Mitochondria in the centre of human eosinophil apoptosis and survival

In . J. Mol. Sci. 2014, 15, 3952-3969; doi:10.3390/ijms15033952 In e na ional Jou nal o Molecula Sciences ISSN 1422-0067 www.mdpi.com/jou nal/ijms Re iew Mi ochond ia in he Cen e o Human Eosinophil Apop osis and Su i al Pinja Ilma inen 1,*, Ee a Moilanen 1 and Hannu Kankaan an a 1,2 1 The Immunopha macology Resea ch G oup, Uni e si y o Tampe e School o Medicine and Tampe e Uni e si y Hospi al, Tampe e FI-33014, Finland; E-Mails: [email p o ec ed] (E.M.); [email p o ec ed] (H.K.) 2 Depa men o Respi a o y Medicine, Seinäjoki Cen al Hospi al, Seinäjoki, Finland and Uni e si y o Tampe e, Tampe e FI-60220, Finland *Au ho o whom co espondence should be add essed; E-Mail: [email p o ec ed]. Recei ed: 17 Janua y 2014; in e ised o m: 20 Feb ua y 2014 / Accep ed: 26 Feb ua y 2014 / Published: 5 Ma ch 2014 Abs ac : Eosinophils a e abundan ly p esen in mos pheno ypes o as hma and hey con ibu e o he main enance and exace ba ions o he disease. Regula o s o eosinophil longe i y play c i ical oles in de e mining whe he eosinophils accumula e in o he ai ways o as hma ics. Se e al cy okines enhance eosinophil su i al p omo ing eosinophilic ai way in lamma ion while o example glucoco icoids, he mos impo an an i-in lamma o y d ugs used o ea as hma, p omo e he in insic pa hway o eosinophil apop osis and by his mechanism con ibu e o he esolu ion o eosinophilic ai way in lamma ion. Mi ochond ia seem o play cen al oles in bo h in insic mi ochond ion-cen e ed and ex insic ecep o -media ed pa hways o apop osis in eosinophils. Mi ochond ia may also be impo an o su i al signalling. In addi ion o glucoco icoids, ano he impo an agen ha egula es human eosinophil longe i y ia mi ochond ial ou e is ni ic oxide, which is p esen in inc eased amoun s in he ai ways o as hma ics. Ni ic oxide seems o be able o igge bo h su i al and apop osis in eosinophils. This e iew discusses he cu en e idence o he mechanisms o induced eosinophil apop osis and su i al ocusing on he ole o mi ochond ia and clinically ele an s imulan s, such as glucoco icoids and ni ic oxide. Keywo ds: as hma; eosinophil; apop osis; su i al; mi ochond ial pe meabili y ansi ion; eac i e oxygen species; mi ochond ia; glucoco icoids; ni ic oxide; mi ogen-ac i a ed p o ein kinase OPEN ACCESS In . J. Mol. Sci. 2014, 15 3953 1. In oduc ion Eosinophils a e cells o he inna e immune sys em in ol ed in he pa hogenesis o alle gic, gas oin es inal and hype eosinophilic diso de s, in an i-pa asi ic de ence and in umo immuni y [1–5]. Eosinophils accoun only o app oxima ely 3% o blood leukocy es in heal hy indi iduals bu hei numbe is ele a ed in subjec s wi h eosinophilic condi ions such as alle gic as hma. Howe e , sho ages o cells o en hampe s udies on eosinophils. In as hma ic ai ways, eosinophils a e d i en in o an ac i a ed s a e e.g., by p o-in lamma o y cy okines such as IL-5. When ac i a ed, hey elease oxic and p o-in lamma o y media o s able o induce b onchocons ic ion, mucus hype sec e ion, and damage o ai way epi helium as well as con ibu e o T helpe 2 cell pola iza ion and ai way hickening [6–8]. Recen da a om clinical s udies wi h an i-IL-5 an ibody and eosinophil-de icien mice sugges ha eosinophils a e impo an o as hma exace ba ions and ai way emodelling. An i-IL-5 ea men led o dec eased exace ba ion a e in pa ien s wi h se e e eosinophilic as hma and hese pa ien s we e able o educe hei glucoco icoid dose in esponse o an i-IL-5 ea men [9,10]. The eby, eosinophils a e impo an o main