Kate Kaiser,* James J. Foley,* Dulcie B. Schmidt,*. Kyung Johanson,*. Cohn Macphee,t. Kitty Moores,t. Dean McNulty,*. Gilbert F. Scott,* Robert P. Schleimer,$.
Cloning
and functional
chemokine human
characterization
of a novel human
that binds to the CCR3
receptor
CC
and activates
eosinophils
John R. White,* Christina Imburgia,* Edward DuI,* Edward Appelbaum,* Kevin OeDonnell,* Daniel J. OShannessy,* Mary Brawner,* Jim Fornwald,* John Adamou,* Nabil A. Elshourbagy,* Kate Kaiser,* James J. Foley,* Dulcie B. Schmidt,* Kyung Johanson,* Cohn Macphee,t Kitty Moores,t Dean McNulty,* Gilbert F. Scott,* Robert P. Schleimer,$ and Henry M. Sarau* of iliopharrrla(’euti(’al
*DeJ)(lrt,lle-,1ts
Penn,SN’ln’ania
Abstract:
t!O/)Io’ifls
Eotaxin
has
to a single
chemokine
sequence
tag
brary,
and
system
MPIF-2,
appears
cytes.
from
MCP-4,
but
completely on
same
these
receptor
was
the
the
chemokines
most bound
eotaxin
CHO
cells
with
1251-Eotaxin-2
and with
eotaxin
equal
with
high
cold
eotaxin-2
Eotaxin
the
response
to
potent
response
LTD,1.
The
and
eosinophil
activity
solely
Biol.
62:
667-675;
the
[5. 6].
the
eotaxin-2
anti
EST
Words: 1IPI!”-2 .
[3. 4]. [8].
Re-tent
tiata
halo- too \iCh#{176}-1in its ahaihitv
jahiils
ligand
promote
[0). 1 0]. bl:N\’l’ES
art-
all pntotlt’t-ti
poonting
that’
other
the
thnooughu
indicate
that
is a
o’oomuipbo’to’ly. ro’stnicto-oi
but eotaxin-2
chemokine CCR3
not
exerting its J. Leukoc.
receptor.
to) inioiut-e-
.
anioong hortoto’i1i:
CCR3.
s’hio’h
o-oataxin
to! o-oosinoa-
(hit- (XC
anti
(C
suggo’st
[ I 3. thus
14] 50h0-
is jaantio’olanly
ao’tivatos
too o-asinoobahuils
[7].
is o-oamlabaara-
naay’ lot’ invoolvooi
‘Flue’ ahaoavt’
anirmmanily’
it) art’
(\ICP-3)
i’hao-rmmotaxis
ira (tmnra is pninmaarib-.
too ao’t throtmgha
fat’-
I a (Ni I ja_ a)
(hut-so’ o-ho’nioakine-s
o-ootaxin
I n adtii-
that-so’
inflammatioori.
to!’ e-oasirueo1ohils.
boo-o-ause-
Rooh shaowna
thuat
to!’ albo’rgio’
that
thotr’o
the
iflilaoonlanit
[3. 4]
Human anti aniimnal oiata ntow stro)miglv [1 1 . I 2]. \ICl#{176}1 [0)] antI o-ootaxina at si(t-s
is oaf
o’hmo-niootat’tit’
I)ntoto’itl
inuoiio-ato’
human
o’ytookino-s
to!’ (lao-i n i nvo olvt’miio-nt
o-hao’miaootao’tit’
MCP-4.
that
ooho’n
m-mioos( imeo(abolo irifianiamaton’y’
neotabaly too tIme- o-n-
o-xaamasioon
(I I -5)
anti
boo-ta uso-
rnonooo’o-
o-noioatbmt-lial
so-lo-o’tive-
kin-S
tof’ naoo(
o’oosimioaplails althooughi
naoo
[IS].
o’hmoniiookino
i’animilio-s
so-i c-n tranismo-nitboranit-
haavo-
bao’i’n
ne-o-c-1otoons o-x-
1997.
(‘/ldfllOh’lXj,S .
it ‘atc-oi
miuao’roophiage
Niabonc’
Key
boy’ in(orloo
in
still
mnalo-cult-s.
tab’ haninaan\
tlao-
oaf’ o’hiomiaoekinios
o-ht-mataxis.
(a
boo’o-n
boor Ioo’aliza-
o-oosinoophaibs
imivoolvo-miio-rat
hoo-o-n i mpl
utibizo-ti
in te-tlao’ning
ino’buding
ntimnbers
toors haavt R:\TES
eosinophils
displace
‘Flit’
art-
rmaio’roovaso’olan
of
tel aoiho’sioonu
ko-y c’ve’nts
a nunuihaor
bias
oaf’ ko-y poaints
nmao-(liatoors
o’xaro-ssitn
mi that’ no-c-no itnae-n
chemotactic through
to
o-xat’t :(‘ti’a(itora
cells stably transfected and both ligands crossof
data
and
affinity
a Ca2 transient in RBL-2H3 with CCR3 (RBL-2H3-CCR3) desensitized
isolated
otasirataphuib
l251
in
a numi)boo-r tin-
o’t-bls.
be-ukooo-yo-s
joroogno’ss
2]. In
.
roo’ruitniorat
It o thmo sitt
tbtao-unuio’nit-ol.
of all
expressed
to eotaxin
tin-
I . art’
to!’ ootbao-r
[I
oiiso’aso-
rag to!’ o-asiniooiohm i I no’o’nuitrno’n(
imuo-luoii rig anob
baoawci
hao’o-n niatio’
to) (hat-so’ sitos.
e’oosinoophail
Eotaxin-2
displaced
stably
to freshly
similar
and
affinity.
also
CCR3
and
affinities
MCP-4.
chemoattractant
cloned
binds
both
and
Ising(!o,n;
has
titan
titoni.
ahose-no’t
inoolvc-nao-n,
oloothiebial
can
( mi/ed
that- so-bo-o’tivo-
situatioonas
t’oansioienalolo-
tb-ar,
vo-ll
inuflami-aniaoory’
thao
unoierstanitii
N(-\I-
eotaxin
in
:lthoaogh
to-lbs
calcium response to that eotaxin-2 shares
Eotaxin-2
(CHO-CCR3)
eosinophils
low
or MCP-3,
nooted. albo-rgk-
experthat
MIP-la,
eosinophil
to the
Eotaxin-2, with
oaf’ ooasinoo1ahils
tour
culture
mobilization
by eotaxin
tested.
li-
cell
indicate
indicating
potent
ohiso-ase
other chemokines. and Ca2’#{176}’ mobilization in neutrophils or mono-
the
used
arid
0)1 that-so’
to
RANTES,
cells,
nhainitis.
niian’
CC chemokine
cross-desensitize
eotaxin-2
be-ngy,
expression it was
(Of PTlI,S,Si(l.
Harlan’,
Park,
Science
exclusively
CCR3.
calcium
I’ron1ur,s
-\d’
King
Ikeeha,n.
.IJafl’/(Ifl(/
bind
identified;
activate
I*o’(’Ium,n,
Ilalti,nore.
monocyte
been
eosinophils
not
Ce,zter,
Using
a Drosophila
identity chemotaxis but not
purified
S,nithKline
,‘iller,’-%’
activated
is a human
eosinophils
using
and
the
an has
Cross-desensitization
iments
to
CCR3.
to only
or CK3-6,
human
of
purified
amino acid sequence Eotaxin-2 promotes in
found
been
chemokine
and
(1(1(1
Smillikli,w
Pharrna(’ologv,
(111(1 PU!lflOfl(lr’0’
of I’Ilar(nl(l(’o!og’
A,Sthd’fl(l
receptor,
screening
a second
expressed
t I)eparimeni
;
(1(1(1 *,IoIiii,s
S(-ien(’e,s
CKJ3-O
. .
(l.StIlllla
CCR3
. .
tag
e-V/)flsSed
(Ot(1Vjll
‘
St”(/lWfl(’ilIg
eolaxin-2
atie enes:
I 1,-a.
mmmyc’boeioi areegc’neitoen
ticeni:C1IO.
iniaiioiteon
o)g\.
nm-5:
Ni C P-3.
innllaniermeatcen fac’toen-2:
i(
nmmcenmooc’y te’ o’iie’rmeoetao’t iencetc-int-
II.
I a:
it’
NI P1 F-2.
el nmee’rase- t’ieainm no-ac’-
Chine-se- imammmsto’r cear’o.
l)n.,Jceimnm it. \\ hide’. b)ept.
(:oerrc-s1aoenmelo’rmc’t-:
I NTRODUCTION
i nmte’rlo’uki
NI I 1#{176}1a. neao’nctjeimago-
janeetc’mne-3:
SnmmithKlinmo’
lte’e’c’imanmm.
Pt).
ccl \Ioele’o’rmlar
Ittex
I 53#{176}). Kineg
.1unto’
I 7. 10)0)7:
cob
InmmnnuneoelI#{176}ntmssia. 1#{176}-N
I 0)1(16
Ftosimuopluils
art’
knoowra
pahoage-no’sis
tel a nunualaer
to)
bat’ o’o’rarally
oaf tliso’aso’s
invtalvo’ti
including
asthnua.
in
the’ ab-
be’c’o’ivo’ol
J unmo’
2.
1 0)0)7:
re’visc’d
ac’c’o’Iete’cl
J merle’ 2(1.
lqt)7.
Journal
of
Leukocyte
Bioloagy
\oabunme
62,
Noavtnmeiaon
1 0)0)7
667
pre-sso’d CXC
on and
thuo- sunface-
cc
o-ight
amid
ido-ntifIo-ob
her
of
[lb.
“orphan
cause-
true-
17].
O)f
ro-ceptors
[21].
thut’ CXC
family
kino’ re-o’o’ptoors anti kino-
binds
o moore
than
ceptoors
with
In
high
this
ernied
sutiy.
inhibitoary
shown
to CC
there
tthuo’n
also
myeboid
knoown
new
to bethat
Tht-
taxin,
selet’tivo’by
pne-sso’d
(NIPIF-2)
N i1-to-rmimiab
stanptioon
Mass
Sat’e’tnometry
lay Bunkout
is receptor tone
che-mo-
To date-,
tonly
eotaxin
[28]
ditmmnm was
tan
[25]
via
purifie’ti
characto’rization.
obuct’s
o’t)sin(aphuil
receptor
throough
migration
eo-
(CCR3)
cx-
Eotaxin-2 by Human
protein
was
expressed
culture
syste-m
for
hue-re- that
and
calcium
b’cebboowing
nc’o’cenmbeinmamit \I(:I’-3. N1l1-1
ce’no-
iatnnthase-oi
mobilization
R-NN’l’i:S.
IL-8.
& I) Systo’ntms.
(H
Inoenmi tlae ecetaxin
inmolio-ate’d
(Peprote’cim.
Nlinmieapcelis.
2-bmycInooxye-thy’lpiao’nazinmc--’\-”-2-e-thane-subIonic
(N-
glutamine--T.
