WIP and WASP play complemen a y oles in T cell
homing and chemo axis o SDF-1a
Ma ia Dolo es Gallego
1
*, Miguel A. de la Fuen e
1
*, Ines M. An on
2
, Sco Snappe
3
,
Robe Fuhlb igge
4
and Rai S. Geha
1
1
Di ision o Immunology, Child en’s Hospi al, 300 Longwood A enue, Bos on, MA 02115, USA
2
Cen o de Biologı
´a Molecula Se e o Ochoa, Uni e sidad Au o
´noma de Mad id, Mad id 28049, Spain
3
Gas oin es inal Uni and he Cen e o he S udy o In lamma o y Bowel Diseases, Massachuse s Gene al Hospi al,
Bos on, 02115,
4
Ha a d Skin Diseases Cen e and Depa men o Pedia ics Medicine and Depa men o De ma ology,
Ha a d Medical School, Bos on, MA 02115, USA
Keywo ds: WASP, WIP, chemo axis, SDF-1a
Abs ac
Homing o lymphocy es o issues is a biologically impo an mul is ep p ocess ha in ol es selec in-
dependen olling, in eg in-dependen adhesion and chemokine-di ec ed chemo axis. The ac in
cy oskele on plays a cen al ole in lymphocy e adhesion and mo ili y. Wisko –Ald ich synd ome
p o ein (WASP), he p oduc o he gene mu a ed in Wisko –Ald ich synd ome, and i s pa ne ,
he Wisko –Ald ich synd ome p o ein-in e ac ing p o ein (WIP), play impo an oles in ac in
e-o ganiza ion in T lymphocy es. We used mice wi h dis up ion o he WASP and WIP genes o
examine he ole o WASP and WIP in T cell homing. T cell homing o spleen and lymph nodes in i o
was de icien in WASP
ÿ/ÿ
and WIP
ÿ/ÿ
mice and se e ely impai ed in WASP
ÿ/ÿ
WIP
ÿ/ÿ
double knockou
(DKO) mice. De iciency o WASP, WIP o bo h did no in e e e wi h selec in-dependen olling o
in eg in-dependen adhesion o T cells in i o. Chemo axis o s omal cell-de i ed ac o -1a(SDF-1a)
in i o was mildly educed in T cells om WASP
ÿ/ÿ
mice. In con as , i was signi ican ly impai ed in
T cells om WIP
ÿ/ÿ
mice and se e ely educed in T cells om DKO mice. Cellula F-ac in inc ease
ollowing SDF-1as imula ion was no mal in WASP
ÿ/ÿ
and WIP
ÿ/ÿ
T cells, bu se e ely educed in
T cells om DKO mice. Ac in e-o ganiza ion and pola iza ion in esponse o SDF-1awas abno mal in
T cells om all knockou mice. Ea ly biochemical e en s ollowing SDF-1as imula ion ha a e
impo an o chemo axis and ha included phospho yla ion o Lck, co ilin, PAK1 and ex acellula
egula ed kinase (E k) and GTP loading o Rac-1 we e examined in T cells om DKO mice and ound o
be no mal. These esul s sugges ha WASP and WIP a e no essen ial o T lymphocy e olling and
adhesion, bu play impo an and pa ially edundan oles in T cell chemo axis in i o and homing
in i o and unc ion downs eam o small GTPases.
In oduc ion
Cell mig a ion is a undamen al biological mul is ep p ocess
in ol ing memb ane pola iza ion and changes in he cy o-
skele on (1, 2). Lymphocy e eci cula ion in o lymphoid and
non-lymphoid issues begins wi h blood lymphocy es in e ac -
ing ansien ly and e e sibly wi h ascula endo helium
selec ins in a p ocess called olling (3). Rolling b ings
lymphocy es in o con ac wi h he endo helium whe e hey
a e ac i a ed by issue-de i ed chemokines displayed on
he su ace o endo helial cells. Chemokine ac i a ion up-
egula es he a ini y o lymphocy e in eg ins o hei ligands on
endo helial cells, esul ing in i m adhesion o he lymphocy e
o he essel wall and subsequen mig a ion ac oss he
endo helium whe e issue-associa ed chemokine g adien s
may also di ec localiza ion (4, 5).
