1Jou nal o Cell Dea h 2014:7
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Jou nal o Cell Dea h
Regula ion o Spon aneous Eosinophil Apop osis—A Neglec ed A ea
o Impo ance
Pinja Ilma inen1, ee a Moilanen1 and hannu Kankaan an a1,2
1The Immunopha macology Resea ch G oup, School o Medicine Uni e si y o Tampe e and Tampe e Uni e si y Hospi al, Tampe e, Finland.
2Depa men o Respi a o y Medicine, Seinäjoki Cen al Hospi al, Seinäjoki, Finland and Uni e si y o Tampe e, Tampe e, Finland.
ABSTRACT: As hma is cha ac e ized by he accumula ion o eosinophils in he ai ways in mos pheno ypes. Eosinophils a e in lamma o y cells ha
equi e an ex e nal su i al-p olonging s imulus such as g anulocy e mac ophage-colony-s imula ing ac o (GM-CSF), in e leukin (IL)-5, o IL-3 o
su i al. In hei absence, eosinophils a e p og ammed o die by spon aneous apop osis in a ew days. Eosinophil apop osis can be accele a ed by Fas liga ion
o by pha macological agen s such as glucoco icoids. E idence exis s o he ele ance o hese su i al-p olonging and p o-apop o ic agen s in he egula-
ion o eosinophilic in lamma ion in in lamed ai ways. Much less is known abou he physiological signi icance and mechanisms o spon aneous eosinophil
apop osis e en hough i o ms he basis o egula ion o eosinophil longe i y by pa hophysiological ac o s and pha macological agen s. This e iew con-
cen a es on discussing he mechanisms o spon aneous eosinophil apop osis compa ed o hose o glucoco icoid- and Fas-induced apop osis. We aim o
answe he ques ion whe he he ex e nal apop o ic s imuli only augmen he ongoing pa hway o spon aneous apop osis o uly ac i a e a speci ic pa hway.
KEYWORDS: as hma, eosinophils, spon aneous apop osis, glucoco icoids, Fas
CITATION: Ilma inen e al. egula ion o Spon aneous eosinophil apop osis—a neglec ed a ea o Impo ance. Jou nal o Cell Dea h 2014:7 1–9 doi:10.4137/JCD.S13588.
RECEIVED: no embe 6, 2013. RESUBMITTED: Decembe 12, 2013. ACCEPTED FOR PUBLICATION: Janua y 5, 2013.
ACADEMIC EDITOR: Ga y Walsh, edi o in Chie
TYPE: e iew
FUNDING: This s udy was inancially suppo ed by he Compe i i e S a e Resea ch Financing o he Expe Responsibili y A ea o Tampe e Uni e si y Hospi al ( Tampe e,
Finland), g an numbe s VTR 15 and VTR 224; Compe i i e Resea ch Funding o Seinäjoki Cen al Hospi al (Seinäjoki, Finland); Tampe e Tube culosis Founda ion
( Tampe e, Finland); and Finnish An i-Tube culosis Associa ion Founda ion (Helsinki, Finland), which is g a e ully acknowledged.
COMPETING INTERESTS: Au ho s disclose no po en ial con lic s o in e es .
COPYRIGHT: © he au ho s, publishe and licensee Libe as Academica Limi ed. This is an open-access a icle dis ibu ed unde he e ms o he C ea i e Commons
CC-BY-NC 3.0 License.
CORRESPONDENCE: pinja.ilma inen@u a. i
In oduc ion
Eosinophilic g anulocy es accoun only o app oxima ely 3% o
blood leukocy es in heal hy indi iduals. Simila o neu ophilic
g anulocy es, hey a e cells specialized o kill pa hogens by he
sec e ion o oxic media o s bu also able o egula e unc ion
o o he immune cells. Al hough e olu iona y, he unc ion
o eosinophils is hough o be he inna e immune esponse
agains pa asi ic helmin hes;1 hey a e also c i ically in ol ed
in he pa hogenesis o alle gic, gas oin es inal, and hype eo-
sinophilic diso de s and in umo immuni y.2–5 Alle gic as hma
is cha ac e ized by he accumula ion o eosinophils in he ai -
ways. The cu en e idence sugges s ha eosinophils a e c i ical
media o s o as hma exace ba ions and ai way emodelling.6–9
The biology o eosinophils di e s om many o he
immune cells o malignan cell lines in hei equi emen o an
ex e nal s imulus o con inua ion o su i al. In he absence
o any such s imulan (eg g anulocy e mac ophage-colony
s imula ing ac o (GM-CSF), in e leukin (IL)-5 o IL-3),
hey die by spon aneous (also e med as “passi e”) apop osis
in a ew days.10 Apop osis is cha ac e ized by cell sh inkage,
nuclea coalescence, ch oma in condensa ion, and DNA ag-
men a ion leading o he o ma ion o apop o ic bodies and in
i o, o hei inges ion by mac ophages o o he phagocy es.