1)e-nmtbbmm/s(rt’Iattotnyoin o’alf
soebtmtioan (Gll3C0-itRL. (;aithe-rsiaung. (Hyo’boermc’): Lymph-pnc’p (Accurate. Westbury.
serunm
ac’iol)\aj.
oubleoxide’
(Sigma
VS
Ccobommmrm type’
(ltaxtc-n
con \‘V’R Ci/mimmoab)
mrminoe’-’I’
b’oetaxin-2
so-ic-mitifie’
I,ouis.
lonooduots);
(spo’o’ifio-
brie’..
CA):
2000
He’igis.
Ci/mrnoal)
itie’nitifiod
Inoona ao’tivattoi
stibsc’otit’rmtly
ioio’rmtiuie’oI
a !)rosopirila
hen semb-oboning
no-gion
was
human amid
into)
arim1alifiod
c-boeno
as
ao-tivity Chloana-
Scie’nuo’es
Fo)taXifl-2
an
e’xpno’ssioian
lay’ polymmie-nase’
suiastnate-
and
chain
3’ (Sa’II
Biology
Voalume
laair site’
62,
o’xpno’sso-d
by Ronkout ht, otataxin-2
roa’tion
time- lobloowing
in
a
o’Ioane
was
veoton.
toy’ hy’jaoatoonic
cell
lysis
fealloawe’d
anol
ne-suspended
ma PNCCM
buffer
or
varioaus
we’re’
wene
placod
hate-d
foar
o-hamht-n.
of
Coan1a.. Cabin
X
100
Iltayden 25
p1
bigands
ito tnilalicate. pore--size’
Jtoian. MI))
(2
nitodifio’oi lint-fly,
agoanist
chamber
(neutnoaiails)
Keosinoaphils
a
[32].
concentra(ioans sparse
(Nucle-opoone-
bo)wt’n
using
pne-vioausby
in tho’ bowt’n
1abaee-d
on a 3-m
and (1.5
perfoonnat’oI
de’so’rihe’d
of
in theA 5-pm
a(oIy(-anbonate
separate-ti
o-elbs/mL)
timo- upper
tan nc’utnophils
nt-suspended ma P.-NGCM ton RPMI. rospe-ctively. oaf the uapon o’hambe-n. Samples woro’ incu(eosimmoaphils) eon 45 mimimu (netmtnoe1ohils) at 37#{176}C in 5%
x 10#{176}#{176} ce-lls/mL) in 30
after
was re-mowed
tach
s’ebl
which
the- o’hambe-r
arid washo-d
neon-migrating
cells
froenm
l)iff-Quik.
Eosino1ahils
ide’nttfioi’oi
and
some-
the’
iSo
tiaan
experiments (100
upper
surface.
eon ne-utrophils
o’ounto-tl. rather
The’
the
froom
oonduo’teoi
nmmo-mhrane
and
stained
in
a
induced
he’ absenceoaf the’
with
uio-bds were
o’o)taxin-2
o’ffe’o’t eon eosinoophib
t’haml)t-r
the
to nt-move
10 lmigh-power
wlmether
assess
tipper
saline scraped.
a o’hemoatao’tie
went’
muM) in
oiisasse’rmihled.
was
in phosphate--buffe-red
migra-
ocr prese-nce
of
apparatus.
(PCR)
us-
oef 5’
TC-
and
November
initiator
1997
pnime’ns
5’-C((.N1Vfl’CC((A(jjACANCCT-
c1Nc’rk(;ATAcAG-3’ (sonse) (EcoR I and immitiathon coadean underlined) and 5’-ccATc(;TNA(;cvr(;(;(;NcGTc(;TATcccT\NAA-3’ (antise-nsc’) (Hind III umide’rlimied) we-nt- synthe’size-d hased con a fullbt’ngtlm cl)N-N o’honmo- (HI)CNHO9) oeiataino’d froem Human Ge-noamo’ Scioniet’s and foound tie lat ioit-ntical in sequence toe the previoously pub-
(Roao-k-
tags
A full-length
[26, 27] as olescnihod
washo’d
we-reas
Obigoonuclt-ootide
Nnimt’rsham
stquenoi’o
moaneecytes.
ti-en-
minimize
Preparation of Chinese hamster ovary (CHO) cells and rat basophilic leukemia (RBL-2H3) cells expressing human CCR3
stain
the’
fnoom
Gt’noame-
soquo’no-e’ol.
c(;c(;c(;GccNccAT(;cc1N(;ucc’FcN
of Leukocyte
Hunmanu
we-no
i)uffe-r
e-(ataxin-2
II’.
oaf t’xiane’sso-d
c’tiliimne’ system
to-Il
time o-I)N,’N
at
sc’odie-nt’ing
nomimove-d
a( 4#{176}C to
gradi-
Alto-n
mag-
(siae-cifioby
we-re’
cc-lls
expe’ninients
buffer
tion,
So-panation
I)iff-Quik
‘I-o’otaxin
bloated
nun).
experiments
ciae’mokine-tit
of eotaxin-2
biianary
Journal
.\nti-Cl)16
o-ustoonmm lahelt-d
activity
“Nnlingto)n
\IO):
NI E) : Nlagnae-tic
Sumanyvale.
2I-o-oetaximm-2
was initially by nanoioern
t-t ab. 1281.
668
St.
I3ioatoc.
NI I ))
o-ooelirmg
Co..
(Nliltoniyi anti
nmme’(heool
Soio’nmo’es.
Expression
ing
Cho’mio-al
(I mmmitmnote’o-im. Irat’. Westianootek.
he’ads
Ml)): NY);
Piscataway. NJ); P1 PI’S [pipenazinio-N’. ‘V’-bis(2Ftmna-2 (Cabhioao’lae’m. San I)icgoo. C-N): dinmoi’-
l#{176}o’no’tobb (i#{176}iaarrumac’ia. otimammstibb’oemiio-
taut
te-o’lmniquo’
CO.,/ain.
Hill.
HEPES
acid).
o-annie’d
Eosinopimils
chamber
statmncts:
Beet-ky
RPMI,
MN):
wene-
g/mL)
g. 30
in-
METHODS
ne’age’rmts
aneecoduno-s
in-antio’ooagulated (1.090
oof CI) I b-positive o’ells (nme’utnta1ahiis) using a MACS stiper naagnet and inanmuneonaagnc’tioho-ads [20)]. Fosimiophil purity based on c’xamiiination cief l)iff-Quik-staine-d cytea-o’entrifugatioan lart-panations was 9#{176}) ± 1% an(i viability (haso-d oh to)ludine blue dye e’xcbosion) was
PNGCM
AND
he-pan
lay Percoll
(1000
Chemotaxis
bio-
eo(axin-2
lnoom
voabunte’e’rs
roaM NaCI, 5 muM KCI, 20 mM PIPES, ph 7.4, with 0.03% homan serum abbuniin. O.10/o glucooso’. 1 muM CaC12. I mM MgCi2) for o’iiemotaxis studies as de-scriiae’d be-bow. Ne’utrophils and monoocytes werepurified lay the methoaois oaf Hoooove’n [30] antI lloayumn [3 fl. nt-spe’ctiveiy.
Reagents
in
medium.
te’mpe’rattmro’
Rol
nnombnamie-
was
do’-
(147
(;CR3.
MATERIALS
o’l)N’N
coanditioned
isoabate’oI
alIcrgio-
at nooam
all
(easino1ahils)
vilbo’,
previously
lay ne-moval
same-
tile
as
analysis. arid Mau’ix-iNssisted Laser liefor ei-otaxin-2 was carrie-ti taut as dc-scribe-d that eoataxina-2 (-000ditio)ne’oI 1)rosophila me--
we’re-
from
Cho’nriotaxis
2200
sohdono’d
characterization of conditioned medium
loon N-IC P-4
eosinoapiails blood
>9803.
like
sequencing
We- re-aoart
to call
and
receptor
cell
many
We- choose-
o-osinophils.
The’
ef-
by
Eotaxin-2,
c1)N,’ [25].
Uunman
a(’(iva(ion.
inhibitory
sharo’d
oaf e-otaxin.
a Drosophila
froam
boogical
nt-tic’
s’as
Cell purification
tnil’tigation.
progenitoan
its biology
oaf human
Inc.
mye-ltaid
or CK3-6.
large-stale
Scio’no’o’s.
chemokine-
af its
to) a single
thut- surface-
novel
activity
he’cause
binds
a
oo as an
too thtase
disctvere’oI
tla’b
t’xo’ept
substitute-of
c-nit e’t’ntrifugation
he-cause-
o-oaaxin-2
similar
Ce-noomo-
fetal
pnoduct
Purification and analytical eotaxin-2 from Drosophila
vt-noous
ro’fe-nro-d
t’hao-moakines
art’
Ni)
PCH
[28].
Pemnitication.
chemoa-
usually
iolo-ntifmo-d
ban(oliferation.
factoor
usage-
‘hit’
anti 5 ‘ GC1’CI’NG,N’VI’-NVI’,NGCAG3’ (Xhab site- underlined) toligonucle-
ho’-
[22-24].
havo’
vo’
fao’toan-2
ton
this
be-c-n
In addition. akhoough
ono- receptor.
affinity
o’ootaxin-2,
fe-ct
out
bind
scniho-d
re-
turning
also
doa not
named
unde-nlino-oI)
ocaoion
Ianimt-rs.
O)titie
num-
chemokine
now
It has
vo’rsa. family,
so
nime-thionine-
(;T(;crni;(;rn;cc-N(;cNTN
(0)
o-xtremo
anti
the-
are- a barge
too known
are
ftour
cloned
lao- ro-stnicted too a single re-ceptor. At the- o)ther is RNTES. which binds multiple chaemokine re-
apoo’ars
was
vice
withini
bee-n
receptors.
similarity
date-
lio
have
the-re
soome oaf which
chemoakino
choriuokino-s spoo’iuici(y
In addition
se-oiue-nce-
[ I 8-20],
cells.
receptors
o’ho-mokino’-like”
of the-in
o’o’p(oors
oaf le-uktocytic
che’mookino’
bished
CC
cho-mokino-
too add o-loning
sites
pno)duct
O)f 1.1
kh
intoo
oqui’alo’nt
the’
ne’o-eptor.
was
CCR3
[33].
the’ lull-lengthm
to)
oiigestcd
silt’s
ccl tiae
s’iti-i
The-se
pninmens
ro-ceptoar by PCR. Eo’oaR
Hind
III anti
inserted
e-xpro’ssion
‘eo’tton
pCDN.
I and
manamalian
wo-ro’ used
Tho- expec(ed
The- inst-nt (af the- resoling consiruot was compleely se’quonccd to o-oanfirnn its ide’ntity arid oonie-mmtatioonm. ‘1’ oohtain a stalabe- tie’11 line’ cxanossing was
the-
o-lectroporatod
CCR3
rc’o’o-p(oor.
intoo
CHO
thc-
on
1aCl)N:CCR3
RI1L-2H3
oxpro’ssioan
o’e1bs.