Chemokines a e a class o molecules ha binds o ecep o s
coupled o he e o ime ic G p o eins o induce he mo emen
o immune cells owa d a concen a ion g adien o he
cogna e ligand (6). S omal cell-de i ed ac o -1a(SDF-1a)is
hough o be he p imo dial chemokine (7). I is highly
conse ed in mammals (wi h a homology o >95% be ween
*These au ho s con ibu ed equally o his wo k.
Co espondence o: R. S. Geha; E-mail: ai .geha@child ens.ha a d.edu Recei ed 27 Janua y 2005, accep ed 15 June 2005
T ansmi ing edi o : K. Rajewsky Ad ance Access publica ion 1 Sep embe 2005
In e na ional Immunology, Vol. 18, No. 2, pp. 221–232
doi:10.1093/in imm/dxh310
ªThe Japanese Socie y o Immunology. 2005. All igh s ese ed.
Fo pe missions, please e-mail: [email p o ec ed]
a E ns May Lib a y o he Museum Comp Zoology, Ha a d Uni e si y on Augus 26, 2013h p://in imm.ox o djou nals.o g/Downloaded om
human and mouse), and is he only chemokine ha is equisi e
o su i al. Dis up ion o ei he SDF-1ao i s ecep o CXCR4
is le hal in mice and is accompanied by se e al de ec s (8, 9).
CXCR4 is exp essed on all hema opoie ic cells (10) and has
also been iden i ied as a p incipal co- ecep o , in addi ion o
CD4, o he en y o he T cells- opic HIV in o i s a ge cells
(11, 12).
The ac in cy oskele on plays a cen al ole in cell mo ili y,
mo phology, phagocy osis and cy okinesis (13). I is spa ially
and dynamically o ganized, p o iding o ce o he shape
change and su ace mo emen in mos euka yo ic cells (14,
15). Rea angemen o ac in is e oked apidly by ex acellula
s imuli, including chemokines (16). Wisko –Ald ich synd ome
p o ein (WASP), he p oduc o he gene mu a ed in Wisko –
Ald ich synd ome (WAS), plays an impo an ole in ac in
polyme iza ion in hema opoie ic cells (17, 18). Lymphocy es
om pa ien s wi h WAS ha e abno mal cy oskele al a chi ec-
u e (19). T cells om hese pa ien s and om WASP-de icien
mice ha e de ec i e ac in polyme iza ion and impai ed p o-
li e a ion in esponse o engagemen o hei TCR (20–22).
WASP has an N- e minal Ena/VASP homology domain 1
(EVH1) domain, a Cdc42/Rac GTPase-binding domain (GBD),
a p oline- ich domain, a G-ac in-binding e p olin homology
(V) domain, a co ilin homology (C) domain and a C- e minal
acidic (A) segmen . WASP in e ac s wi h he Wisko –Ald ich
synd ome p o ein-in e ac ing p o ein (WIP) ia i s EVH1
domain (23), wi h Cdc42–GTP ia i s GBD domain, wi h
mul iple SH3 domain con aining p o eins ia i s p oline- ich
egion (24) and wi h ac in and he A p2/3 complex ia i s VCA
domain. WASP exis s in cells in a closed inac i e con o ma ion
due o in amolecula in e ac ions ha p e en he C- e minal
acidic domain om in e ac ing wi h he A p2/3 complex.
Binding o Cdc42–GTP o o SH3 domain o p o eins such as
Nck, G b2 and co ac in is hough o cause a con o ma ional
change in WASP, which allows he VCA domain o in e ac wi h
and ac i a e he A p2/3 complex (25–27) o egula e ac in
polyme iza ion.