Gene ally, apop osis can be induced ia wo di e en pa h-
ways, ex insic ( ecep o media ed) o in insic (mi ochond ial
cen e ed) (Fig.1).11
A majo i y o he s udies on he egula ion o eosino-
phil apop osis ha e used alle gic as hma as a s a ing poin ,
ie hey ha e ocused on he signi icance and mechanisms o
su i al-p olonging cy okines.10,12,13 Gi en he impo ance o
eosinophils in ce ain pheno ypes o as hma,14,15 his app oach
is e y sensible. Howe e , eosinophils, albei in low numbe s,
Ilma inen e al
2Jou nal o Cell Dea h 2014:7
Eosinophil apop osis can be induced by se e al agen s
acili a ing clea ance o eosinophilic in lamma ion. Eosino-
phil apop osis can be induced eg by liga ion o Fas o by
liga ion o umo nec osis ac o ecep o (TNFR) amily
membe CD30and by many pha macological agen s, such as
glucoco icoids, heophylline, and leuko iene modi ie s.21–27
Plen y o e idence exis s suppo ing he occu ence o s e oid-
induced eosinophil apop osis in he ai ways o s e oid- ea ed
as hma ics.28–31 Many cell ypes o he ai ways, such as b on-
chial epi helial cells, b onchial smoo h muscle cells, ib o-
blas s, Tcells, and eosinophils exp ess Fas ligand (FasL),29,32–34
and in T cells, he exp ession is educed by Th2 cy okines
GM-CSF, IL-5, and IL-4.35 Neu aliza ion o FasL enhanced
ai way eosinophilia in a mouse model o alle gic as hma p o-
iding e idence ha FasL is a ele an p o-apop o ic agen
o eosinophils in i o.36 T ea men wi h an i-Fas mAb was
shown o enhance apop osis o ai way issue eosinophils in
mice. Howe e , his ea men esul ed in agg a a ed ai way
in lamma ion due o cy olysis and p og ession o apop osis
in o seconda y nec osis.37 This emphasizes he impo ance o
e icien phagocy ic clea ance o apop o ic cells.
To comba he eosinophilia associa ed wi h se e al dis-
ease condi ions, unde s anding he signaling pa e ns ela ed
o eosinophil su i al and apop osis is ex emely impo an .
Ideally, a no el pha macological agen aimed speci ically o
deple e eosinophils by inducing eosinophil apop osis could
be a ge ed o co e all he ollowing op ions: (1) inhibi he
ac ion o signaling o su i al-p olonging ac o s, (2) mimic
he ac ion and/o signaling o known ex e nal induce s o
a e also p esen in he blood and issues in heal hy indi iduals.
I has been es ima ed ha he mean u no e o eosinophils
is app oxima ely 2.2×108cells/kg/day. Once in he ci cula-
ion, eosinophils ha e a hal -li e o app oxima ely 8–18h and
a mean blood ansi ime o app oxima ely 25–26h.10,16,17 In
wo ecen s udies, mig a ion o adiolabeled eosinophils was
ollowed in heal hy indi iduals in eal ime. Eosinophils, a e
lea ing he blood ci cula ion, ansi ed h ough he lungs and
accumula ed in he li e and spleen.16,18 E idence was also
a ained ega ding he e-en ance o eosinophils om he
li e o he ci cula ion.16 Howe e , 48h a e injec ion o he
adiolabeled eosinophils, app oxima ely 50% o hem esided
in he li e , 30% in he spleen, and he es in o he o gans.