enance and exace ba ions o as hma [11]. In heal hy indi iduals eosinophils a e sho -li ing cells. In in i o condi ions, blood eosinophils unde go spon aneous apop osis in a ew days bu in a physiological si ua ion hey end o mig a e and accumula e in o li e and spleen, whe e hey a e likely o li e longe han a ew days [12–15]. Apop osis o eosinophils can be delayed o accele a ed by a ious agen s [14,15]. Eosinophil longe i y may be enhanced up o 1–2 weeks by p o-in lamma o y cy okines such as IL-5, IL-3 and GM-CSF p esen in in lamed ai ways [16]. Indeed, blood and issue eosinophils om pa ien s wi h as hma ha e been shown o li e longe when compa ed o eosinophils om heal hy indi iduals [17,18]. Eosinophil emo al om he ai ways is bene icial o educe eosinophilic in lamma ion and alle ia e symp oms o as hma [19]. Apop osis is a non-in lamma o y way o cell dea h comp ising a bene icial means o cell emo al. Memb ane in eg i y is e ained h oughou he p ocess and he ha m ul cell con en main ained inside he cell. The immunological silence o apop osis is ensu ed by o ma ion o smalle apop o ic bodies ha a e apidly inges ed by phagocy es. Apop osis may be execu ed ia wo di e en main ou es, ex insic ( ecep o -media ed) o in insic (mi ochond ion-cen e ed) pa hway [20]. Ex insic pa hway is ac i a ed e.g., by liga ion o he dea h ecep o Fas/CD95. This leads o o ma ion o a mul ip o ein complex called dea h-inducing signalling complex (DISC) ha egula es ac i a ion o ini ia o caspase-8. Ini ia o caspase-8 may ei he di ec ly ac i a e e ec o caspases ha execu e apop osis o clea e BH3-in e ac ing-domain dea h agonis (Bid) esul ing in ac i a ion o an addi ional mi ochond ial loop. In insic pa hway can be ini ia ed by se e al in acellula s ess condi ions such as DNA damage, oxida i e s ess and cy osolic Ca2+ o e load. Membe s o he Bcl-2 amily a e c i ical in moni o ing in acellula damage and aim o media e ac i a ion o po e- o ming Bax and he ollowing mi ochond ial memb ane pe meabiliza ion (MMP), a cen al e en in apop osis [20,21]. Mi ochond ial memb ane pe meabiliza ion can also be media ed ia mi ochond ial pe meabili y ansi ion (mPT) [21,22]. MMP esul s in loss o mi ochond ial memb ane po en ial (∆Ψm), hal ed mi ochond ial ATP syn hesis and elease o p o-apop o ic p o eins such as cy och ome c o he cy osol. Cy och ome c s imula es o ma ion o he apop osome, a pla o m ha ac i a es ini ia o caspase-9 [20,21]. Ini ia o In . J. Mol. Sci. 2014, 15 3954 caspase 9 ac i a es e ec o caspases 3, 6 and 7 esul ing in deg ada ion o cellula componen s and apop osis. Eosinophil apop osis can be accele a ed by physiological ac o s such as Fas ac i a ion [23]. Fas ligand is a signi ican p o-apop o ic agen o eosinophils in i o because i s neu aliza ion enhanced ai way eosinophilia in a mouse model o alle gic as hma [24]. NO is p oduced in high amoun s in he lungs o as hma ics and has been shown o egula e eosinophil apop osis in a complex manne . NO has shown bo h an i- and p o-apop o ic e ec s on eosinophils [25–27] and bo h enhancing and educing p ope ies ega ding lung eosinophilia [28–30]. The eby, he ne e ec in esponse o NO can be di e en in di e en pa hophysiological si ua ions and is no known a he momen . Also many an i-as hma ic agen s such as glucoco icoids, heophylline and cys einyl leuko iene ecep o an agonis s enhance eosinophil apop osis in he absence and p esence o eosinophil su i al-p olonging cy okines [31–36] and he p o-apop o ic e ec s o hese d ugs may con ibu e o hei clinical e icacy [37–42]. An i-in lamma