WIP is exp essed a high le els in lymphoid issues (23). WIP
binds ac in ia i s VH domain loca ed a he N- e minal end o
he molecule (a.a. 1–151) and binds WASP ia a sequence in
i s C- e minal end (a.a. 460–485). In lymphocy es, >95% o
WASP is complexed wi h WIP (28). WIP plays an impo an ole
in he ec ui men o he WIP–WASP complex o ZAP-70
ollowing TCR liga ion. TCR liga ion causes p o ein kinase
Ch-dependen WIP phospho yla ion and disengagemen o
WASP om he WIP–WASP complex, allowing WASP ac i a ion
by he small GTPase Cdc42 (28). A ole o WIP in Tcell ac i a ion
was demons a ed by he s udy o WIP knockou (KO) mice.
T cells om hese mice ail o polyme ize F-ac in, p oli e a e,
pola ize and ex end p o usions ollowing TCR liga ion. Fu he -
mo e, he ac in cy oskele on is dis up ed in WIP
ÿ/ÿ
Tcells (29).
Monocy es om WAS pa ien s ha e a educed chemo ac ic
esponse o MCP-1, MIP-1aand FMLP (30, 31) and hei
T lymphocy es display an abno mal chemo axis in esponse
o SDF-1a(32). These da a sugges ha WASP is impo an
o chemo axis. Gi en he ole o WASP and WIP in ac in e-
o ganiza ion in T cells, we ook ad an age o he a ailabili y o
mice de icien in WASP, WIP o bo h o examine he ole o
WASP and WIP in he homing o T cells o pe iphe al lymphoid
o gans and in T lymphocy e olling, adhesion and chemo axis.
Me hods
Mice
The gene a ion o WASP- and WIP-de icien mice has been
desc ibed (21, 29). WASP
ÿ/0
WIP
+/ÿ
males we e b ed wi h
WASP
ÿ/ÿ
WIP
+/ÿ
emales o gene a e WASP
ÿ/ÿ
WIP
ÿ/ÿ
double
knockou (DKO) mice. All mice we e om mixed 129S 3
C57Bl6 backg ound. Wild- ype (WT) li e ma es we e used
as con ols.
An ibodies and FACS analysis
Fluo och ome-labeled mAbs [an i-CXCR4–PE, an i-VLA-4–PE
(CD49d), an i-VLA-5–PE (CD49e) and an i-CD62L–PE] we e
ob ained om BD Biosciences (San Diego, CA, USA) and
used o s ain cells which we e analyzed by FACS. Exp ession
o E-selec in ligands was de e mined by low cy ome y using
ecombinan mouse E-selec in Fc chime a (R&D Sys ems,
Inc., Minneapolis, MN, USA). Cells we e incuba ed sequen-
ially (30 min a 4C) wi h Fc-block (CD16/CD32, BD
Biosciences), E-selec in chime a (5 lgml
ÿ1
), goa F(ab9)
2
an i-human IgG–bio in (3 lgml
ÿ1
; Cal ag, Bu lingame, CA,
USA), s ep a idin–PE (2.5 lgml
ÿ1
, BD Biosciences) and
conjuga ed an ibodies, an i-CD3–FITC o hams e IgG–FITC
(BD Biosciences). Analysis was pe o med on a Bec on
Dickinson FACScan IV using CellQues so wa e. Aliquo s o
cells we e p ocessed in HBSS con aining 10 mM HEPES and
5% FCS plus ei he 2 mM CaCl
2
o 5 mM EDTA, o con i m
calcium-dependen binding. As an addi ional nega i e con ol,
aliquo s o cells we e s ained wi hou E-selec in chime a
(seconda y an ibody, s ep a idin–PE and conjuga ed an i-
bodies only) in 2 mM CaCl
2
.
In i o homing o T cells
Tcells we e pu i ied (usually >90% CD3
+
cells) om spleen by
nega i e selec ion using he T Cell En ichmen Colums ki
(R&D Sys ems). Pu i ied Tcells om WT mice we e labeled o
15 min wi h 10 lgml
ÿ1
o Alexa-Fluo 488 (Molecula P obes)
o o 10 min wi h 0.8 lgml
ÿ1
o e ame hyl hodamine-5(and
6)-iso hiocyana e (TRITC, Molecua P obes), a 37C. T cells
om WASP
ÿ/ÿ
, WIP
ÿ/ÿ
and DKO we e labeled wi h TRITC.