I is easonable o hypo hesize ha he li e may be he p i-
ma y si e o eosinophil clea ance h ough apop osis, possibly
spon aneous apop osis. No di ec e idence, howe e , exis s
abou he occu ence o spon aneous (o passi e) apop osis in
i o. As s udying he mechanisms o eosinophil su i al and
apop osis in issue samples is la gely impossible, a as majo i y
o eosinophil s udies ha e used eosinophils isola ed om he
pe iphe al blood by he CD16-nega i e selec ion. Indi ec e i-
dence o he appea ance o spon aneous eosinophil apop osis
in blood was ob ained ecen ly in a s udy e alua ing he aging
o cells in blood samples, whe e eosinophils showed a dec ease
in o wa d side sca e (FSC) alues indica i e o smalle
cell size ypical o apop osis.19 Fu he mo e, he occu ence
o blood eosinophil apop osis, e en hough no spon aneous,
was demons a ed in mice in i o a e he adminis a ion o
Siglec-F an ibody.20
EXTRINSIC PATHWAY
Ini ia o caspase-8
Fas ecep o
DISC
E ec o caspases-3,-6,-7
BID
BID
APOPTOSOME
Ini ia o
caspase-9
INTRINSIC PATHWAY
In acellula damage
Bax
mPT po e
Cy och ome c
APOPTOSIS
Bcl-2 amily membe s
E ec o
caspases-3,-6,-7
Mi ochond ial
memb ane
pe meabiliza ion
ENDOG
AIF
Figu e 1. The main ea u es o ex insic and in insic apop osis pa hways. Fas ecep o -media ed pa hway is shown as an example o ex insic apop o ic
pa hway. Ex insic apop osis is ini ia ed by liga ion o dea h ecep o Fas leading o he o ma ion o DISC and ac i a ion o caspases. Some imes, BID
clea age in o unca ed BID ( BID) and mi ochond ial ou e is equi ed o caspase ac i a ion in ex insic apop osis. In acellula s ess condi ions ini ia e
in insic pa hway o apop osis, whe e Bcl-2 amily membe s and MMP play majo oles. MMP can be media ed by po e- o ming ac i i y o Bax and/o
BID o by mPT. I caspases a e inhibi ed, apop osis may be execu ed by apop osis-inducing ac o (AIF) and endonuclease G (ENDOG).
Regula ion o spon aneous eosinophil apop osis
3Jou nal o Cell Dea h 2014:7
a e 48 h o cul u e. The mechanism o CD40L-induced
su i al p olonga ion in ol ed induced exp ession o cellula
inhibi o o apop osis p o eins (cIAPs).71 CD40L- de icien
mice showed dec eased eosinophilic lung in lamma ion
72h bu no 24h a e alle gen challenge72 sugges ing ha
CD40–CD40L in e ac ion a ec s main enance o eosino-
philic ai way in lamma ion. In he absence o any o hese o
o he su i al-p olonging ac o s, eosinophils p oceed in o
spon aneous apop osis.
P og ession o Spon aneous Apop osis
When eosinophils isola ed om human blood a e cul u ed
in he absence o any induce s o inhibi o s o apop osis,
app oxima ely hal o hem unde go spon aneous apop osis in
2days.64,73 We ha e applied se e al me hods o he de e mina-
ion o eosinophil apop osis p o iding basic in o ma ion on he
p og ession o apop osis and he cascade o apop o ic e en s in
spon aneously dying eosinophils. Acco ding o ou combined
da a o e ime, apop o ic alues ob ained wi h Annexin-V/
p opidium iodide double-s aining a e con inuously highe
when compa ed o hose ob ained wi h o he s anda d me h-
ods o apop osis de e mina ion (DNA agmen a ion assay,
mo phological examina ion, DYm
dissipa ion) (Fig.2, unpub-
lished obse a ion). This sugges s ha cell su ace exp ession
o phospha idylse ine (PS) p ecedes many well-known mani-
es a ions o apop osis in eosinophils. Ea ly occu ence o PS
exposu e has been p e iously demons a ed in eosinophils,74
and e idence exis s also o PS exposu e as a caspase-dependen
e en .52,74,75 Mi ochond ial e en s such as cy och ome c elease
and DYm dissipa ion we e shown o occu a e PS exposu e in
eosinophils, which was in con as o lymphocy es.74 I seems
ha he o de o e en s is s imulus dependen as well as cell-
ype dependen .74,76,77 To u he suppo ea ly ime-cou se
o PS exposu e, i was shown ha PS exposu e p eceded cell
sh inkage and DNA agmen a ion in a lymphoma cell line by
using h ee di e en s imulan s o induce apop osis.78 Du ing
he p ocess o apop osis, ea ly appea ance o PS is logical since
PS unc ions as a cell su ace signal o phagocy es o inges
he apop o ic cells and a ac ion o phagocy es and phagocy-
osis may be conside ed as one o he mos impo an e en s
o occu ence o apop osis in a non-in lamma o y ashion.37
Indeed, inhibi ion o PS exposu e du ing apop osis led o mo e
han 50% educ ion in engul men o apop o ic cells.79 How-
e e , om he me hodological poin o iew, i is ecommended
o measu e apop osis using a combina ion o di e en me h-
ods, no solely Annexin-V-assay despi e he ea ly appea ance o
PS in apop o ic cells. All me hodologies o analyze apop osis
ha e hei d awbacks.80,81
Media o s o Spon aneous Eosinophil Apop osis
Bcl-2 membe s and mi ochond ial e en s du ing spon-
aneous eosinophil apop osis. Membe s o Bcl-2 amily a e
c i ical in moni o ing in acellula damage and impo an o
mi ochond ial memb ane pe meabiliza ion (MMP) o occu ,
apop osis such as FasL o glucoco icoids, and (3) enhance he
in insic p o-apop o ic signaling pa hway du ing spon aneous
(passi e) eosinophil dea h. This e iew ocuses on he signaling
o spon aneous eosinophil dea h, a phenomenon ha is la gely
neglec ed, and compa es i wi h he mechanisms o known
induce s o eosinophil apop osis, FasL, and glucoco icoids.