o y glucoco icoid medica ion is he co ne s one in he ea men o as hma and unde s anding i s ac ions is o c i ical impo ance. Glucoco icoids modula e longe i y o many immune cell ypes and he sensi i i y o glucoco icoid-induced cell dea h depends on he cell ype. Fo example, CD4+ T cells bu no CD8+ T cells a e sensi i e o glucoco icoid-induced apop osis [43–45]. In con as , glucoco icoids inhibi ed neu ophil apop osis [31,46–48], e en hough no in an en i onmen wi h se e e hypoxia [49]. In eosinophils, glucoco icoids accele a e apop osis [31–33]. Unde s anding he signalling ela ed o eosinophil su i al and apop osis is ex emely impo an o unde s anding he pa hogenesis o eosinophilic in lamma ion and o he de elopmen o no el d ugs o ea diseases associa ed wi h eosinophilia. S udies using p ima y human eosinophils a e hampe ed by se e al issues. Low numbe s o cells a ailable o he s udies es ic s ca ying ou expe imen s wi h long- ime se ies and di e en ea men s. Addi ionally, he sho li espan o eosinophils excludes use o mos mode n molecula biology me hods such as ans ec ion and RNA in e e ence. Because o he es ic ions, knowledge o p ima y human eosinophil unc ions is based mainly on he use o pha macological inhibi o s and me hods a ailable o di ec measu emen s o cellula unc ions and in acellula media o s. In eosinophils, mi ochond ia play a cen al ole in apop osis and su i al [50]. Mi ochond ial e en s ha e been shown o be c i ical o spon aneous, glucoco icoid-, ni ic oxide- and an i-Fas-induced apop osis o eosinophils [51–54] and he eby media e bo h ex insic and in insic o ms o eosinophil apop osis. Mi ochond ia a e o ums whe e p o- and an i-apop o ic signals me ge and he a e o he cell is de e mined. Because o he e iden impo ance o his cell o gan o eosinophil su i al and dea h, his e iew concen a es on discussing he mechanisms o eosinophil apop osis and su i al ocusing on he ole o mi ochond ia and he clinically ele an p o-apop o ic s imulan glucoco icoid. 2. Mi ochond ia and Bcl-2 Family Membe s In mos cells, mi ochond ia unc ion as “ene gy ac o ies” p oducing ATP ia unc ion o he elec on anspo chain main aining mi ochond ial memb ane po en ial (∆Ψm). Eosinophils ha e been sugges ed o con ain only a low numbe o mi ochond ia, 24–36 pe cells [50] bu his esul needs o be con i med by cu en echniques. I has also been epo ed ha eosinophils main ain mi ochond ial In . J. Mol. Sci. 2014, 15 3955 memb ane po en ial a he by hyd olysis o ATP han ia espi a o y chain [50], e en hough e idence also exis s o unc ional espi a o y chains in eosinophils [55]. I is clea ha eosinophil mi ochond ia a e able o elease cy och ome c om he in e memb ane space and by his mechanism ac i a e caspases and induce apop osis [50]. 2.1. Bcl-2 Membe s and Po e-Fo ming Ac i i y o Bax and Bid Membe s o he Bcl-2 amily a e c i ical in moni o ing in acellula damage and he balance be ween an i-apop o ic and p o-apop o ic Bcl-2 membe s ( a io o p o-apop o ic Bax o an i-apop o ic Bcl-2) de e mines he suscep ibili y o cells o apop osis. Sho -li ing g anulocy es ha e high a ios o Bax/Bcl-2 while he co esponding a ios in monocy es and lymphocy es a e ela i ely low. This a io de e mined he suscep ibili y o leukocy es o an i-Fas-induced apop osis, g anulocy es being he mos suscep ible and lymphocy es he leas suscep ible [56]. Eosinophils exp ess high le els o p o-apop o ic Bid and Bax, which a e p o eins capable o oligome ize and o m po es o he mi ochond ial ou e memb ane [53,54,57,58]. Bid is p ocessed du ing spon aneous apop osis and