Stahbo
plasmid o’o’ll lines
were isolat-d by growing o’ells in the- absence of nue’le’easide-s (C 110 CCR3) or s-ith G418 se’Iection (RBL-2H3 CCH3). Thceso’ cell line’s that grew toot af se’le-rtioorm ve-re- (lao-n analyze-of Ion CCR3 c-xaro’ssieon using
a
5I-e-ootaxin
Analysis
binding
assay.
of intracellular
Eosinoaphils
ton RBL-2H3
CCR3
as describe-of
previously
Fura-2
For
the
reach
tXi)enimtni(aI
cated
conuctntratiorm.
the
unme of 10 p.I.
and
ing
late-r.
first
Maximumal
Chemokine
was
by
binding
mL)
vo’re
loadeol
30
we’re’
were’
alter
s
adoieol
and
binding
‘51-eoaxin-2
ence,
Inc.
cells
30-(oO
agonist
oar eoosinitaiahils have
‘1-eoataxin
x
1o
mm
at
37#{176}Cin
the-
wene
cells/niL
used.
‘ester
using
or
2200
o’oonu’entnationm
oef biganad
iainmoiinmg.
alto-n
end
ro’o’conmieinmamit
injo’o’ticari
toa
cob’ the’
KL1I
o’etaxinm-2
ol a synmtiaetie’
i-ia
a
lno’e’
e’xpne’ssioonm
o’cetaxina-2
c’vsto’ine’
(‘ys3#{176}
aoholo’d
tee
thc-
acjotiobe-.
is related
Se-quencing
taf’ cl)N
to MCP-2
and eotaxin
o-ation
the-se
most
St’i-
tivate-d
CCR3
etasinophils.
0.15
5l-t-otaxin-2
azioie’.
Tho
s’c’ro’
that
was
in
and
Into’
nM
%‘itia 2
re-ached
Brarmole’ll
bloo’ke’oi
nt’sulte-d
1oroateins
a number
lean
(o
MCP-4
mtontacyte
chuemokino’
mu\I
has
of new
are-
notably
[35]
the’ co’11s in a that
Life CHO
exe’o-pt
libraries
of a number
in the-
frtam
identili-
varitous
oaf new
cho’mtokino
10,
36-38].
So-quenoimug
was
c(amplo-(e’d
families. me-mbo’rs,
Ci/mnmcel)
with
bimiding
filters
nimconitorinmg
Eotaxin-2
abaeovo. ‘Flit’ o-xperinmmo’rmtal 0.27
(if seadium
GE/C
the’
siaeo’ilio’
s
stimimolatiten
Intern Amersham
Eo1uilihrium
i)y har’t’e’sting
‘hatman
act.
peri’oormed
pre-viously
prt’so’n(’t’
went’ so’para(ed
[9,
library
was
identifie-d
that
Eotaxin-2
MCP-2
and
eoaxin
the-
similar
acts
anti
specifically
is a CC
chemtakine
(39
34%,
and
of an
a haote-nt
ac-
novel
ton human
with
huomoloagy
respectively,
1).
Fig.
3()
Base-d
hotmnmd
tan
o’e-ll
han-
eootaxin,
1 h
with
mole-cu
the-
l)iobogical
name-
activity
etataxin-2
is
of e-ooaxin-2
pntaaso’ti
foar
%‘ith his
new
le.
polyetbiyleniminio’. ‘251-eotaxin
binding
cab tea those o-uhatioan
wih
‘Flit-
Binding binding,
ai)sene’e
amount
of
tells
1O
X
shake-n
nadioaactivity
we’re
ioie’nti-
cells
we’re’
( I 50
qonm) at roonma 6-nm-
l)000nol
ha
ustd
the’
antI
Eotaxin-2
in-
data are prose’nited as specific 23l-eeotaxin oar ‘2I-ecataxinwhich is define’d as the difference’ lae’tween binaoiinmg in the-
(em
so-intillation
prt’st-ne’e
oaf I 00
CCR3
br
using
as
0.15
nM
the’
lC5.
nM
onlaiae’led
ligand.
s’as
dete’rnmuimied
251-o-otaxin
on
1C5()
Eotaxin-2
was
i
Tho’
mu oeampetitiomi
0.27
riM
A G L M K
MCP-2
AQ
-
-
f’rammi
I
V T
5
V
5
AJJWLL
A
F
signal
sequence-s
transfe-cte-tI for
L
0
V
c
L
I
rabbi
H H I A
F
o’xpenimo-nus.
using
Drosophila
tta co)rno’ctly [26.
lJro.soJ)hila
e-toaxin-2
JA
for hioakagical
se-crete-ti
shown
ptobyclonal
and purification
protein and
previously
vectoar
amid
o’o)mio’o-nutratioamm-
M T -
bly
iainohimig
-
punt’
expressed
human
aIim-
3l-eotaxin-2
(ietenmuaimie’(i
Eotaxin
binding
expression
‘lo ohtain
was
filto-rs
was
oaf agonists
expnessed
CHO-CCR3
4
spectronue-try--.
and
assays.
osinig
except
mm on an orbital
toy gamma
quantitated
ity
sttidies
ee)sintapiiilS
loon 90
was
it’ratune.
2
iO
were
liganol O.14
oheseribed
7 X
cells/muiL
h1)
isoabato’d as described
bee-re
a’itiu
(spa.
Ci/mmol)
He-ights.
tootal
RESULTS
reo’eond-
studio-s.
and CHO cells
231-eootaxin
(sp. ao’t. 2000
(‘Nrlingtoan
coanoiitions
using
the’
o- al. [34].
to eosinophils
assays
fear
ira rahabaits
Phe’7’1) (‘OU[obc’(i
‘mong Ro-ct’1ator
mit-ti as
defi of
for
in a
comme-nmo’ing
alter
;mmkie--iez
oso’ci
(1011)H-to’rnninal
tee
ss’as 5(YVo
to eotaxin-2
raise’ci
thnoaugh
alleawo’oi
arid
at (ho’ math-
nmmeanuito)re-d continually
agoanists
Ca2#{176}attained
as (iescnihe(h
were
claemokine
loon desensitizatioan
intracelltmlar
was calculatt-d
(2 with
being
ad(hition.
1O#{176} ce-lls/mL)
cells
stiniulation
adohition
sec’eamid
toe oiisplat’o’
,-Nmitilatadies
[28].
In all expe’nimo’nts.
the
the’
o’e’lbs (2 X
‘Flie’ flooresceno’e
lime-.
indicated
in eosinophils
toroceolore-,
37#{176}C loon 5 mimi bofore-
t’tmn-t-t’s
rc’cjtmirc’d
Antibodies
calcium
with
ro’si)eonse
was
ro’coagnizo’
36]. S2
Crowth
tells
by
raised
I
-
T
the-
froam
V
-
5th-
o’xpre-ssion
inumumobaboat,
A
a sys-
anti
o a synthetic
-
sLi#{176} G L
cells.
medium
carrying
analyzed
antiso-num
e-toaxin-2
52
using po-1aiide-
a ole--
28 27
PD5V7
Eotaxin-2 Eotaxin McP-2
QJ38
Eotaxin-2 Eotaxin
A
GV
I F
T T
K
I
F
K T
K L JRD
I
I F
K T
K RJJE
VC
Q KKV
MCP-2
ELA_V
KQ
Eotaxin-2
AQKKAJ1RARAVAVKGPVQRYPGNQTTC
Eotaxin
Q K 5
P
T
P
K
MCP-2
Q
Q N
L
K P
I F
Q
F9JD
P
KQ
C A D P KK A
D PjE
90
K WV
Q D 5 M KYL
RJSM
D
KHjLD
89 69 119
A
P
I
77
2 Fig.
1.
Amimmoe acid
respectively)
weight
to)
table.
seqoence
eotaximi-2.
:Nnnow
1 marks
aligminmie-nt Identical
the
start
of o-ataximi-2
amino
ao’i(hs
cal the
putative
with are
MCP-2
booxo’d.
mature
and
Alignnucnt
sequeno’c
e-otaximi. cas
while’
The’se
twoe
o’oanm1aleed
annow
2 marks
chenmokines lay using
have
a pote’ntial
JTliite
time’ highest
time-’ c’ustnal
e-i (ii.
heanmmoabogy
mehath
with
N-glyo’oesylationm
site’.
Characterization
P-NM
(30.)
and
34%,
I 00
nc’sioitmo’s
of eotaxin-2
669
rive-ol
!‘rooiau e’ootaxin-2 vas
to-ira
s’as
osso’titially
o’o’lls (Fig.
213).
1oe-ak with
a niaass
anti
tiaat
that
Iontottiri.
ruoon-initiuo’o-d
fitual
naiass.
NH2-te-nliairial
tine’
moalo’o’uI
)
anti
(oiata
cob’ hauniiama
stuoiy’
Effect
of eotaxin-2
o’o)mllhoaro-ti loaoiooi
‘itla
trationas
oatho’r
too thuo muglvo’oosy-
o’ht’mnotaxis
o’homriookimuo-s
knuown
Ftira-2
shioowra
l)t’t\%
wo’ro
mi Figure
amaoh \iCla_,l
3.
:S
[ I 0]
stimuuulatooi ‘l’ho-
o’ootax
0,0-ni
riaootaxis
are’
i
h)y- eataxin-2
hunuana with
‘arioaus
ti-2
antI
ro-I000nto-oi I000tt’mat
oaho’n
c-onto-n-
curvo’s
inohuo’o’rs
A
o-oataxin
cast-
Eoataxin-2
o’abo-iuni
at o-oono’e-mitrationis
diti
in
up
(to ao’(iva(e’ this
tatlier
hao’two’o-n
o’oosinoophils
o’hambao-r
for
withu
and
human
too I 00
analysis
0.5
anti
100
ro-spo’o-tivo-lv).
Atitli(io)n
art’
ruM
inig
is
that
this
is
=
(FC-11
[39.
o’hoiuioakinetio’
no’slaonse-
oob’ [Ca2#{176}] tranu-
\%‘ithi hunian
nie-utroh)biibs
(oiata
mi Figure
exhuibaiteoi
4. o’bue-
a
was
ypical
the
moast
I n NI) o-tamiapareti with = 3. 10. anuol 6 nNl. too tbit
ooasinieoobiib
o’hio’moatao’tio-
other
imuolticeti
l’oataxin-2
to!’ o’otaxima-2
a
sluoawnu
miNi anti
assesse’oi with
Coomuo-e-ntnation-
o-oataxin-2
(‘un’vo’.
pret-mitttl
was
o’oanujaare-oi
o-oosinoophils.
o’hio’naokino-s.
oloaso-nespoanso
to!’ that- apparatus
no-la-
o-iao’miioakinio-s
pro’vioausly’.
[101.
e-’oataxin
anioaho-n
mu
intrao’e-llulan
poott’rut t-hue-rmaoatactic f’ao,tor (E( NiCI#{176}4. e-totaximi. anti R-Vl’ES
ooosinophils
o’oonio’o-ntratioan-ro-slooonse
sho’ro-as
tan hiumuian
in a 48-we-Il
Coona1aare-ti
intracellular
o’hionioakino-s.
tab’ o-hio’miaookino’s.
tit onmsha i a
-‘1
%‘itha
miNi) in o’oasimuo-
on cell chemotaxis
That- o’!’fe-o’t oaf’ o-totaxin-2
so’o’n adjao’tnit
t’oosinio)phails
iii
o
\ICP-
!‘oon NICI#{176}-4amid
o-quipoao-nt
ton nilonoot-yto’s
hell-shiapo’ei
that- ao’tivatiooni
increaso-
30
than
niot shoown).
ro-spoonse
]h
an
1ate-mit
(EC41
we’ro’
a po)te-n(
is o’oonlj)arah)lc-
o’oan-
stab’.
on eosinophil
moore’
o)r R:NTES
o-oataxini
nuo-utro)phibs
at Jaoosi-
so-tue-mut’imig
oo vaniationis
Effects of eotaxin-2 Ca2 concentration
t’learly
noo( lanoonitoto’
ii)a huighao-r
oaf that-
ioroobaahlv
vhuio’hi
I 170
loomis
are-
lout
inuoluo’etl
2 nINI).