Inhibi o y Signals o Spon aneous Apop osis
Many eosinophil su i al-p olonging in lamma o y agen s,
such as GM-CSF, IL-5 and IL-3, a e p esen in he lungs
o as hma ics, and eosinophil apop osis has been shown
o be educed in he ai way submucosa o pa ien s wi h
s e oid-un ea ed as hma when compa ed o heal hy
con ols.29,38–40 Eosinophil su i al may be p olonged up
o 1–2 weeks in esponse o hese cy okines, and IL-5 is
he mos po en .41 GM-CSF, howe e , seems o be he
main eosinophil su i al-p olonging cy okine in as hma ic
ai ways.39,40 Pa hways ac i a ed by IL-5/GM-CSF include
Lyn/Syk–Ras–Ra -1–ex acellula signal- egula ed kinases
(ERK) 1/2, Jak2-STAT1, and PI3K-Ak in eosinophils.42–49
O hese, he ERK pa hway does no seem o be in ol ed in
he su i al-p olonging ac ion.50,51 In addi ion, inhibi ion o
Bax ansloca ion o mi ochond ia by IL-5 and GM-CSF has
been shown in eosinophils.49,52 TGF-b, in e es ingly, ab o-
ga ed IL-5/GM-CSF-induced eosinophil su i al, and his
mechanism in ol ed inhibi ion o y osine phopsho yla ion o
Jak2, Lyn, and ERK 1/2 as well as inhibi ion o phospho yla-
ion o STAT1 and Ak .53–55
In addi ion, o he signi ican su i al-p olonging ac-
o s seem o exis because delayed apop osis o blood and
nasal polyp issue eosinophils was only pa ly p e en ed by
an i-GM-CSF, an i-IL-5, and/o an i-IL-3 an ibodies.56,57
Many pa hogenic componen s, cy okines (eg TNF-a, lep in,
in e e ons (IFNs)) and alle gens also p olong eosinophil
su i al.10,58–65 Gene ally, NF-kB may be he mos impo an
ansc ip ion ac o media ing eosinophil su i al, as i s inhi-
bi ion u ns eosinophils in o he apop o ic cascade.64,66
TNF-a, which can be p oduced locally by mas cells,
was demons a ed o be an an i-apop o ic ac o o eosino-
phils i NF-kB was no inhibi ed. This e ec was p oposed
o be media ed ia TNF ecep o s, NF-kB, and induc ion o
GM-CSF p oduc ion.60,67 IFN-g is p oduced by T helpe 1
cells, and i s e ec s on eosinophils seem o be complex. IFN-g
inhibi ed IL-3- o IL-5-induced di e en ia ion o eosinophils
om co d blood mononuclea cells68 bu p olonged eosinophil
su i al in i o.69 Lep in is a cy okine ha is mainly p o-
duced by adipocy es o whi e adipose issue wi h he main
unc ion ela ed o inhibi ion o appe i e. Lep in has been
shown o inc ease eosinophil su i al e en hough i emains
unclea whe he he su i al-p olonging concen a ions may
be eached in i o.70
In addi ion, CD40–CD40L in e ac ion has been shown
o p olong eosinophil su i al. F eshly isola ed blood eosino-
phils did no exp ess CD40, bu he exp ession was s ong
Ilma inen e al
4Jou nal o Cell Dea h 2014:7
o Bcl-2 was ound in he lung eosinophils o pa ien s wi h
as hma and child en wi h se e e exace ba ions when compa ed
o eosinophils o heal hy indi iduals o child en wi h mild-
o-mode a e exace ba ions, espec i ely.91–93 An i-apop o ic
Mcl-1L is deg aded du ing spon aneous apop osis and in an
accele a ed manne du ing glucoco icoid-induced apop osis.90
In addi ion o he po e- o ming ac i i y o clea ed Bax
o Bid, MMP can be media ed ia mi ochond ial pe meabil-
i y ansi ion (mPT) po e.11,94 I is a channel o med o he
me ging poin o inne and ou e mi ochond ial memb anes
in esponse o Ca2+, oxidan s, o p o-apop o ic Bcl-2 am-
ily membe s leading o ee passage o solu es and molecules
up o 1.5kDa.94,95 mPT does no seem o be impo an o
spon aneous apop osis o Fas-induced apop osis bu is a c i i-
cal media o o eosinophil apop osis induced by glucoco i-
coids.73,96,97 As discussed abo e, po es o med in he ou e
mi ochond ial memb ane by he clea ed Bax and/o Bid a e