in enhanced manne du ing glucoco icoid- and an i-Fas-induced apop osis bu i is he mos c i ical media o o Fas-induced apop osis [53,54]. Also Bax was demons a ed o spon aneously ansloca e in o mi ochond ia in un ea ed eosinophils and in an accele a ed manne in esponse o glucoco icoid ea men [51,52]. Pep idyl-p olyl isome ase Pin1 may be he key egula o o Bax ansloca ion. Bax ansloca ion in o mi ochond ia was p e en ed by GM-CSF, which ac i a ed ERK1/2 o phospho yla e he h eonine esidue o Bax. This phospho yla ion enabled in e ac ion o Pin1 wi h Bax p e en ing i s mi ochond ial a ge ing [58]. Pin1 was also shown o be a key egula o o apop osis induced by an i-Fas ia he Fas-associa ed dea h domain (FADD) in ac i a ed eosinophils. In he p esence o su i al-p olonging IL-5, Pin1 quenched phospho yla ion o FADD a Se 194 and p e en ed apop osis [59]. These mechanisms may be highly impo an in de e mining whe he alle gic in lamma ion is con inued by IL-5/GM-CSF o diminished by Fas ecep o signalling. An i-apop o ic Mcl-1 has been ound in eosinophils and was deg aded du ing spon aneous apop osis and in an accele a ed manne du ing apop osis induced by glucoco icoids o by an inhibi o o cyclin-dependen kinase [57,60–63]. In HeLa cells, Mcl-1 was demons a ed o inhibi Bax downs eam o i s mi ochond ial ansloca ion. Mcl-1 p e en ed o ma ion o Bax oligome s a mi ochond ia, equi ed o po e o ma ion, bu his inhibi ion equi ed no di ec in e ac ion [64]. The eby, deg ada ion o Mcl-1 in apop o ic eosinophils p obably enables po e- o ming ac i i y o Bax and mi ochond ial ou e memb ane pe meabiliza ion. The mechanism o Mcl-1 deg ada ion accele a ed by glucoco icoids emains unclea bu may o example in ol e ansc ip ion o p o eins in ol ed in he deg ada ion [63]. In neu ophils, glucoco icoids, in con as o eosinophils, induced Mcl-1 exp ession, which may play an impo an ole in he mechanism o p olonga ion o neu ophil su i al [65]. Con adic ions exis conce ning he exp ession o an i-apop o ic Bcl-2 in eosinophils; exp ession seems o depend on he o igin o he eosinophils [39,57,60,66]. 2.2. Mi ochond ial Pe meabili y T ansi ion Mi ochond ial pe meabili y ansi ion (mPT) is one mechanism o he mi ochond ial memb ane pe meabiliza ion. Du ing mPT, pe meabili y o he inne mi ochond ial memb ane is inc eased o In . J. Mol. Sci. 2014, 15 3956 solu es and molecules up o 1.5 kDa. A channel sensi i e o Ca2+, oxidan s and p o-apop o ic Bcl-2 amily membe s is esponsible o his phenomenon [21,22]. Mi ochond ial pe meabili y ansi ion esul s in mi ochond ial ma ix swelling, mos likely due o he in lux o ions ha a e accompanied by wa e . The mi ochond ial ou e memb ane is up u ed due o ma ix swelling and apop osis-inducing p o eins a e eleased o he cy osol [67]. The mPT channel is hough o be a mul ip o ein complex bu i s molecula s uc u e is s ill unknown. Glucoco icoids and ni ic oxide induced apop osis in eosinophils ha was media ed by mPT [68,69]. Howe e , mPT had no c i ical ole in media ing spon aneous o an i-Fas-induced apop osis [23]. Wha de ines he mechanism o mi ochond ial memb ane pe meabiliza ion in esponse o di e en p o-apop o ic s imulan s in eosinophils emains unclea . Oxidan s a e gene ally impo an media o s o eosinophil apop osis and known induce s o mPT bu do no , howe e , always media e mPT. I seems ha ROS ha e se e al mechanisms o media e apop osis, one o which is s imula ion o mPT. Fo example, Bid has been shown o engage a ROS-dependen bu mPT-independen mechanism o mi ochond ial memb ane pe meabiliza ion and cy och