Cells we e hen cen i uged, washed and e-suspended in
RPMI. A o al o 8 310
6
TRITC-labeled WT, WASP
ÿ/ÿ
, WIP
ÿ/ÿ
and DKO T cells we e mixed wi h an equal numbe o Alexa-
luo -labeled WT T cells and injec ed in a enously in o
ecipien mice. A e 1 h, ecipien mice we e killed and
spleen, inguinal lymph nodes (LNs) and mesen e ic LNs we e
ha es ed. Single-cell suspensions we e analyzed by low
cy ome y, ga ing on 3 310
5
li e lymphocy es and he
pe cen ages o TRITC
+
and Alexa- luo
+
cells we e de e -
mined. T cell homing index was calcula ed as he a io o
TRITC-labeled o Alexa-labeled cells.
Lamina low assay o T cell a achmen o selec in
Selec in ligand unc ion was de e mined in a pa allel-pla e
low chambe using subs a es p epa ed wi h E-selec in o
P-selec in IgG chime as as desc ibed (33). T cells we e
washed in HBSS con aining 10 mM HEPES (H/H) and aliquo s
we e suspended a 1–2 310
6
ml
ÿ1
in H/H con aining 2 mM
CaCl
2
o indi idual assay uns. Cells we e loaded in o he low
222 WIP and WASP in T cell homing and chemo axis o SDF-1a
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chambe and allowed o se le o 2 min. Flow was hen
ini ia ed a 0.28 dyne cm
ÿ2
o 1 min ollowed by s epwise
inc eases in wall shea s ess e e y 15 s up o 1.40 dyne cm
ÿ2
.
Cells exp essing unc ional selec in ligand emain a ached o
he subs a e and oll in esponse o he applica ion o shea .
Calcium-dependen binding was con i med a he end o each
un by pe using he chambe wi h EDTA (5 mM in H/H) a 1.40
dyne cm
ÿ2
. Expe imen s we e obse ed in eal ime and
ideo aped o subsequen analysis. Pe cen age o cells
bound was de ined as (cells emaining bound in shea low
minus cells emaining in EDTA di ided by he o al numbe o
cells se led on o he pla e su ace a he onse o shea ) 3
100. New cells a aching o olling in o he ield o iew du ing
he applica ion o shea we e no included in he analysis.
Adhesion o ib onec in
A o al o 5 310
5
pu i ied spleen T cells om WT o WASP
ÿ/ÿ
,
WIP
ÿ/ÿ
and DKO mice and suspended in 2.5% BSA in PBS
we e added o 96-well Nunc Maxiso p Immuno Module la -
bo om pla es (Cos a , Camb idge, MA, USA) coa ed wi h 10
lgml
ÿ1
ib onec in (FN) (R&D sys ems) and allowed o se le
wi h ea men [medium alone o SDF-1a(100 ng ml
ÿ1
)] o 1 h
a 4C. Samples we e incuba ed a 37C o 15 min ollowed by
washing wi h 2.5% BSA in PBS. Adhe en cells we e eleased
wi h 10 mM EDTA in calcium- and magnesium- ee PBS and
we e coun ed by T ypan blue exclusion.
Chemo axis assay
In i o chemo axis was assayed using answell chambe s
(diame e , 6.5 mm; po e 5 lm) ob ained om Cos a . A o al o
5310
5
pu i ied splenic T cells in 125 ll RPMI 1640 wi h 1%
FCS we e added o he uppe chambe and 500 ll RPMI 1640
wi h 1% FCS medium wi h o wi hou SDF-1a(P epoTech,
London, UK) was added o he bo om chambe . A e 3 h a
37C, cells ha mig a ed o he lowe chambe we e collec ed
and coun ed. The expe imen s we e pe o med in duplica e
and we e epea ed a minimum o h ee imes.