p obably esponsible o he MMP in he pa hways o spon a-
neous and Fas-induced eosinophil apop osis.84,90
Caspases and calpains. Caspases a e cys eine-dependen
aspa a e-speci ic p o eases in ol ed in he execu ion phase o
apop osis and a e u he di ided in o ini ia o s (caspases-8, -9,
and -10) and e ec o s (caspases-3, -6, and -7). Ini ia o caspases
a e syn hesized as inac i e p oenzymes and equi e dime iza-
ion o ac i a ion ha is enabled by pla o ms such as dea h-
inducing signaling complex (DISC) o apop osome. E ec o
caspases occu as inac i e dime s and equi e clea age by ini-
ia o o o he e ec o caspases o become ac i a ed. When
ac i a ed, caspases clea e cellula componen s in o e apep ide
sequences op imal enough o i hei ca aly ic si e.98,99
Eosinophils ha e been shown o exp ess caspases-3, -6,
-7, -8, and -9.52,75,87 Many apop o ic e en s du ing spon ane-
ous o induced eosinophil apop osis a e educed o p e en ed
by pan-caspase inhibi o s sugges ing ha eosinophil apop osis
is media ed by he ac i a ion o he caspase cascade.52,75,96,100
Caspase-9, accoun ed as he ini ia o caspase ac i a ed in
esponse o mi ochond ial apop o ic pa hway, has been shown
o be p ocessed du ing spon aneous and induced apop o-
sis.52,84,101–103 Howe e , i s inhibi ion by Z-LEHD-FMK did
no p e en spon aneous apop osis o FasL-media ed apop-
osis, sugges ing ha i may no unc ion as a c i ical ini-
ia o caspase in hese pa hways.84,101 Howe e , a possibili y
exis s ha he inhibi o used was ine icien , because acco d-
ing o ou da a i inhibi ed only 65% o caspase-9 ac i i y
in eosinophils. I can be suspec ed ha he esidual 35% o
caspase-9 ac i i y was enough o ac i a e e ec o caspases.101
Also caspase-8 ac i i y has been de ec ed in spon aneously
dying eosinophils in some bu no all s udies. Bu simila o
caspase-9, i s inhibi ion did no p e en apop o ic e en s du ing
spon aneous apop osis.52,84,101–103 Al oge he , he ini ia o cas-
pase esponsible o he p oceeding o spon aneous apop osis
is no clea . In neu ophils, ac i a ion o caspase-8 was shown
o be dependen on ini ia o caspase-9104 and may be ac ually
ac i a ed by e ec o caspase-3, as p e iously desc ibed.105 In
especially in he in insic pa hway o apop osis. The Bcl-2 amily
consis s o a g oup o an i-apop o ic p o eins and wo g oups o
p o-apop o ic p o eins.82,83 Because eosinophils unde go apop-
osis qui e apidly, he exp ession o p o eins egula ing lon-
ge i y is balanced owa d p o-apop o ic membe s. Gene ally,
p o-apop o ic Bcl-2 amily membe s Bax and Bid a e s ongly
exp essed in un ea ed human eosinophils.49,84,85 Clea age o
Bax and Bid in o po e- o ming agmen s enables pe meabi-
liza ion o he ou e mi ochond ial memb ane and elease o
cy och ome c. I was shown ha du ing spon aneous eosino-
phil apop osis, Bax is clus e ed and e-localized in o mi ochon-
d ia, independen om caspases, and his leads o he elease o
cy och ome c o he cy osol and ac i a ion o caspases.52,86 An
accele a ed Bax ansloca ion is obse ed in dexame hasone-
ea ed eosinophils.86 Also Bid is p ocessed du ing spon aneous
apop osis and a a as e a e du ing Fas- and glucoco icoid-
induced apop osis.84 Spon aneous, FasL-, and dexame hasone-
media ed eosinophil apop osis we e educed by 30, 50, and
25%, espec i ely, in cul u ed b onchoal eola la age (BAL)
eosinophils om Bid-de icien mice, sugges ing ha Bid has a
lesse ole in spon aneous and glucoco icoid-induced apop o-
sis and is a mo e c i ical media o in FasL-induced apop osis.85
I seems clea ha ex insic (FasL-induced) apop osis equi es
an addi ional mi ochond ial loop in eosinophils.