ome c elease [70]. Mi ochond ial pe meabili y ansi ion may unc ion in wo di e en modes. In addi ion o he i e e sible sus ained opening o he mPT channel occu ing du ing cell dea h, he channel may also luc ua e be ween open and closed s a es ( licke ) [71,72]. In NO- ea ed eosinophils, ea ly licke ing mPT p eceded pe manen mPT and mPT-dependen eosinophil apop osis [68]. The ea ly licke ing mPT was no necessa y o apop osis o p oceed and may ac ually ep esen a cell su i al mechanism [73,74]. I has been demons a ed ha licke ing mPT may ac as a mechanism o elease ROS o calcium [75–77] and by hese mechanisms, licke ing mPT may pa icipa e in cell signalling. Fo example, in NO- ea ed eosinophils licke ing mPT media ed ac i a ion o JNK [68]. 3. Reac i e Oxygen Species (ROS) and P o-Apop o ic Signalling Pa hways Reac i e oxygen species such as supe oxide O2•− and hyd ogen pe oxide H2O2 we e demons a ed o media e induced eosinophil apop osis as well as spon aneous eosinophil apop osis [52,68,78–81]. In mos issues, he mi ochond ial elec on anspo chain and especially he complexes I and III se e as he p ima y sou ce o supe oxide (O2•−) e en hough O2•− can also be gene a ed by unc ion o NADPH oxidase o xan hine oxidase in ce ain immune cells such as eosinophils, neu ophils and mac ophages ollowing hei ac i a ion [82]. App oxima ely i e- o en- old highe s eady s a e concen a ions o O2•− exis in he mi ochond ial ma ix when compa ed o he cy osol, acco ding o one es ima ion [83]. Mi ochond ia ha e a di e se an ioxidan de ence sys em including supe oxide dismu ases (SODs such as Manganese (Mn)-con aining SOD), glu a hione, glu a hione pe oxidase, ca alase, pe oxi edoxins e c. In he p esence o supe oxide dismu ase (SOD), O2•− is con e ed in o a mo e s able non- adical oxidan , hyd ogen pe oxide (H2O2) ha may also unc ion as a signalling molecule [84]. Ca alase unc ions by decomposing H2O2 o oxygen and wa e . 3.1. ROS H2O2 has been shown o induce eosinophil apop osis and ca alase has been demons a ed o dec ease spon aneous eosinophil apop osis [55,85]. Eosinophil apop osis induced by exc e o y-sec e o y p oduc s om helmin h was associa ed wi h inc eased le els o H2O2 bu no In . J. Mol. Sci. 2014, 15 3957 supe oxide and e e sed by ca alase bu no by mime ic o supe oxide dismu ase (SOD). Inc eased H2O2 p eceded mi ochond ial inju y [86]. I is possible ha H2O2 o en ac s as he ac ual media o o eosinophil apop osis ins ead o O2•−. H2O2-induced apop osis also equi ed p oduc s o he mi ochond ial espi a o y chain because inhibi ion o mi ochond ial espi a ion by o enone dec eased H2O2-induced eosinophil apop osis [55]. In hymocy es, glucoco icoids induced p oduc ion o H2O2 and o e exp ession o ca alase in hese cells esul ed in hei esis ance o glucoco icoid-induced apop osis suppo ing a media o ole o H2O2 in he p ocess [87,88]. In eosinophils, e idence exis s also o he impo an ole o supe oxide i sel as he media o o apop osis. Glucoco icoids inc eased le els o supe oxide in eosinophils a e 24 h o ea men [52]. Spon aneous apop osis as well as glucoco icoid-induced apop osis we e associa ed wi h dec eased le el o he mi ochond ial an ioxidan MnSOD bu no ha o he cy osolic an ioxidan CuZnSOD a 24 h [52]. Dec eased le el o MnSOD would lead o inc eased le els o supe oxide adical and dec eased o ma ion o H2O2. Also, ni ic oxide-induced eosinophil apop osis was educed by a SOD mime ic, sugges ing ha supe oxide is an impo an media o [68]. Why a e he le els o ROS ele a ed when eosinophils a e on hei way owa ds apop