NICP-4
that-nt’ s’o-ro- nato oothao-r sbaoorto-n oar bongo-n thec
I nM)
9 riNi)
=
Eoataxinu-2
=
a pncoioinant
\‘\-‘l P
1oo-aks so-ro-
(EC5
joliils. ,similar carder to!’ Ptote-nit’y havo- boo’on no-poonted pre-vioously [0)],
acids
aoioiitioonal
s’itha
eosinoolahuils.
(EC-11:=
4 (EC5)
sizo’
that’ ariuino
i’soo
starto-ol
huniian re-slaoonse-
ooataxin
coan(ainioti
2. Fig.
(anroow
in
o’alo-iunma
1aoly’ao’n’lamio1o-
‘Ehuis aoioiitioanal
N-glyo’oos’latioon
rmoo( shoawn).
anti
Iano-para(itan
tori a saimit-d
s’hait’lu was
sit’mits
1)rosopliila
lay- ioon e’xo’hange-
oaf I I .(a2 1 l)a.
e-otaxin-2
muaaum 1ooak: latioon
s’hio’hi
shioowoti
tie
I I S (oiaa
!innno-oi
tho’
muioalo-o-tilar
is tInt’
l)rto
cells,
slot-o’trtonio’t’
thio larotiitto’ti nimass
titan
I’romia that-
as visualizo-oi
Mass
Phuo’T’#{176}). : 15-klia
sulhito--intluo’o’ol
-l’to’r Iat1ni11o’1tioami
sao-o’io’s
go-I (Fig.
Ian
o-oolaboor
o’lanoamaograjahiy’.
I S-kl)a
thara
flit-
abaso-mat
2A).
t’xt’ltisiooni (I
(o-y’s58 thiroetigh
in
obo’to-o-teob
uapo’r
chamber
chuonuotaxis,
ro’slaoonso-
ntot shioawn).
indicat-
ratho-r
than
In similar
oar mooiaoao’yte-s,
a
studies
o-oataxin-2
‘as
B 1
1
2
2
-29
-18 -14 Fig. 52
2. cc-bbs
(N)
Expre-ssionm anti
nific-el
eotaxin-2
+
11621
1500C Cl) C
1000c
apomyoglobin 8476.75
+
+
5000 (1
0oo
7000
8000
9000
1O#{212}OO 11#{212}0012#{212}0013#{212}0014#{212}0015000 Mass
670
c’cepee’r
I ammo’ I . (:()o)rmmasmo’
20000
0
52 toIls:
1)rosojilnila
rime-cl i ti rim lrermm
ens.
Joaurnal
oaf Leukocvte
Biolog
Noolumo’
(m/z)
(a2. \eavo-nmlao-n
10)97
in
I)rosoplmila
eel ieunilie-oh
pneeto’ini.
banmo- 1 . inmmnmmtmnmcelahet eel’nmme’chitmnmm l’n’cermm neerm-
inmdtrc’c’ch
C
ed o’oetaxinm-2
e’imarac’tc’nizatioen
oceaximi-2: (C)
trceimmo-tr’
lanmo’ 2.
2. ci enmohitiermo’oi
nmoltic’e’ch
ieltie’-staiime’el
\Iatnix-assisto’ol analsis
lane-
stmllate’-i
ge’l
nmicebo’c’tibar cbo’’ceniet
eel joiiril’io’el
cells.
ieenm nmmass
o-otaxin-2.
(13)
(120/o) eel it %%o’igimt naarkslee’c’-
R:\TES 150
nioa tiosonsitizt-oi
s’ero-
tEat-v have
be’o-n
ah)ly’ loet-atisevt-mit
C
thie-
they’
0) C Co
with
100
0 E
lb
50
0
[ I 5J;
alsoa
1
himiol CCR
(CR3.
oaf
do-senusitizatioui
eotaxin-2
Binding
C.) CO
tan
h)y’ ooo(axitm-2.
too hainioi CCR3
doawn-regulatioan
ho-to-roalogous
a)
shuoawn
utilizing
anti
‘Flit’
oqahuils
studies
tlo’o-rniine-
no-o-oatoor
we-no’
usage-
0.1
1
100
10
Chemokine
Concentration
(Fig.
at 37#{176}C in thoton
[nM]
3.
pe’ak
Iiost--ne’siaoense
[CaJ
in
Fora-2-ltaaded
e-(a(axini-2
(fiIIo’d
sqtmare-s).
R-NN’l’FS
added
deto’nnim
1aeaint is the
iioniman
circle’s).
e’oataximm
(ceioen
at that’ inohio’ae-d
tratie)nm
ben rhaennokimie’-siinmmolatedi
our-ye’s
as
a’erago
c’irc’Ic’s).
of
deso’ri loon
lad-ti
\l C P-4
1 ox (filled
anti
time- maximal
i n Nlate’nia
triangle’s)
(eo}oc’n
Methcocls.
( Fig.
Eac’
(a-N), o’otaxin
h mug
e-xao’ninmie’nts.
site-s
n\1. to toe- inactive
at oonce-ntrations
nNl (tiata
too 30()
U[)
[ I 5. anti
anti
(EC5
to
3 nI\i
substituo-ol
tiio’se-
cells
eootaxin.
too lore%’o-rut
anti
oar
ro-o’o-p-
o-ootaxin
Coonu1o’titioani
eoasinoa1ahils
analysis
shioaweoi
R-NTES
that
t’oouboi o’tamn-
o-oormulae-to’oi e’tpially’ at absto
1251-ooaaxin
hiraoi-
N-II P I a up
too I Ut)
if CHO-CR3
(Fig.
to-bbs
#{212}C).Stuoiie-s
re-so-alt-ti
h000tora(lY’
fear the-
wlae-ro’as
o-oano’o-nitratioons obataino-ti
fear e’oosinuoo1ahils
amiol NIC P-4
6A) nao’asuro-oi
hay’ oothuors.
!‘oor hoothi ligamiols).
we’re
using
(Fig. s’as
5. 6. 10. anti 70 nNi. ro-spo-o’was laoound too ooosinutojohiils (Fig.
o’ootaxin-2
ro-suks
azitio
33].
oiis-
usimig ratiioababoo-lo-oi oaf’ oootaxin-2. ooasina-
birmoiirmg
N1CP-4.
t-oataxin-2 =
oiisplao’e-
Similar
vo-ro’
not shown).
Homologous cross-desensitization human eosinophils
too CCR3
e’otaxin.
o-omisisto-nut
lay’ o-oaaxima-2.
ole-niioanstrato-oi
‘25l-eotaxina
binds
#{212}B) usimag
f’aibo’d found
oaf’ stadium
Pott’ fear this binding (FC501 tivo’ly). \\-‘he’ni 251-o-ootaxin-2
%c’ne’ c’cemmconi-
Ca2
Is arid
pro-se-net’
coabol e-oataxin-2,
Chenmieekinic’s
sqtmares).
ocr N-I IP-
t’ont’t’nitnaiion
inc-el
o’cesinmeaiahmils.
(Ii lle’d
in
ine’roaso’s
Fo1uilibriunn
,-s
are
251-e-ootaximi-2
foB).
intennalizatioan.
spo-o’ifically Fig.
with
ino’uhaatid
25l-eoaxin
o’iro’tmniaoaf’ thiose’
in t’oasiiuoo1ahibs.
1abao’t’nuient stuobio’s s’eroo’oonoiuctt’ol toataxini-2 anti oeataxini. ‘lb assess lainoling 0
is baro-stmmiu thius
ro’sults
o-xao-niniie-nats
CCR3
competition
further
o-vo’ni thonighu this
thuat
oiislalao’ooi
that-
%‘ithi
o-ooaxiri-2. naoiioolaboe-le-tb
in 40
Exposure to
of chuemookine-
lead
(ion
ta rapiol
through on
We- have
used
the-
the’
bloooi’ked
as eataxin
and
also
true
sa
j).
taxin,
amid MCP-4
The-seIn
e-osinophils.
second
anti
share
C).
to
with
with
did
e’otaxiii-2
010
with 5.
eotaxin-2.
neat
three
and
11)
(Fig.
tiesensitizo’
on
nespoanse
e-(asinophils alter
MCP-4
luavc
desensitization that s’ithu which [42].
eo)taxin-2 this.
boe’ni
of nuediating Be-o’ause
showni
too interact by the-se
alsta bee-mi
is
capable of’ CCR3.
oaf’ etataxin-2 interacts
eootaxin-2
has It
are
activation
withu
failed shown
inte’ro-sting
with
a calcium
this
re-ceptcor.
too cnoss-de-se’nsitize tea utibizo-
toio nott-
CCR that
1 tan booth
anti
CCR3.
chemokines
croosssuggests
In ko-t-ping M I P- 1 a, e-tosinoa1ohils N-ICP-3
Concentration
[nM]
5. oloa
re-ce-p(oars
eoaxin
100
10
Chemokine
Fig.
4.
ielcoeci
amid
(ho’niickinme’-inmeltmo’o’cl
c’c esiticol)bm
iae’ns.
present
I
a
(Fig.
totluer
0.1
human
o’ho’mokino-s
that
0
o’oa-
oar \IIP-la
tlut’se-
E
as vo-ll
ro’o-o’hotoar on
RANTES. that
0
The
ha eoataxin-2
consise-n
N-ICP-3.
suggesting
E).
0)1 o-oosinuophuils
o-o)(axin-2
20
homologoaus
too o-oataxin-2
(hut’ same
coantras,
re’o’e’p(tars
response-
30
0 C C.)
are
too eoaaxini-2
5. A. I, anti
are
sharing
response’
1), F,
shioow
a resptanse
results
Ca2#{176}
ligamids
coonucentratioons
that
pretreatnuent
blocked
B and
not
that
or eoaxin
x
01)
[41].
oaf’ cvtosolic
oaf eosinophils
(Fig.
serine’
receptoar
Chemokine’
a seconoi
MCP-4
internaliza-
‘ho-thuo-r
those
were-
Pre-exposuro-
conipletely
o)f the’
do-sensitization
rece-pto)n.
studies
desensitization.