As expec ed in cells p one o unde go apop osis, he
exp ession o an i-apop o ic Bcl-2 membe s Bcl-2, Bcl-xL,
and Mcl-1L is gene ally low in eosinophils.87–90 Howe e , he
le el o Bcl-2 exp ession seems o depend on he s a us o he
pa ien and o igin o eosinophils, because highe exp ession
Figu e 2. Compa ison o pe cen ages o spon aneous eosinophil
apop osis ob ained by di e en apop osis de e mina ion me hods.
Apop osis was de e mined by Annexin-V FITC/p opidium iodide
double-s aining (Anx, n = 12), Dna agmen a ion assay ca ied ou
by p opidium iodide s aining o pe meabilized eosinophils (PI, n = 56),
mo phological analysis o May-G unwald–Giemsa-s ained eosinophils
(Mo , n = 23), o de e mina ion o mi ochond ial memb ane po en ial by
JC-1 s aining (MMP, n = 5) a e 40 h o incuba ion. Desc ip ions o he
me hods used can be ound a e . 73.
No e: ***Indica es P  0.001 as compa ed o all o he columns by using
anoVa analysis.
Regula ion o spon aneous eosinophil apop osis
5Jou nal o Cell Dea h 2014:7
possible ha loss o mi ochond ial an ioxidan de ence a leas
pa ly explains he inc eased ROS du ing spon aneous and
glucoco icoid-induced eosinophil apop osis.
Mi ogen-ac i a ed p o ein kinases (MAPKs) and
mammalian s e ile 20-like kinase (Ms ). MAPKs a e
se ine/ h eonine kinases mainly ac i a ed by 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 kinases (JNK) 1-3, ERK
1/2, 3, 5 and 7, and p38 amily membe s and a se ial o phos-
pho yla ion cascades 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 unc ion o
nume ous o he p o eins ia phospho yla ion.120,121 JNK has
been p e iously shown o media e apop osis h ough se e al
pa hways: AP-1-media ed ansc ip ion o FasL and TRAIL-
ecep o 1,122,123 phospho yla ion o Bcl-2 amily p o ein
membe s,120,124,125 mPT induc ion,126,127 and phospho yla ion
o his one H2AX equi ed o DNA agmen a ion.128
Some e idence has been ga he ed ega ding he ole o
JNK as a media o o spon aneous eosinophil apop osis, e en
hough esul s a e con adic o y. Spon aneous eosinophil
apop osis was dec eased by a pep ide inhibi o o JNK bu
no by he o he JNK inhibi o s es ed. Fu he mo e, mod-
es o no ac i a ion o JNK and lack o ac i a ion o c-Jun has
been demons a ed in spon aneously dying eosinophils.73,129
Ins ead, JNK was in ol ed in glucoco icoid-induced eosino-
phil apop osis and i s ac i a ion was dependen on oxidan s.86
Indeed, inc eased le el o ROS is one possible gene al ac i-
a ion mechanism o JNK in eosinophils p oceeding owa d
apop osis. Addi ionally, ac i a ion o JNK pa hway has been
p e iously demons a ed o occu in esponse o FasL in lym-
phocy es,130,131 and some e idence poin s o he ole o JNK
in FasL-induced eosinophil apop osis.132 The o he MAP
kinases ERK 1/2 and p38 seem o media e eosinophil su -
i al, no apop osis.49,50 In e es ingly, p38 MAP kinase seems
o be ac i e in isola ed eosinophils, and i s inhibi ion by a
pha macological inhibi o induces apop osis.50
Ms 1 belongs o a g oup o ge minal cen e kinases (GSKs)
ha is in ol ed in many unc ions o immune cells such as a -
icking, p oli e a ion, and apop osis.133 Ms 1 has been shown o
be in ol ed in he ac i a ion o MAPKs such as JNK.134 Cas-
pase-media ed clea age and elease o 36kDa agmen o Ms 1
was demons a ed o co ela e wi h eosinophil apop osis bu no
wi h neu ophil apop osis. Clea age o Ms 1 was inc eased by
FasL and dec eased by IL-5, sugges ing an impo an ole o
his kinase in media ing eosinophil apop osis.135
Summa y and Conclusions
Eosinophil apop osis induced by FasL o glucoco icoids is
a physiologically o clinically ele an mechanism o eosino-
phil clea ance. Plen y o e idence exis abou he clinical el-
e ance o s e oid-induced eosinophil apop osis in he ai ways
o s e oid- ea ed as hma ics.28–31 Mos likely, spon aneous
eosinophils, ac i a ion o bo h ini ia o caspases (8 and 9) has