osis? Gene ally, i is hough ha mos o en inc eased le els o ROS esul om comp omised an ioxidan capaci y a he han inc eased p oduc ion o supe oxide [82,89]. Indeed, as discussed abo e, glucoco icoids we e shown o dec ease le els o MnSOD in eosinophils [52]. The mechanism o glucoco icoid-induced enhancemen o ROS has also been s udied in many cell ypes o he han eosinophils. In hymocy es, glucoco icoid-induced p oduc ion o H2O2 was dependen on complex III o he mi ochond ial espi a o y chain and hese e en s media ed glucoco icoid-induced apop osis. In isola ed mi ochond ia, glucoco icoids ha e also been shown o inhibi membe s o he mi ochond ial espi a o y chain [90,91]. In neu al s em cells, ea men wi h dexame hasone esul ed in down- egula ion o 72% o he in es iga ed genes in ol ed in he mi ochond ial espi a o y chain, as well as 29% o he genes encoding o an ioxidan enzymes [92]. Al oge he , he sou ce o ROS p oduc ion may a y depending on he p o-apop o ic s imulan . Glucoco icoids may ele a e ROS by di ec ly modula ing unc ion o he enzymes in he mi ochond ial elec on anspo chain as well as by ansc ip ional egula ion o an ioxidan o espi a o y chain enzymes. ROS a e signi ican media o s o eosinophil apop osis induced by many p o-apop o ic agen s bu how do hey ac ually media e eosinophil cell dea h? A some poin , excessi e mi ochond ial ROS may each a h eshold ha leads o mPT po e opening esul ing in ROS elease o he cy osol [76]. In he cy osol, ROS may ac i a e se e al p o ein kinases as discussed below. Acco ding o he hypo hesis o Zo o e al. he eleased ROS migh igge a simila phenomenon in neighbou ing mi ochond ia and lead o ampli ied oxida i e s ess signals, mi ochond ial inju y and possibly cell dea h [76]. Al e na i ely, a mPT-independen mechanism was demons a ed whe e ROS was equi ed o mi ochond ial memb ane pe meabiliza ion and cy och ome c elease induced by Bid [70]. 3.2. Kinases Ac i a ed by ROS Mi ogen-ac i a ed p o ein kinases (MAPKs) a e se ine/ h eonine kinases mainly ac i a ed by bac e ial p oduc s, p oin lamma o y cy okines, g ow h ac o s and en i onmen al s ess. MAPK amily consis s o c-jun N- e minal kinase (JNK) 1–3, ex acellula egula ed kinase (ERK) 1/2, 3, 5 and 7, In . J. Mol. Sci. 2014, 15 3958 and p38 kinases. A phospho yla ion cascade conduc ed by MAPK kinase kinases (MAP3K) and MAPK kinases (MAP2K) leads o ac i a ion o MAPK. MAPKs phospho yla e ansc ip ion ac o s esul ing in ansc ip ion o genes in ol ed in apop osis, su i al, p oli e a ion and di e en ia ion. Addi ionally, MAPKs a ec he unc ion o nume ous o he p o eins ia phospho yla ion. MAPKs a e inac i a ed by phosphop o ein phospha ases (MAPK phospha ases (MKPs)) [93–95]. Ge minal cen e kinases (GSK) such as mammalian s e ile 20-like kinase (Ms ) 1 a e also ac i a o s o MAPK pa hways and a leas some amily membe s unc ion by ac ing as MAPK kinase kinase kinases (MAP4K) [96]. 3.2.1. JNK ROS a e known ac i a o s o MAPKs JNK, p38 and ERK 1/2, known o egula e cell su i al and dea h pa hways [97]. O hese MAPKs, JNK has been shown o media e glucoco icoid-induced eosinophil apop osis as well as spon aneous apop osis, apop osis induced by ni ic oxide and se e al d ugs [27,52,68,98–101]. Ac i a ion o JNK by ROS seems o be indi ec . E en hough no shown in eosinophils, he ac ual a ge s o oxidan s such as MAP3K (MEKK1) and apop osis signal- egula ing kinase 1 (ASK1 o MAP3K5) eside ups eam o JNK, and lead o JNK ac i a ion ia unc ion o MAP2K [97]. In addi ion, pe oxyni i e led o ac i a ion o JNK in eosinophils bu was ound o equi