NICP-4
ro’co’latoor
o deermine
same-
is kmuoawn
of intrao-e-llular tail
he-to-roltagous
l’tar the-so-
was
and
COOFI-terminal
in eosinophils
utilizing used
ole-sensitizatitan
to chemoakines
thue phoasphorylation
residues
transients
re’ce-}ato)rs
‘I’hc
s as
e’imo’nmmcetaxis
nmme-astm re-cl
c’iicnicekinmo’s
i no 18-we’ll
e’ctaxinm-2
(Ii he’d
NIC P--f (ce1ee’nm sqemaro’s).
sqtiano’s). aohohe’cl
iI
tee timo’ Ieo’cs’e’r c’hanmmioe’rs
(‘conc’o’nitraticonls.
cimorc’as
iac’r c’imanmmbc’r
as
circle’s).
con 11 N \‘l’I’S
eel time’ c’imenmmctaxis
ietmnifie’ci
to esimicqhils
in \Iato’nials
described
ccl’ 1mtmn’ilic’cI
aimel
jae’n’iioime’ral
rim ie’rc eo’ime’nmmcetaxis
c’ima rim-
e’cetaximi
(Ii he’d
(colic-nm c’iro’Ie’s)
we’re’
tmnmit at time’ indicated s ore’
placed
\Io’timceehs.
in
t lie’ up-
NIignaticenm
is
ox-
}ane’sso’tl as niie’a n titi rim i)o’r 0 of migrate’oh cc’I Is i ii t ut’ lent’so’nmc’e’ col c’imo’nmicekino’ ohiviehe-ol i-oy- time’ nmmc’anmntmnmmheo’r eel the’ nmmigrate’cl ce-hIs in time’ aiaso’nc’c’ the’
‘control
tel ciie’nmmcekirmo’. c’cumchiticenms
tel fe eti r e-x1ac-ri
Ntmnmmbe’n
vas
ccl e’eesinmco1ahils
6 ± -I-. \Itie’s
timat
ro’prese’nmto’cl
nmmigrate-cl ire’
u rider
nime-anm ±
Si’:Ni
immo-mits.
If/nile’
e’i a!.
Characterization
oaf eotaxin-2
671
250
A
zoo 210
II
190 170
250
250
210
210
190
190
170
170
150
150
130
130
110
110
90
go
70
70
50
50
240
D
Eotaxin-2
MCP-3
240
Fi2.
22
“.“
200
180
180
c1tmc-ntially kinie’s
imiohic’atc-eh
c’hc’mmme-
with
I 0 rmM eel
cotmslv ntt-eeroic-oi
1
1
inmoiie’ato-s
100
100
resent mime-nits
50
Eotaxin-2
RANTES
i’’i’-’-’’’’i’’’-i’’’’i’’-i’’’’i-’’-i’-’i
J
672
‘toitim
160
70
Journal
of
Leukocyte
Biology
Volume
62,
November
1997
I,.
into-rvals
140
30
ho’te’nooleacreous
htmniman e’eesinmeoiaiails. with Funa-2 wo’ro’ so-
(0-s
too
160
Z]GZH
and
ol
stinimtmlate’d
at 3()-
140
c’at’h
,
.r,
200
,‘
(_,)
Hcenmmcdoo’;eotis ‘.
“-‘
.
5.
e’ncass-do-s-nmsitizatieon losinophiIs looadod
.
o’he’niokino’:
a 1-nun
the
dcining
I(.a”]
,,
was
time- aeiditiconms.
timno- inte-rval.
ceantmniti-
l’ime lean
Re-suIts
ne-p-
tracings ccl l’oeur inmci1eae’rmoio’nt e’xioe-nipe’riearnime-ci tmnmelc’n iciemitio’aI ()naoiitidens.
A
100
B
100
C ‘D C
80
C
0
C cc
cci
U
0
60
0
60
0 C)
40
C)
40
0
0.1
1
10
Chemokine
Fig.
6.
0.27
nNI) was incubate-ti
Binding
vas
used
test
displace-mime’nt.
and
compt’te’d
(C)
s’itia
(EC5()
CCR3 RAINTES
to
the
sqoart-s).
In
CHO
cells
specificity
[nMl
and
or
RBL-2H3 kines 45]
cells when
have-
by elevating
anti
riot
o’lear
for
different
CCR3
cells
calcium
intracellular
were
\ICP-4
with
with
(Fig.
but
Fura-2
Eootaxin-2,
cells
in a concentration-dependent 70 nM,
MCP-4,
and
are-
and
sably
R,ANTES
(FC5()
=
respectively).
(ion
oaf a new
identified
human
by EST
brary. bind
has
This
including
other
CCR2B
eotaxin
1)roduced
and
This
is a rather
pared
with
oathuer
tor. they
For
instance-,
share
activity
che-mokine-
CCR5.
ilignmen
low
sequence-
at
tha is 62%
CCR2B,
etataxin
but
este-oi,
to the
ide-rifle-al
where-as
with
identity
identity to
liarid
ol’ e-tothxin-2
bind
was
dooe-s not
receptors
aciol alignment
chenutokine-s MCP-1
to
withu CCR3
known
that moanooo-yto’
simnilanities
interacts
an aminoo
34%
activato’d
of only
tan that
54
oaf eoataxira
mature the
calcium
human specific
cyte’s.
In a re-tent
human
[25],
oaf’ thus
clae-miimookine-
in
he-
uniliko-
soome
and
(iioi n(at
the-
cho-mnokinue’
samo’
antI boae
anti
ligands
share
was
further
suppoorto’d
(ho-
Displacemen NICP-4
agrees
ao’tivate
and with
e-o)simuo)phil
chemeataxis
the-
range moost
reo’e-ptoor(s).
tea that data
CCR3
a to
[7.
}aoto-nt
hrllit()
that
Thus.
that-se-
oobse-nvatioan
too CHO-CCR3 o-otaxin-2.
eoosinoaphils. N1CP-4
This binols tiaa
to) ant-
CCR3. NiCP-4
moabaibizatioanu Hoawo’vo’n.
calciunia-moahibizing
(-1 (11.
l25l_
that
that’ binuoling
anti
o’alcium 49].
in
ooosin-
dis1abacoti
hay’ e-oataxin,
utilizing
10. 46.
attt-miae-oi
This
see-n
RAINTES. and
wt
studies
shuoawing [37].
etoaxin-2
reco-p-
was
a sup-
foornnatioara, an ability
inmoiicatirug
h)y binioiinig
identical
same-
nanitoniolar
hintiing. samo’
published
CCR2B
consistent
moanta-
haoi
In hauman
(‘onnlabo’(o’ly
to!’ ‘231-o’eataxin
was
with
btath
nanoomoolar
CC-o’ho-moakino-s
assays
Lay e’oaxin-2.
cotaxiri
1251-t-taaxin-2
two
or
in-
incro-aso-ol
oitao’s nuoot acti-
lanootein
binding
used
anti
e-othxin-2,
anti
but
tho’ ne-copteor(s) e-otaxin-2
oells.
anti
eoosinto1)hils.
labele’d
Eootaxin,
is 59%
inunine-.
arossi’eeffect on nuiunineH PP-CFC o’oboany’ small o-ffo’ct oan nesting I ce’bl o’ho-moataxis. anti
com-
N1CP-4
hiuman.
e-ootaximi-2
etosinophils study
and
o’eonso’cu(ive-
tat e’oasinoophuils
miuobilization
for
acAn-
o’oathxinu-2
oaf thro’e
in tho
CC-che-natakine’s.
ne-u(no)phihs
it s’as
aminta
acfivatioon.
[46-48].
migratitan
tother
is coonse-nved
humara
(ho’ back
56
hainoi (to structure-
crucial
anti
IO#{176})-amninta-ao-ioi form
che-motactio’
Like
dooo’king
is
to
liganois few
Thois not
t-ootaxin
(hue- tertiary
l)otWooii
shaecie-s
at CCR3. ioio’nutiy
anti
that oaf (ho-so’
d iffore-nce
when
eotaxin
Va c’oommtrcol
i niohc’pe’niolc’nmte-x-
at-tivity so-quono’o
ro’laiively
fear re-ce-litton
foarms
etataximu
chuaracleniza-
e’otaxin-2.
taf an
sequemice-
che-mokine-
too several
anti
CC-chemokine-,
sequencing
Eotaxin-2
MCP-2.
identification
imsc’oI tea
Binmohimmg was
ant’ the’
eel’ Ic our
o’oaaxin-2
se-que-nt-o’
sie-.
positions
taphils, the
that
at
due-es
share-
rect-I)toar.
pig
too oio’hne
DISCUSSION describe-s
(fibbc’d
e’oesimieo1oiiibs/mL)
ionc’st-mitoi ohata
low
inolit’ae-
lysinues
vato’
alsoa aofivaeoi
tho-y with
guimie’a
tTsing
article
l)ata
cef the-
oaf’ tiiffene’mu
ao-tivate-
This
10
7 X
ton \1#{231};1a_( (oolot’nm soltiare’s)
Parts
into-re-sting
range.
(EC5(,
manner
e’conc’o’nmtratieemm
o’eotaxima-2
with time’ 231-o’cetaximm.
t’t)Uld
intrace-llular
a robust
(af eotaxin
anti
re-oiuire-ol
‘The
RBL-2H3
shtaweoi
addiiioan
(final
square’s).
anti
Re’pnc’sc’rmtative’
activity
>
[44,
the-rekreThue-so’
his
at (hue binding
too che-more-ce’la(e)rs
CCR3.
7) to the
nM).
1, 2, and
o respeand
0. I .5 tiM
(eoioe’ma sqtmane’s).
too NICP-4 fear
eotaxin
approapniate
(bIle-ti
100
[nM]
231-e’cetaximm-2
ine’uboate’th
\Ie-thcoois.
bout float (ho’ pnima’)
cells
calcium.
tells
loaded
transient
these
the
(-N)
e’climxirm
(00100-mi circle’s). wtrc’
oo
bindirug
systems.
shown
with
RBL-2113
transf’e-cted
0.4
he-en
transfected
o’onc’e’nitratiton
o)n \ICP-4
ne-asoon
itis
in RBL-2H3
cells.
(final
Materials
oathe-r
Calcium mobilization expressing CCR3
CCi1t3
ehonimekinits
itiemutical
additioan.
>
c’ino’lts).
10
Icon e’ac’im pceinit.
eoataxin-2
e-o(axin
(FlO
al die-
((jIb-cl o’iro’les). R-NN1ES (4 X I 0 c’elIs/mmmI) in
1
chemokine
2l-o’cotaxinm
(filbod
olo’so’ribt-d
(results
of
0.1
100
c-c-hIs
CCR2B
chemokino’ =
in all cell
as
pro’so’nteeh
is
cloned
R3
o’ootaxin-2
o’ho’nmcekinmt
the
for
etotaxinm-2
C 110-CC
respectively).
e-otaxin-2
MIP-la
as (-N) c’xe’ciot
oX(’ept
sammmples
bind
oaf pootency
there-lore>>
(B)
(tilIed
in
confirnuing
10
tto hmtmmrmanmecasinicopiiils
conditions
eel o’oold
2 nM,
(‘ailed
order
was
as
ecotaxima
expressed
thus The-
1
in the- abso’mmc’e- or prt’stno’e-
t’oataximu (f’mlleti sqtiano-s).
commclitieons
3, 7, and
rece-ators
CCR3.
(B) Same
c’onict’nitratioani
also
25l-cotaxinm-2
eosimicopiuils/nmL
time’ rime’ati (of eitiiahio’ate
‘25l-e-otaxin-2
shown),
triangle’s).
fear eat-li
arid
I0
X
duo’ o-iie-nimeakimies
is shci-n.