been de ec ed du ing Fas- and glucoco icoid-induced apop-
osis.84,106,107 The e idence indica es ha caspase-8 unc ions
as he c i ical ini ia o caspase in FasL-media ed eosinophil
apop osis as i s inhibi ion was epo ed o p e en Bid-clea -
age and educe apop osis.84
Ac i a ion o e ec o caspases-3 and -6 seems o be a gen-
e al ea u e o eosinophil apop osis. In ol emen o hese cas-
pases has been ound in spon aneous eosinophil apop osis and
apop osis induced by a ious s imuli.52,75,87,96,103 Lamin deg-
ada ion and DNA agmen a ion a e caspase-6- dependen
e en s in eosinophils, and inhibi ion o caspase-6 delayed o
hal ed apop osis a he le el o ch oma in condensa ion bu
did no p e en apop osis.75 This is consis en wi h he esul s
in o he cell ypes.108–111 Also PS ex e naliza ion was shown
o be pa ly dependen on caspase-6.75 Inhibi ion o caspase-3
pa ially p e en ed DNA agmen a ion in eosinophils.75,96
Calpains a e calcium-ac i a ed (papain-like) neu al p o-
eases ha a e in ol ed in he execu ion o bo h apop osis and
nec osis. A leas 14 iso o ms o calpains exis . Simila o cas-
pases, calpains a e cys eine p o eases; bu in con as o cas-
pases, hey equi e no pa icula amino acid in he subs a e
pep ide sequence. Calpains a e ac i a ed by inc eased in a-
cellula calcium and hei subs a es include X-linked IAP
(XIAP), Bcl-xL, Bid, and p o-caspases-3, -7, -8, and -9.112,113
DNA agmen a ion du ing spon aneous apop osis was
p e en ed by inhibi ion o calpains 1 and 2. Un o una ely,
no in o ma ion exis s on he ole o calpains in FasL- o
glucoco icoid-induced eosinophil apop osis. Howe e , lack o
ole o calpains in ni ic oxide-induced apop osis sugges s ha
calpains a e no ac i a ed in analogous si ua ions o caspases.
Calpains ha e been shown o be in ol ed in he clea age o
Bax in spon aneous eosinophil apop osis. Bax clea age is a
p o-apop o ic e en leading o i s mi ochond ial a ge ing.49
Reac i e oxygen species (ROS). ROS induce apop osis
o human eosinophils and a e o en in ol ed in he mi ochon-
d ial pa hway o apop osis.114,115 Thiol-an ioxidan glu a hi-
one is conside ed o o m he mos impo an an ioxidan
de ence in mi ochond ia.116 Spon aneous eosinophil apop osis
was educed by an ioxidan s ha ele a e in acellula le els
o glu a hione and by hypoxia86,115,117,118 sugges ing a ole o
ROS in media ing spon aneous apop osis and impo ance o
glu a hione in he egula ion o in acellula oxidan le els in
eosinophils. In a simila manne , he an ioxidan s inc eas-
ing glu a hione educed Fas-induced apop osis.115 Also,
glucoco icoid-induced apop osis was p e en ed by mime ic
o supe oxide dismu ase (SOD) and hypoxia, indica ing he
in ol emen o ROS.86 ROS seem o media e also eosinophil
apop osis induced by many o he s imulan s.73,115,119 Howe e ,
he exac mechanism o inc eased oxidan le els emains
unclea . Dec ease in he le els o an impo an mi ochond ial
an ioxidan MnSOD was demons a ed du ing spon aneous
apop osis as well as in glucoco icoid- ea ed cells.86 Le els
o a cy osolic an ioxidan we e no simila ly dec eased. I is

Ilma inen e al
6Jou nal o Cell Dea h 2014:7
DISCLOSURES AND ETHICS
As a equi emen o publica ion he au ho s ha e p o ided signed con i ma ion o hei
compliance wi h e hical and legal obliga ions including bu no limi ed o compliance wi h
ICMJe au ho ship and compe ing in e es s guidelines, ha he a icle is nei he unde
conside a ion o publica ion no published elsewhe e, o hei compliance wi h legal and
e hical guidelines conce ning human and animal esea ch pa icipan s (i applicable), and
ha pe mission has been ob ained o ep oduc ion o any copy igh ed ma e ial. his
a icle was subjec o blind, independen , expe pee e iew. The e iewe s epo ed no
compe ing in e es s. P o enance: he au ho s we e in i ed o submi his pape .
