of apoptosis by LTB 4.PMN were incubated for 1h in the presence of (A)wortmannin (0.1and 1A M),(B)graded concentrations of PD98059,or (C)both wortmannin (1A M)and PD98059(10A M)simultaneously,or their respective controls (DMSO).Cells were then incubated for an additional 19h in the presence of LTB 4(100nM)or its vehicle (control).Cells were analyzed for annexin V-FITC binding in conjunction with propidium iodide.Results are illustrated as annexin V-positive,propidium iodide-negative cells,and represent the mean T SEM of at least four independent experiments.Statistical analysis was assessed using two-way ANOV A (*p <0.05).D.Pe ´trin et al./Cellular Signalling 18(2006)479–487483As the PI3-K inhibitor affected the effect of LTB 4on neutrophil apoptosis (Fig.2B),we studied the effect of wortmannin on the expression of the anti-apoptotic protein Mcl-1.A preincubation with wortmannin (1A M)inhibited the increase in Mcl-1expression induced by LTB 4.Because the inhibition of the MEK/ERK pathway also influenced thedelay of neutrophil apoptosis induced by LTB 4,we testedwhether the specific MEK inhibitor had an effect on Mcl-1protein expression.Similarly to the results obtained with wortmannin,PD98059(10A M)inhibited Mcl-1protein expression induced by LTB 4(Fig.4D).Hence,both PI3-kinase and ERK,via this effect on Mcl-1expression,may participate in the LTB 4signaling pathway leading to a delay in spontaneous neutrophilapoptosis.Fig.5.Downregulation in Bax mRNA and protein expression.(A)PMN were stimulated with LTB 4(100nM)or ethanol (control)for 1,3or 6h.RNA was extracted,reverse transcribed,and complementary DNA was used for real-time PCR amplification,performed in triplicates.The triplicate data were averaged before normalization over GAPDH,and controls were set to 1.Results are expressed as mean T SEM ratios of four separate experiments.Logged data were subject to statistical analysis using Student’s t tests (*p <0.05).(B)FACS analysis of PMN incubated in presence of LTB 4(100nM)or ethanol (control)for 24h.Cells were then stained with an isotype control Ab or anti-Bax Ab,followed by a secondary FITC-conjugated anti-rabbit Ab.Results are illustrated as mean T SEM percent of Bax-positive cells for five separate experiments.Statistical analysis was determined using Student’s t test (*p <0.05).(C)PMN were preincubated for 1h with 1A M wortmannin,10A M PD98059,or DMSO prior to LTB 4(100nM)treatments for an additional 23h.Flow cytometry was performed as in B.Results are presented as mean T SEM of five separate experiments.Statistical analysis was determined using two-way ANOV A (*p <0.05).Fig.4.Induction of Mcl-1mRNA and protein expression by LTB 4.(A)Human neutrophils were incubated for 1,3,or 6h in the presence of LTB 4(100nM)or ethanol (control).Following RNA extraction and reverse transcription,complementary DNA was amplified and analyzed by real-time PCR.Reactions were performed in triplicates,and the averages of the replicates were normalized over GAPDH.Controls were set to 1.Results are presented as mean T SEM ratios for at least 5independent experiments.(B)Representative western blot analysis of Mcl-1from freshly isolated PMN (fresh),or PMN treated with medium,GM-CSF (10ng/ml),ethanol,or LTB 4(100nM)for 24h.Equivalent loading was verified with vinculin.(C)Cells were incubated for 1h in the presence or absence of wortmannin (1A M),or (D)PD98059(10A M).PMN were then incubated with LTB 4(100nM)for an additional 23h prior to cell lysis and Western blot analysis.Illustrations are representative of three independent experiments.D.Pe ´trin et al./Cellular Signalling 18(2006)479–4874843.5.Effect of LTB4on BaxThe balance between pro-and anti-apoptotic members of the Bcl-2family is thought to control neutrophil fate.For this reason,we investigated whether the pro-apoptotic protein Bax could be modulated by LTB4treatments.First, Bax mRNA expression was examined using real-time PCR. In contrast to Mcl-1mRNA,Bax mRNA was diminished following LTB4treatments,reaching a maximum of4-fold reduction at3h(Fig.5A).Down-regulation of Bax mRNA was accompanied by a decrease of the protein expression,as observed by flow cytometric analyses(Fig.5B).Moreover, wortmannin(1A M)as well as PD98059(10A M)reversed the decreased expression of Bax induced by LTB4(Fig.5C). These results indicate that PI3-K and MEK are also involved in the modulation of Bax expression by LTB4. 