CCR5
2
Samime’
0.1
Chemokine
e’eotaxin
with
time’ cobol e’imemokines
binoiinmg
penimlients
100
[nM]
col ‘25I-ialao-led
or NI I P- I a (fillcol
eotaxin
0
0
0
spet-ifie-
20
20
20
ciro’Ii’s).
40
C)
0
0
60
C 0
C
C 0
C
80
0
80
0
cc
=
100 0) C
0) C
0)
Characterization
all
proamiltatein tho- low
whao-ro-as liganti
in
of eoataxin-2
e-eotaxin human
673
o’he-makino-s
twa
300
...
primarily
tiemo)nstrateti
by the-
RANTES
in
ulilizo’
we-ak
RBL-2H3-CCR3
This
CCR1.
calcium
was
response
l’urthe-r
e-xhihaite-d
ce-bbs (EC1-,
70
=
by
nM)
com-
C
paro’tl a)
witlu its ptatent eak-ium mnoalailization in human eosin(EC-00 = 6 nN--I). In addition, NICP-3 anti RANTES
to1ahils
0) C CO
200
0
E
were
we-ak
anti
e(asintopbuils,
lowo’r
CO
evor,
0
o)r
0.1
1
10
Chemokine Fig.
I )cose’-re’sjeeenmso’
7.
l( :a2]
1ee’jk eel
(filled
circle’s)
so’rc’
late-cl
leer
e’ac’lm
tc e cells
anicl
c’imo’nmmoekinio’
ro’sioeonmso-.
1000
[nM]
‘c-ll
(coioe’nm soluaro’s).
o’eonmc’e’ntraticenm
n’c’siooense
i!]
do-so-nbc-tI
as
sttmcl
in
ccl a so-rio’s
nioo time inmoost was
cat-itatis
and
1000(0-rat
o’hae-iuiookinie’
t)J)huils.
The’
ro’asoani
boor this
vioatis.
Hoowoor.
cotho-rs
using
lonm
ol the
human
have-
been
oaf o-haeiumoakino’
[0),
1 ()].
alsoe
ooo(o-no-y
simnilar
-
oiive-rgo-muo’e’
and
o-oataxin.
apoo’ars
reflect quantities
the-
fact or
tively. e’x-
R3
in
ef’fietasin-
is noat (oh-
be-c-n
tabse-nvt-oi
anutong
f’oor thie-se’
H3-C(
age-nit
thio’ relative-
(to diff’e’r
tanoio’r ooote-nt’y
ira R 13 1-2
oohataino’oi
has
whit-re-
assay.
In
higbio-r
niaxintial
thoeugla
o-oataxima-2
\%‘ith
simuailar
(l’(-()
protiuced
that-se-
toa that
obtaint’oi
in
aanfial
ageamiist
nesptonse
l)y
ne-o-o’}otoor, i.o’. (:du3. with
t-roass-deso’nsiiizatiton
lii
t-henuiookint-s
‘eonutrast,
RN’FFS-.
\IIP-
o-ootaxin-2 I a-.
an
uitohoilizatioon.
cabo’iuria
naoobilizatiton
lizinig
(:(.:R
R-iNTFS
anti
:i,
kirios
cc:
alsoo
o’iro’umiivo-n
R3.
I ii fio-,
MIP- ha anti
674
utilizo-
‘lhuis
tan
Journal
is o’oansise-n
a e-eammtanu cab-
CCR3.
yt’t
is pno-snnaahaly ao-tivato(hit-
R\IVFES
of Leukoaey(e
vo-re-
CCR
1 })ro’sonit
oho’se-nsitizinig
hio-to-raloogoous iii
NI I P- 1 a solo-ly
neat
be-o’ause
5,
Biology
suggo-sts
monocyte-
that
has
small
Alterna-
in a very
tran-
iii
library.
thus it was
m(anocyte
However.
potent
ao’tiviy
to
the
so)uro’e-.
through
with
ctample-e-ly
the-
e-o)sinophil
mediated
experiments
tarder
and
Be-cause
a very
its
expre-ssionu
needed
diseased
tis-
understand
in e-osino)phil-me-(liate-d
twea
the
disease-s.
anti
C. it..
\.,
Isimizaka.
J,
Exp. Ncescc,
8.
K..
the-se
-6(1(1.
‘I’..
t!(’(!,
and
j9t)7
,J,, Nlieicllc’tccni,
(.
1. lie ., Eltisse’.
( 10)88)
‘I’.
F.. V..
J’ ‘dI((!,
I,’,nrI,
11cc-cl, C. F.,
(Ic)))3)
o’els,
I d)33)
Selective’
induced
Ellis,
Ecsirie-
J..
clitli’rc’nmtimiiceni
and
in- rc’c’c)nmmi)inlanmt Imonmiani in-
85. 2288-22#{176})2. Ni.. Nliimra.
( .S1
‘t .. ilaracla. N.. 5 simicieerts time ter-
l’;guc’bi.
lnirific’c1
i)rcolte-r(ctcm
initc’rlc-okinm (of nmonimic’
‘I’.. ilrommnmc-r. ‘l.
Ilaggieclinmi.
-\.
Ni.
leaso1oimil-
7.51-7.56. l#{176}rcocesi. 10,, Nan
N..
Se,, L1dIa.
lx. (
a rmmcost effective’
1
e’csino1ohilic’
lro-’tmr-
(I d)d)4)
Norm ‘l’sc’imarno’r. Nlcumcec’vtc’
1).,
N .. ( :alelmt.
(‘imo’flicctae’tic’
anmd c’cesincelelmil-ac’tivatinmg
c’Emc’nmcokinc’,
179,
lOanmnmo.
i0rct0’i52
J..
Sciirocie’r.
J,
.3 (N1(I#{176}-2and
and
N-I. ( 1 O)1)
Human
NIC1#{176}-3)attract
ium-
clc’so’nisitizo’ time’ rimenimcotac’tic’ resfoenmso’s tecisarchs LtmO(Im(-’,Im,!Jm()/)!mi.’, Rs, (oiimiimun. 200. I -(701476. and
t-eesinmceolmiIs
time’ nmiigratiton
cOos. J, I0alfernm.
ac’tivatiecn
cci ueornmal
imtinmman c’osineophmil
grariuko-
t!’d.
Icor imtmmmmanm e’cc0iflc)0Iiils
cot nme’utrcio1ohils
tgtmc’e’icnmi.
and
176. 1-1W)- 141)5, l. .1.. i1c’ifc’r, 1, ii.. Emmmioc’r.j.
E-j.
181. 21 I)-2 (),
\,
R;N’I’ES. Hot. -N.. Krie’gc-r. NI.. l3ronmrme’r. ‘L. llisc’imolf. . (.. Schall. ‘1’. J.. I)ahindeni. C, A. ( I e)c)2) lt-N’l’ES armoi nmacrocimago’ inilanmmnmatecr ircetc’imm I mlioima iii-
lationm
l)t’tweeli
o-esinioclmils,
&csieo-(’ts. lii 41/org) PrdFm(i/)1c.s (111(1 Pr(I(’lIce, l6#{176})-200, A. NI.. I,e’ilc’mmamm. K, NI.. I)onmnmc-niiocrg. \. 10..
Isimizaka,
--k.. Geiso’r.
(;.
1#{176}.. NIinmt.
c’lmc-nime)taxini
at
cf
th’(I, 167. -(3-56.
Lxjo,
1 Oaiminiclo’nm.
nmmanm
7.
(.Io-ic’im,
‘1onginmgo-r.
cc! Imc-mmiatccioc)ic’ti(’ tolls
to-rlc’okimms. Proc. NUll, Nanimagtmc’lmi. ‘0.. Soda. ‘I’toniminmaga. -i.. ‘I’akatso.
lure’
amid
immmnmmtmniccbicclog’o
1ii-iil Ieiccc’imc’nmiistr\ omicl cellular -(tim ccl,, St. I_onus. Nit): Nlcsln. Sajtce, El.. Uatako’. 14,,,. I Ovcerak.
teifl
l)y
Noalunmme (a2, Ntovenimboo-r
I ) ‘l’lmc
-Ndolphseonm.
J,
o’hemoa-
that
1#{176}. F. ( I d)c)
l’errara.
uti-
cal e’eotaxin-2
(‘nooss-d(-se’nsitiza(ion o’oasiraoahiils
NICIP-3
tan eoasiriopliiis
action
very
stimuli
difficult.
is clearly
mmmconcoc’ytc-c’imcnm(Ota(’tic’
tlo-se’rusitizo-ti
the’se-
in cells.
0)!
may
1 1 Ill-I I 18.
-riu.
N-II l- I a-mtdiae’ti htatbi
be
\1ce’llc’r.
ors.
o’roass-
Hoowevo’r.
pat-
This
l)e- produced
as oarue- potential
nmminal clifli-re’ntiaticemm
not
with
activated
factoar
Furthe’r
prolife’m’atiomi
is
oar N-ICP-3-induo’oti
too oiio-so’rasifize-
I tori o-oosinioo1ahibs. tan
ooataxin-2. thus
Faibtmro-
mature
(hue- expre-ssie)n
by unknown
particularly
F, F.. -Ncikimmsccnm, ,J. IN. I”..
(o.
ciurn
human
the
che-mokine-.
is pr(oduce-d
may
eotaxin-2
o)f eotaxin-2
o’oosinoa1ahils
oiid
thewith
re-pe-nt(oire-
induced
fram
role
o’xfaeni-
sharod
eotaxin-2
detection
will
4,
(lire-c’
exactly
0)1 this
a restricted
being
sue-
ce-lbs
tan
that
be- consioiere-d
CCR3.
al-
I nNl)
MCP-4
the- f)ro.sophila
[50].
localization
their
2 .Sur.S,..
o-e)taxin-2,
RBL-2H3-CCR3
anti
using
two
(or MCP-
expresso-d
to de-te-riuuine-
o’luomoakine-s
manner,
o-bae-mnotactie’
I,
muitobili-
-o’lls is neat e’le-ar
o-ooaxin,
woork,
oeathxin
was
inve-stigatitami.
o-ootaxinu-2.
that
do’so’musitize-
=
!‘oan this
o-hie-miiookimio’
Losing
inielicato-oi
(ho-
MCP-4
for
with
within
REFERENCES
laharatoanio’s
than
starts
by
chmo’rnookimue-s
and
RI3L-2F13-CCR3
that-
ci nob-n
Ho’to-rodoagoaus
nit-ruts
eotaximu
How-
e-otaxin-2
also up
bias
5o)urce-.
oardo’r
mu thio’ o’abo’iumn
no-spoanso’s
stiniiulate-ti
i’lao’ ro’asoon ira
pro’so-n(by
to-hIs.
oi-abo’itmnii
jooto-no’y
e-easinioalabails. eootaxini-2
these’
or MCP-4.
oaf eoataxin-2
human
sie-s
line-s
proate-in
by
some
sient
324.
zatioomi
start
have
unable-
oar the- tissue-
(copc’rm
thie’ most
oiiscrt’pamucy
e-otaxin-2,
e-otaxin-2
In parallel which
o’ale’tm-
eel thre’e’
ago-mit
have
tee stinm-
t-he-niioatactit’
c-honaootao’tic
apparo-mut
a similar \ICP-4
Ptotonit
o-oonsis(en(ly
the
HCC-1.
che’mokine Etoaximi-2
that
0)!
che-mokine
flutist
sas
cells
woa ligands
Drosophila-derive-ti
acids [46].