e Fas. Fas was demons a ed o be a di ec oxida ion a ge o eac i e ni ogen species (RNS) bu he mechanism o JNK ac i a ion ia Fas emains unclea [102]. Kine ics o JNK ac i a ion seems o be an impo an de e minan in whe he he ac i a ion leads o su i al o apop osis. Ea ly JNK ac i a ion has been desc ibed o ep esen a s ess esponse esul ing in cell su i al signalling while delayed and sus ained JNK ac i a ion has been ypically ela ed o apop osis [97,103,104]. Indeed, ea ly and s ong JNK ac i a ion was demons a ed as a ea u e p eceding glucoco icoid-induced eosinophil apop osis, as well as apop osis induced by se e al o he ac o s in eosinophils [52,68,101]. Eosinophil apop osis induced by glucoco icoids and ni ic oxide exhibi many simila i ies such as ea ly and la e JNK ac i a ion, mPT and caspase ac i a ion [52,68]. Ea ly JNK ac i a ion induced by ni ic oxide, was howe e , no c i ical o NO-induced apop osis and migh ini ia e a s ess esponse aiming o cell su i al [68]. In he s udy o Ga dai e al., ea ly JNK phospho yla ion s imula ed by glucoco icoids was p e en ed by an ioxidan ea men sugges ing in ol emen o ROS [52]. Fu he mo e, in NO- ea ed eosinophils, ea ly JNK ac i a ion was dependen on pa ial mi ochond ial pe meabili y ansi ion (mPT) [68]. By combining hese esul s wi h indings o Zo o e al. i can be sugges ed ha ea ly JNK ac i a ion in esponse o glucoco icoids could be media ed by mPT s imula ed by ROS and he ollowing elease o ROS o he cy osol [76]. Ac i a ion o JunD by JNK migh p o ide a link o cell su i al [105]. Ins ead, an addi ional la e and sus ained ac i a ion phase o JNK seems o ake place in eosinophils unde going glucoco icoid- and NO-induced apop osis [52,68] and e idence exis s ha he la e phase media es apop osis [68,103,104]. In e es ingly, i was shown, ha ROS oxidizes he inac i a o s o MAPKs, MAP kinase phospha ases (MKPs), he eby inhibi ing hei ac ion and enabling p olonged JNK ac i a ion [106]. I is possible ha only high le els o ROS lead o oxida ion and inac i a ion o MKPs and p olonged JNK ac i a ion while low le els o ROS may no in luence MKP ac i i y esul ing in apid JNK inac i a ion (Figu e 1). This could explain he kine ics o JNK ac i a ion in In . J. Mol. Sci. 2014, 15 3959 eosinophils. In eosinophils, JNK may be mainly in ol ed in egula ing DNA agmen a ion, because i s inhibi ion p e en ed DNA agmen a ion bu no e.g., phospha idylse ine exposu e o mo phological signs in induced apop osis [99,101]. 3.2.2. ERK The e emains some con o e sy whe he ERK has a ole in media ing cy okine-a o ded eosinophil su i al [57,107,108]. Howe e , e idence exis s o i s ole in media ing eosinophil cell dea h e en hough i was no in ol ed in eosinophil apop osis induced by dexame hasone [109]. Ac i a ion o ERK1/2 (bu no ERK5) p eceded H2O2-induced caspase ac i a ion and eosinophil apop osis [55]. Addi ionally, Siglec-8 induced ROS-dependen cell dea h in IL-5- ea ed eosinophils ha was media ed by enhanced ac i a ion o ERK1/2 [81]. Howe e , siglec-8-induced cell dea h ended o be mo e nec o ic han apop o ic. Recen ly, pai ed immunoglobulin-like ecep o A (PIR-A) was demons a ed o d i e eosinophils in o apop osis in he absence o i s supp esso PIR-B. The p o-apop o ic ac i i y o PIR-A was ound o in ol e G b2 associa ion and ERK1/2 phospho yla ion [110]. Fu he mo e, an i-CD30 an ibody induced eosinophil apop osis ha was pa ially p e en ed by inhibi o s o MAP/ex acellula signal- egula ed kinase kinase (MEK) 1 and MEK1/2 ha lie ups eam o ERK1/2 [111]. 