4.DiscussionMature PMN are terminally differentiated cells that rapidly undergo spontaneous apoptosis.However,their progression through the cell death program can be delayed by LTB4,a powerful inflammatory mediator[3–5].Upon binding to its heptahelical,G-protein-coupled receptors, LTB4stimulates a number of cellular functions,including cell adhesion to vascular endothelium,transmigration, oxygen radical generation,and degranulation,among others [10,44–46].The present study showed that LTB4reduced sponta-neous apoptosis within10h of incubation.Our group has previously reported that the delay in PMN apoptosis induced by LTB4was still present after48h,although the percentage of cell death could reach60%in presence of LTB4[5].Moreover,U75302and LY255283,BLTR antagonists,effectively blocked this effect.Here,using pharmacological inhibitors of signal transduction,we further examined the mechanisms underlying the anti-apoptotic effect of LTB4in human neutrophils.PI3-K figures among the signaling molecules that are often associated with the regulation of cell survival.For example,it was previously reported that GM-CSF delays neutrophil constitutive apoptosis,in part,through the PI3-K pathway[29].In our study,we observed that inhibition of this kinase reversed the effect of LTB4on neutrophil apoptosis.Although wortmannin is a potent PI3-K inhibitor, it is not isoform-specific.However,data from PI3-K g knockout mice suggest that PI3-K g is the sole isoform that is coupled to GPCRs in neutrophils[47–49].PI3-K g isoform is known to be activated primarily by the h g subunits of G proteins[22].Accordingly,our results revealed that the delay in PMN apoptosis following LTB4 treatments is dependent on PTX-sensitive G proteins.This observation is in agreement with the fact that most actions of LTB4are generated via PTX-sensitive G proteins[4,19].G a i subunits may presumably be also involved here,as other PTX-sensitive G-proteins such as G o and G t,which are considered cell-specific,have not yet been identified in neutrophils.We found that inhibition of MEK significantly attenuated the effect of LTB4in annexin V binding studies.This observation is consistent with earlier reports of ERK activity in response to LTB4and of ERK involvement in the regulation of neutrophil apoptosis[3,29,50].Combined PI3-K and MEK inhibition did not show an additive effect when compared to either inhibitor alone.This possibly suggests that LTB4promotes an anti-apoptotic response that requires sequential action of MEK/ERK and PI3-K.Src family kinases could have been possible intermediates linking PI3-K to MEK/ERK activation[27],but the src-family protein tyrosine kinase inhibitor PP1did not attenuate the anti-apoptotic effect of LTB4.Some protein kinase C(PCK) isoforms could possibly act as intermediates here[24],but prolonged incubations in presence of general PKC inhibitors (H7and GF109203x)severely altered the phenotype of PMN(D.Pe´trin,unpublished data)and thus this possibility could not be properly assessed.The functional significance of p38MAPK in neutrophils is still debated.Recent reports suggest that p38MAPK signals survival in human neutrophils[31,51].Other groups established that p38MAPK activation was required for constitutive neutrophil apoptosis[52].Here,we show that the serine/threonine p38kinase played no role in the anti-apoptotic effect of LTB4,supporting the idea that p38 activation does not necessarily affect to apoptosis.The anti-apoptotic protein Mcl-1plays a central position in the survival of neutrophils[32,33].Some studies have shown that STAT3was necessary for the regulation of expression of Mcl-1,and the inhibition of Jak/STAT pathway by AG490resulted in apoptotic cell death in different cell types[34,53].Therefore,we tested the effect of AG490on PMN apoptosis,even though BLTRs have not, as yet,been reported to activate this pathway.We found that LTB4utilizes a different pathway to mediate its effect on neutrophils,since AG490did not attenuate the delay in apoptosis induced by LTB4.However,we demonstrated that LTB4has the ability to upregulate Mcl-1expression. Additionally,Mcl-1protein upregulation was prevented by inhibition of PI3-K and MEK kinases.The upregulation of mRNA expression indicates that the increase in Mcl-1 protein is,at least partially,translational,although increased Mcl-1protein stability,as it has been shown with GM-CSF [54],cannot be ruled out from our experiments.In parallel,we found that in LTB4-treated PMN,Bax mRNA and protein were diminished.Bax protein down-regulation was also prevented by the use of PI3-K and MEK inhibitors.The presence of both Bax and Mcl-1in PMN has been proposed as an explanation for the constitutive neutrophil apoptosis and for the ability of different agents to prolong PMN lifespan[32].As soon as Mcl-1protein expression is decreased,endogenously expressed Bax has the possibility to dominate and lead to apoptosis.Overall,D.Pe´trin et al./Cellular Signalling18(2006)479–487485。