CFIO-CCR3
form
authentio’
indit’ates
wo-
derived
it
of the
aminoa
making
mu (hit’ sanule- co’bls.
an
system,
tern
is shmcesnm.
ooosiraoo1ahiils
e-otaximu, mature-
expression
We
\lato-nials
nmNl laile-el
than
from
or MCP-2
three-
(lie-se-
in
square’s).
R-NJNFES
on’
[(
tm1o tee :3:30
-N ro’lore’se’nmtativo-
(hiIlooI
CCR3
isolate-ti
2, respectively
iii time’ pro-sc-nec’
c’cotaxini
time’ nimaxinmmti rim
NI I 10_ I to at e’cenme’e’nmtratieenis
tilato’ a ( :o2+ ieo’rinmme’nmts
::l-1:3
(hme’nmmcokinie’s. \l ( ; P-4
c’i role’s).
\ie’timcecls.
Concentration
11 11 1,-2113
(c!.
celolo’ch
100
hoer e’ime’mmuoekinme-stinmmtmlato’oh imme’reaso’s
-s
in Ftin’a-2-Ieoaclc’ch
o’cetmxinm-2
aimci
c’tmr
nmm\i c’xtrac’e’lltilcr
1
for
alignnuut-nt
e-o(axin
from
thuai
time’ biohagicaliy
mu(at lace-n
0
2I-e-otaximu
suggo-sting
affinity
Fca date-.
100
.3
at displacing
nit
-N., Elmigli. riot
1’. F.
( I e)c)5)
Icon mic’utrceptmils.
(ia
is a
I . ( 3a stimmmo-
is se’c’cemmdar-mtce e’osinmeo1elmil ac’tivatiecnm. J, Lip.
%!(‘d.
127. Ni..
I.ccc’tsc’imo-r.
1#{176}.. Ecersonmiammni.
t ., I Oc’walcl,
B..
I .i. II..
Linia.
S. H..
1.i.
( 1996)
Kre’iclc’r.
‘0 ..
Nkenee’yte’
funmctionmal
and
B..
Garotta.
chermmeetactic
anaheguc
U..
Time-len.
prei6’in NI(P-3
of
NI..
4 (NICP-4). and
Baggieolini.
a nmovel
J. E.vp.
c’otaxiim.
NI,
tied.
183. :3 1 .
2379-2384.
to,
Stellato.
(;..
1 1.
C.,
Collins.
‘r.. Sc’hle’imc’r. NIC1#{176}--t by- aincay
to other
(i-C
tof I L-5
Keonno.
S..
K..
lmiati(’
patie’nmts.
lonmg. J.
Adae’hi.
(,.
Mouse
lung
respeomise. Iiiinininii NI. E.. Luster.
allergic
Cconstitimtive guinca
Pomiath.
and
\o.. (;e-rarel.
tenizationm
C.,
4. 1 - I 4. A. 0.. Lilly.
J. Lvp.
Qinm. S..
Pcost.
M’d.
ily. Trend 17.
Nburphy. tant
18.
Plianimticol.
reccptcons.
Raioont.
J. NI..
J.. -tm. Nlole-cular
ing
the-’ ge’ne’
Icon a fune-titenal
Ceonmbadie’rc’.
C..
-\huja.
scmal koe’alizaticon. receptor-like’ germe.
21.
llIeuI.
Sl)F- 1 22.
Ra1oort,
and
is
liganci
a
for
of leukcoc’ytc’
RN1N
e-xpressionm
( I 996)
Loni-
Parohinm.
ireoteimm
1 ahioima
(‘he’nmmcokine
HIN-
I e’nmt.
Ni.
l...
Cray.
I’. )c..
aimd functional
functional
e’imaracterizaticoni
expression.
I. F.
of
anmd signaling
re’e’e’ptor. Ce-Il 72. 415-425. Van Riper. (i;.. Siriliano. S.. Fischer. P. 1N.. N-lc’urer. H.. Reosen. H. ( 1993) t3iaraetcrization and spec’ic-’s distribution ity (;Tl-#{176}-coim1alechre’ce-ptors tractant
25.
Pate-I.
for
I . J. Exp.
prone-in
N-. P., Kre-’ieler.
imunian
R:NN’I’ES
and
Li. II..
atlinm-
K.. Sah’e-eltm.
‘E. Narcle’lhi.
26.
27.
(icon and
-17.
28.
e’onstitutive
cells using 13e’rkimont,
proge’nitors.
e’Xprc’ssieoni
hygromye’ini ‘IL A.. Sarati,
1). B..
lmtourgia,
nmessy.
1)..
and
ciie’nmcokine
tunt’tional
Hansu1.
of time’ nmmconote
T. ‘F.. tOo’ \rie’s,
so-n. K., alke-r, time’ isolation
%Jetlr.145.
ioreocluc’ts
NI.. Nlakwana.
t’iiarae’tenizatieen c,he-rmie)tactir
that binds and signals through (;1m’rcm. 272. 16404i(o410.
105-I
it).
the- CC
I. J.. III, ‘F.. Ribs.
C. (1 0)9 j ) -Nn cot imighl ieurifieci
‘cled. 185.
in culture’
flrosoplmilu
Curr. Mci/n, Mo!. Bin!. 1. 1-8. K.. mite’. j. R.. Elstmceurbagy.
C.. Nle’Nulty. 10.. Nlattiieios. Ni.. (;rotot, P. H. F;.. Macphce’.
Scott.
e’xpression.
29.
eof genie’
B sek’c’tieon. H.. Nlceorcs,
E., Re’a1ee’. 1. J.. Brawner.
pc’Iloaum.
J. Exp.
cof a
nmc)ve’I
prcite-’inm (MCP) clme-nmmokirie
Bps.
(NICP-4)
Ni.. Be’tz.
S., Bla-
ioree’e’oltmre’
leer
J. lomnmunol.
(10)80))
S. ‘C
10)845_
1i
rc’-
19855.
em,!tm,ro-
of
insed(
Ni. E.. Sarah. :N. I).
timrcouglm
0..
ainays
Fim’
CC
I). N,
Hsuan.
c’imc’nmmcmkinme
CC
loascolohils
ineleme’ecl
c’Ime’nmomkine’
tisstme’-sioe’e’ific’
c’yttokinit-
1
J. Evp’
inmtlaimmmaticenm.
P. 10.. Roessi.
1). ‘L. Nlcee1bel.
J. ( 10)0)4)
dc’te’c’te’ei
197. 1 167-1
in
157.
time’ nuonieoc’vtc’
cariocoxyl-nail 56(16-56
I no-fomnmc’-
iereote’in
in re’c’e’iotcer
12.
Berm
P.
L.,
\\illiams.
cesinmeeicimils in Ilioelu’no, ho-
c’ime’nmmcoattrac’tanmt
re’ce-ptcons: (;romcllm
Xci.
J..
P.
B.. (3maro. I. F. ( I 0)0)6) inmioileits sigrmalirmg and
se’ninme’s/timrecenminme’s
Niurpimy. P. Ni. (I 90)6) Cime-rucokinme’ ill nmmic’roebial patimeogenesis. (vtokine-
.J. Ni. ( 10)95)
a
pig
172.
‘I’sceem. C. L.. Cceughlinm. S. a ( iorte’in-c’eenmioIe’tl kinaso’ of
Ecetaxinm:
a guinc’a
tie-cl. 179. 88 1-887. A. C.. Jccse,
J’. by
e’x-
J. I.nn,koe.
reo’e’lotcor-2.
P. 10.. \\-alslm.
Ccehlinms.
11. I..,
N. I.., ‘I’rcong.
iorooteinm-4:
e’he’nimcokinoc-’. c’c)taxinm. activates giminme’a-oig time-in accunmmmlationm mmdcc time- lung iii vivce.
-k..
Hemnmman
in althreenigh time’ CC c’he’imioe157. 5(a 13-5626.
and
J. J.. Williams.
10. -N.. (cellinis.
C., (;c)sling.
Barucim.
Ni. N..
( lt)96)
Ycoung.
II..
structure’. func’ticin and I”aetor Re’n’. 7. -(7-64.
Bo’nmgali.
Nieonmeoe’yte’ c’iie’mcotac’tic’ re’e’e’ietors_
Oioime’nmime’irnm. J. J..
iorcone’inm’
: ( : KR
(:(
K..
node’
(NICI#{176}3)inmtc’rae’ts
I . a re’c’e’letomr fur
inilanimnmmatore i)rcote’inm I alplma/hl-NN1I’.S. N1CP3. J. Bio!, (:1mm,. 270. 22123-22128.
is alsce
a ltmnmc’ticonmal re-
for
Rie’imardsonm.
H. NI.. 1)nBose’.
Snmvole’rnian.
R.
(I e)95)
H. -N.. :\li.
Re-geilatieonm
H.. ‘I’conmmlmave. K. I)..
eel imunman
Hanibabu.
initc’nle’ukin-8
B..
ne’c’e’iettor
iN:
a loimcosioimein’Iaticenm side’ inmvoolve’cl in nimeoclulatinmg nec’ciotcer functions. BiOe1r((1l. 34, I1lt)3_ 1-1201. Ric’imardsconm. R. NI.. Au. ii., ‘Feonimimave. E. I).. liariloalen, B.. Snyde-rrmian. II. d)f
Crcoss-chc’se’misitizaticon
270.
2782927833. S.. Rinmgle’n.
I#{176}cnmatim. P. 1).. Qini, N..
Smith.
j.
:..
col cimc’rmmceattrac’tanot
re’ec’iotcers
cec’c’urs
l’viclc’nmc’t’ leer a rode’ fcer inmimiieiticonm cd Piio)sleimc)Iiioase
U..
NIackay.
Simi. (:.
oootaxini.
X..
R_
I). J..
Conmzalee.
(I #{176})96) Cloonminig
F:xioressioim.
lark
(
J. A,.
ccl the’
W..
anmg.
J..
arid
Kassanm.
Gntierrcz
imunmman e’cosincoiehil
binding.
re’c’e’ptor
a nmme’c’imanisnm for time’ selective’
I..
1,c’wis,
Newnmman,
at nmmnh-
C activity.
funie’ticcnmal
Ranmos, e’ime’nmieoatIariee’rtie’s
rc’c’reiitnmme’nmt ccl e’csinmceioiiils.
J. (un.
97. 6()-1-6l2.
jeese’. P. J..
AeIe’eook, I. NI.. Gritlitims
jcohns(en.
10. A.. 13e’rknimaim. N.. L’lls,
N.,
\Nilliarmms, ‘L J_, i#{176}covo’r. C. A. ( 1 O)()4) i’cotaxirm: c’leenoinmg ccf aim o-cosimicoioimil clme’rmicoattrac’tant (‘te)kiniO anti inen-’ase’el nmRNN e’xierc’ssicen mm allo’rgc’ni-c’imallcnmge’el gmiino-a-pig
48.
lungs.
Biodieni.
N-i.K..
Rcotime’nmioc’rg. illdlu(’e’(l Kamc’yoshi.
( I 0)0)2) 50.
mcetc-nt Schulz loins,
(1
90)6)
A.