3.2.3. p38 In eosinophils, MAPK p38 has been mainly shown o media e su i al a he han apop osis [108,112,113]. Howe e , eosinophil apop osis induced by an i-CD30 an ibody was pa ially p e en ed by SB203580, an inhibi o o p38 [111], sugges ing ha simila ly o JNK and ERK, p38 may also ac as media o o apop osis in ce ain ci cums ances. Whe he ROS we e in ol ed in he ac i a ion o p38, emains o be de e mined. 3.2.4. Ms 1/2 Mammalian s e ile 20-like kinase (Ms ) 1 belonging o he g oup o ge minal cen e kinases (GSKs), is in ol ed in many unc ions o immune cells including apop osis [96]. Release o 36 kDa agmen o Ms 1 co ela ed wi h eosinophil apop osis and was inhibi ed by ca alase and inhibi o o caspases [114], sugges ing ha Ms 1 ac i a ion was dependen on H2O2 and caspases. In emb yonic s em cells, Ms 1 was demons a ed o be in ol ed in ac i a ion o JNK and ch oma in condensa ion du ing apop osis. This e ec was dependen on ups eam ac i a o s o JNK, MAP2K4 and MAP2K7, because when hese kinases we e supp essed, Ms 1 was no able o media e ch oma in condensa ion [115]. In . J. Mol. Sci. 2014, 15 3960 Figu e 1. Hypo he ical model o desc ibe he mechanisms and ou comes o MAPK ac i a ion induced by di e en le els o ROS. I a p o-apop o ic s imulan induces elease o ROS ha is high enough o quench MKP, his may lead o p olonged JNK ac i a ion and apop osis (B); Low le els o ROS may lead o ansien ac i a ion o JNK due o p esence o ac i e MKP and end up in a p o ec i e esponse (A). The speci ic pa hway media ing ac i a ion o Ms 1 is unclea . ROS, eac i e oxygen species; MAP3K, mi ogen-ac i a ed p o ein kinase kinase kinase; MAP2K, mi ogen-ac i a ed p o ein kinase kinase; JNK, c-Jun N- e minal kinase; MKP, MAP kinase phospha ase; Ms 1, mammalian s e ile 20-like kinase 1. 4. Summa y and Conclusions Mi ochond ia a e ex emely cen al in media ing induced eosinophil apop osis and a e in ol ed in many s eps om he ea ly s ess esponse o he decision o he cell o cope o unde go apop osis as well as o he inal loss o mi ochond ial memb ane po en ial. Many clinically ele an induce s o eosinophil apop osis u ilize he in insic pa hway o apop osis and e en he ex insic pa hway s imula ed by Fas ac i a ion in ol es a c i ical mi ochond ial loop. Glucoco icoids and ni ic oxide s imula e an in insic pa hway wi h many simila ea u es in ol ing ROS, ea ly and la e JNK ac i a ion and mPT (Figu e 2). Addi ionally, glucoco icoids accele a e deg ada ion o an i-apop o ic Mcl-1 which migh enable oligome iza ion and po e- o ming ac i i y o Bax. P ocessing o p o-apop o ic Bid in o i s unca ed, po e- o ming agmen is also enhanced by glucoco icoids. S udies suppo ha ROS may ha e a cen al ole in media ing many o hese e en s: JNK ac i a ion, mPT induc ion and Bid-media ed cy och ome c elease. Glucoco icoids dec ease le els o mi ochond ial an ioxidan s in eosinophils, which mos likely enhance hei p o-apop o ic e ec . Unde s anding o hese pa hways in eosinophil apop osis is c i ical o suppo de elopmen o new agen s o ea eosinophilic diso de s such as as hma. Fu he mo e, hese pa hways may also occu in o he immune cells in esponse o glucoco icoids, and inc ease ou unde s anding o he mechanisms behind he di e gen e ec s o glucoco icoids on he longe i y o di e en cell ypes. In . J. Mol. Sci. 2014, 15 3967 78. Wedi, B.; S aede, J.; Wieland, B.; Kapp, A. Eosinophil apop osis is media ed by s imula o s o cellula oxida i e me abolisms and inhibi ed by an ioxidan s: In ol emen o a hiol-sensi i e edox egula ion in eosinophil cell dea h. 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