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hough di ec e idence is di icul o ob ain. The signaling
pa hwayo spon aneous apop osis seems o o e lap wi h he
pa hways o  FasL o glucoco icoid-s imula ed apop osis.
A summa yo di e en pa hways is shown in Table1. P e-
mi ochond ial phases o FasL-s imula ed apop osis ha e
unique ea u es such as Fas-associa ed p o ein wi h dea h
domain (FADD) phospho yla ion and ac i a ion o ini ia o
caspase-8. Howe e , mi ochond ion has an impo an ole in
all o hese pa hways o apop osis as sugges ed by common
dependence on mi ochond ial ROS and p ocessing o p o-
apop o ic Bcl-2 amily membe s. The mechanism o MMP
may di e be ween hese pa hways o apop osis. Bax and
Bid seem o be impo an media o s o MMP in eosinophils
unde going spon aneous apop osis, while FasL-s imula ed
apop osis was dependen on Bid. mPT is emphasized du ing
glucoco icoid-induced apop osis. E ec o caspases seem o
be simila ly ac i a ed in all h eeapop o ic ou es. Ac i a ion
o JNK may also be common o hese pa hways o apop osis,
e en hough addi ional e idence is equi ed o add ess i . In
a physiological si ua ion, some le el o mi ochond ial dis up-
ion may al eady be ongoing a he ime he cell encoun e s
a p o-apop o ic s imulan , and he s imulan p obably adds
p o-apop o ic signals ha me ge a he le el o mi ochon-
d ia o augmen , ampli y, and inalize he ongoing p ocess o
(spon aneous) apop osis. The eby, enhancemen o he ongo-
ing spon aneous apop osis is a ele an he apeu ic s a egy o
ea diseases wi h eosinophilic in lamma ion.
Au ho Con ibu ions
PI w o e he i s d a o he manusc ip . HK con ibu ed o he
w i ing o he manusc ip . HK and EM made c i ical e isions.
All au ho s e iewed and app o ed o he inal manusc ip .
Table 1. A summa y o he mechanisms in ol ed in he egula ion o spon aneous eosinophil apop osis when compa ed o ex insic apop osis
induced by as o in insic apop osis induced by glucoco icoids.
FAS-MEDIATED
APOPTOSIS
SPONTANEOUS
APOPTOSIS
APOPTOSIS INDUCED
BY GLUCOCORTICOIDS
REF.
Caspase-3 ++ ++ ++ 52, 75, 87, 96, 101–103
Caspase-6 nD ++ nD 75
Caspase-8 +++ + + 52, 84, 87, 96, 101–103,
106
Caspase-9 + + + 52, 84, 101, 102, 106, 107
Calpains nD ++ nD 49, 75
mP –++++ 73, 96, 97
Bid +++ ++ ++ 84, 106, 85
Bax nD ++ ++ 49, 52, 86, 90
JnK ++ (id) ++?++ 73, 86, 129, 132
oS ++ ++ ++ 73, 86, 115, 117, 135
Ms 1/2 ++ ++ nD 135
Abb e ia ions: +++, apop osis is comple ely dependen ; ++, apop osis is pa ially (app oxima ely 50%) dependen o clea ly in ol ed in apop osis; +, mino ole in
apop osis; −, no ole in apop osis; ND, no de e mined, id, indi ec e idence; Bax, Bcl-2-associa ed X p o ein; Bid, BH3-in e ac ing domain dea h agonis ; JNK, c-Jun
N- e minal kinase; Ms , mammalian s e ile 20-like kinase, mPT, mi ochond ial pe meabili y ansi ion; ROS, eac i e oxygen species.
Regula ion o spon aneous eosinophil apop osis
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