I)..
inelut’ileht’
(unmoor sul)lcre-ssionm. NY.. lOcorsoimner.
Cteekine
Rcs. (orlltlnIlcr.
Biophvs.
l.ustc’r.
ooioiiil (‘imenm(oattrac’tanmt 4d),
J. Biol.
cof
J. ho!.
at Ie’emkotnie’no’
(Ni ( :i#{176})-4is a ncevc’I
c’lme-nmceattrac’tant
cause’s
Franci.
I,mne’si.
CC
2B.
imorove-el inmmunomagrmc-tie’ Imunmanm i)lc)ce(I ecosincoialmils.
and
suggest
N.. Ao-
imunman
family-
re’ec-iotcor
S.. Vcaimelzilak.
vitro
pins.
trae’tant.
J.. Fcele’y. J. J’. Scimnimiolt. J.. O’l)cenmnme’Il. K.. O’ShanmC. (1997) Ckonming. in vitreo
S.. Crocekc’.
1,tmste’r.
c’hc’rmieeta’iic
Jcoimnmsccnm.
Jcehnmsonm,
J. ho!. 4(a.
cof mye’Ioid
(;niltitims
tiiolc’ levels,
1 163-1 172. Anmgelie’hio. NI. I... Beck. J’ A.. Johansen. H.. Ive-y Hoyle’. NI. (1991) Coenmmpaniston of several l0rnmmte-rs armol polyade’imylationm signals ben use iii lme’terologous genmc’ eXl)ressi(en in cultured Drosophila cells. \7uelcie J(Ids Rci. 19, 5037-5043. Nan I)e’r Strate’n. -\.. Johanso’nm. H.. Rose’rmbe-’rg. Ni.. ct a1. (1t)89) Immtreocltmcclasse’s
23400_2354.
Ierco1oo’ie’s.
t:.. Rcenhnnmioe’rg.
ceosinieeioimils.
Nionmc;c’-te’
eof aIIc’rgic’
(;niffiths
( I 0)95)
B..
Pippalla. NI, Ge-ritz, S., ‘fluotakura. R.. et al ( I 0)97) Nlolecular amid functiconmal c’imarae’te-nizatic)nl eel two novel imunmian C-C c’imc’meikines as inmimileitors ccl t%ce distinct
183.
activity
1#{176}. Ni..
ierc)teinm
signaling
ide’ntitie’atioenm
e’he’nmmcoat-
flid)n000’ytO’
J. J.. Nicong. agonist
i)a’ah
1... Sinco-
c’imarac’to’nizatiooni
rmc’w ge’nmc’rationm
\
and -3. J. IlllfllIllmO!. 10.. itqoort. C. J.. Sc’imwe’ic’kant.
N. F.. Truong.
oimage’
45.
I.eung.
J..
I-.
Totty.
e’e’ptc)r
NI. S..
cot high
and
61. 35:3-360.
\anmg.
44. Springer.
Me(!, 177. 851-8S6.
B. L.. Li, 1.
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NIcecol a
c’imaraetc’nistie’s
250.
flier.
Nlalkcowitz.
\..
llemeorc’sce’nc’e’
N-I.. lolow.
Niuqehy.
% itim nmmtmltiple’ lc’ukcoc’vte’
C-C chemokinme 24.
(1d)d)7)
.
(icon. J. Jni,iiu,mol.
“salur(’
(3mareo.
E.vp.
inflammmnmaticenm timit signals
ior(enicotes internalization ceiotcer. Critical noel.- of
d’Imc’nimceattrmctcnmt
liii-
cloning.
-1-1 .
I.. See-
lewis.
J.
l’
nmeenicec’yte’s.
PimeespheoryIatieon
anici
cloning
P. \\
‘I,. J. ( I 90)3)
e’imreermmee-
a novel
Ic’cemlar
40.
42. Sc’imwcickart,
on
anmel nmconallergic’
nimodel
inmc’kmcl-
‘mi-In
23.
H..
tissuc-’-s1oe’c’ilic
C.. Clark
blcocks
Q..
Hanmicl.
joeote’nmt c’c)sine)1ohil
lynmoImocyio-
and
NI..
kimme- re’(’e3)tors (CCII)-2 (cdiska, R.. Cimantr’.
Bio!.
The’
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F..
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CC c1mcnmmckine rc-(’e’ptor (CCRS) ton RAN’FES, Ni I P- 1 ioetc. Nlli#{176}-lalpha. J. Bicil. C/menc. 271. 17161-17 166. Nc’ote. K.. 1)iGregeonio, 1).. NIak. J. 1.. Horuk. B.. Se’haII. ‘L J. (1993)
Nltolee’ular
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3#{176}). loose,
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K. E.. IOc’rse. C. 1’. cam dc’grarmnlatieon.
l -#{176})37 tells. J. Rio!. (:Ime,im. 264. Nltmrnanme’, A.. (10)0)2) %kdiu /r
nm000nie)e’yte’ c’imc-’nmceattrac’tant
1#{176}. \\.
(3onming.
In
anmol lcernmmaticorm cof le’uko-
o’xiorc-ssioni.
impne)vc’ol
Il.
with
N 28 is in Iy-nmm-
P. Ni. (1995)
nmcoimoo-
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102.
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lOc’Niantirmco.
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irmtlamnmmatcery
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J..
S.
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l.avigne.
17494-17501. Murphy.
with
in difte’rc’ntiato’d
llamcos.
T B.. Cra.
macreoplmagc-
c-ndcetimc’hium nmeutrce1eimils.
and
g raelie’nmt
(i’llS.
chc,mmmoattrac-
ret’e’1oteor-hike’
clonsity
eosinopiiil c’otaxin rec’c’iatcor. ./‘ (;,. Pcoe’nmie’, Ni., ‘Fsic-’n, H. 0 . (I 0)85)
(:Imerem. 260. 3440-3450. Satmssy-. I). l... Jr.. Sarum.
re’ceptor-c’nrodinmg ge-nero-co-lotors and is c’xioressc’dl
chenmokine’
vascular
hunmaim
(‘imarad’-
re(’e’pt(or
granmulcoe’vto’s
-Nc’aele’rmmic l#{176}re’ss. 88-
NI. S. ( 1 d)O)6) Clconinmg.
inmdi(’ators
Io’rgic
R. L. R.. Sheows.
S. K..
J..
the’ unman
12. 5t)3_633.
LESTR/fusin
829-833. C. J.. Gceslinmg.
36.
38. bicilcegy
I)-”e1 (;‘l! Bin!. C. C.. Farzan. NI.. Choc. H.. J.. Seninigc’r. T. !, (1 996)
clroski.
382.
beta
J. Bin!. (:Im(’nm. 270.
receptor.
20.
1#{176}.
nimR NA
on
tel time’ ciie’mokinmc’
V. L.. Eddy.
mouse’
Le’ele’r.
(‘Ie)nminmg anti sele-ctive-ly-
to
B. I... Sicilianmce.
Niechanisnims
a
N. P.. Nc-w-
phoid and nmetmral tissue’s. Go-eme 163. 295-29). 1#{176}). (;aci. J. i... N-hmrphy. P. N-I. (1995) Clonitig anmd oliffene’iitial cof time-c
to
159-165.
onioiman G-protc’in-couoIee1 re-lat-d to gene-s ton cime’nmokine’
e-x1)ression
:35.
37.
1... Gerard,
iainching
(;--nmkio’-ie’z.
(:a
aeon-
eol e’cotaxin
icoehinmate’cl
Ne’w ‘ten’k:
ie- timnitit’cl
IOmugimc.t-. eof
NI.. Cutie’rrcz restricted
using
Ni. J. e’atioons.
J. Cell .Sd. 45. 73-86.
sembstratc’.
d)f Iynmmjeimcoc’te’s.
H. P.. Frc’e’laimel. II. S.. l#{176}e-te’rs. S. P.. Breewni. cot time’ e’ffe’cts eel gIuccot’eorticeoiels
timid. A.. Soringe’r.
12 1 1-1216.
properties
Time-’
closely
riot
is
NI.. lJraze’n.
e-xi)re-sse(h
Rem’. Innnrunol.
C. J.. Seliweickart.
( 19t)5)
it
exprc’ssieenm
181.
[1mo’ nmolec’ular
-Intro.
:33,
N. -\.
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and
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Kco1of.
bloated
B. U..
: roole’s cmf elivalc’nt
An asscssnmment
tric-nmc B4
cot astim-
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imunman
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i-i,
C.
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froni
-.. Vare’.
\\.
tee o’nclcotimc’hiuni
c’heinmmcitae’tic’ age-lots
(1t)84)
A.
c’e’itrs
(10)96)
C. R.
c’otaxin
1ahils
16.
lout
1. \%., Vanmg.
NIae’kay.
c)f a hunian
nm spotuni
paralle’ls
inflanmnmatiton
aIlc’rgenm-incluc’o’el
joig lung.
1)..
P.
32.
clme’nmmeoattractanits
(‘ono’t’nmtrations 73-78.
cxi)re-ssie)nm
charge’.
Boeyunm.
cimnnimctaxis.
Inmnmu,mol. 109.
Ecotaxin
Th2-type’ Rotimenberg.
nman.
Cytokinie
surface
1101110(1) in Lrrzvnmolcigv.
P. (l#{176})96)lcIe’nmti-
cosineoloimil
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cot the’ novel C-C c’hc’its i)ie)Ieogical activity
S.. Vc-nge-.
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R.
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.4lIert.rv
during
in the
L.. Rak.
Hcetooer’,
Rosa.
Korokawa.
(1O)d)(a)
NI.
lent. Arc/i,
mulation
( 1 995) 1.5.
1llcr.’v
.,
99. 926936.
incest.
as the
‘i..
Ranmios, J. C. ( I 90)6)
14.
C/in.
of
J. -N., Jia. (,. Q.. Agimirre-. V. Friend. 1).. (Iole-. S.. Katz. H.. Lic’imtnmmani. A.. Ceo1oeland. N..
Ganzalo. Lin.
J,
R-NNTES
(;conokami.
moto,
L. N-I.. Bickel,
NI.. Hakarissonm.
arid
1).. Newnmmani.
El. P. ( I 997) Production coIls antI (‘onmioarison
c’ime’nmokimio’s.
\e’mmge. J.. Lanmpinen.
La C.. Liu. NI.. Heichner.
i#{176}eonoathm. P. 1).. Scolo-r.
iIliams, nue)kine
in time astimnmatic
13.
P..
J.. S(’im-iehae’,t,
Li. I-I.. White’.
tication
12.
:10).
structural
RNNTES
in eneicotimc’hiah
I#{176}roe’. -Nut!. %.. Nialle’t..
released
205.
788_70)4.
l.c’ole’n. 1#{176}. ( 10)95) Niimninme c’cctaxinm: aim ncesiim‘lead.
ce’lls .‘l(i.
anti
in inmterle’ukinm
4-
ISA 92. 80)(oO_80)64.
-. I,, timnistceioimcrs. F ...S o’imroole’r. J. Ni. by timrcenmleinm-stinmulatc-oI elateIe’ts is a
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Knappe’.
183. 295-2#{176})#{176}).
h/mite et a!.
Characterization
of eotaxin-2
675