Ac in polyme iza ion
Pu i ied splenic T cells we e s imula ed wi h SDF-1a(500 ng
ml
ÿ1
) o he indica ed imes and he eac ion was s opped by
adding 10% o malin (Sigma). The cells we e ixed o 15 min
on ice, washed wi h PBS and s ained wi h 5 lgml
ÿ1
TRITC–
phalloidin in 0.1% T i onX-100/PBS. Cells we e hen washed
and examined by FACS. Values we e exp essed as he a io o
mean luo escence in ensi y in s imula ed e sus uns imula ed
cells ( old inc ease o e baseline).
In acellula immuno luo escence
T cells pu i ied om spleen we e suspended in RPMI 1640
medium con aining 1% FCS and incuba ed o 30 min a 37C.
The cells we e pla ed on co e slips and allowed o a ach o
30 min a 37C. A ached cells we e s imula ed wi h 10 nM
SDF-1a, ixed wi h 10% o malin and hen pe meabilized wi h
0.2% T i onX-100 in PBS o 3 min. A e washing wi h PBS, he
cells we e blocked wi h 2.5% BSA and hen incuba ed wi h
1lgml
ÿ1
TRITC–phalloidin o 20 min a oom empe a u e
and examined using a luo escence mic oscope. A minimum
o 200 cells we e coun ed in each sample.
P o ein y osine phospho yla ion a e SDF-1as imula ion o
T cells
Pu i ied T cells we e le uns imula ed o we e s imula ed wi h
10 nM SDF-1a. Lysa es we e p epa ed in 1% NP-40 lysis bu e
supplemen ed wi h p o ease and phospha ase inhibi o s and
esol ed by SDS-PAGE. P o eins we e ans e ed on o a
ni ocellulose memb ane and p obed wi h he indica ed an i-
bodies. Phospho yla ed PAK1, co ilin and E k we e de ec ed
using abbi phospho-speci ic an ibodies o PAK1 (Biosou ce),
co ilin and E k (bo h om Cell Signaling), ollowed by
ep obing he memb anes wi h an ibody o PAK1 (Cell
Signaling), co ilin (Cell Signaling) o E k (San a C uz Bio-
echnology, Inc.) as loading con ols. Lck immunop eci a es
using mAb o Lck (BD T ansduc ion Labo a o ies) we e p obed
wi h an i-phospho y osine mAb 4G10 (Ups a e Bio echnology)
and ep obed wi h an i-Lck an ibody as loading con ol. Rac-1
GTP was pulled down om lysa es using a usion p o ein o
glu a hione-S- ans e ase and he PVB domain o PAK1 (Pie ce
Labo a o ies), ollowing he manu ac u e s’ ins uc ions. An
equi alen aliquo o lysa es was p obed wi h an i-Rac-1
an ibody (BD T ansduc ion Labo a o ies) as loading con ol.
S a is ical analysis
S uden ’s - es was used o compa e he di e ences be ween
g oups. A P- alue <0.05 was conside ed s a is ically signi i-
can . Time cu es o F-ac in con en in KO mice and WT
con ols we e analyzed by analysis o a iance (ANOVA) using
he G aphPad PRISM so wa e (G aphPad So wa e, Inc., San
Diego, CA, USA).
Resul s
De ec i e homing o WASP
ÿ/ÿ
, WIP
ÿ/ÿ
and DKO T cells o
lymphoid o gans
To examine he ole o WASP and WIP in T cell homing o
pe iphe al lymphoid o gans, equi alen numbe s o TRITC-
labeled T cells om KO mice and Alexa-488-labeled T cells
om WT con ol mice we e injec ed in a enously in o WT
ecipien s. As a con ol, equi alen numbe s o TRITC-labeled
and Alexa-labeled WT T cells we e injec ed in o WT ecipien
mice. One hou la e , spleens, mesen e ic LNs and inguinal
LNs we e emo ed and analyzed by FACS. Tcell homing index
was calcula ed as he a io o TRITC-labeled o Alexa-labeled
cells. As expec ed, he homing index o WT T cells was close
o 1 in all h ee lymphoid o gans es ed (Fig. 1). In con as , he
homing index o WASP
ÿ/ÿ
, WIP
ÿ/ÿ
and DKO T cells was
signi ican ly dec eased in all h ee o gans. These esul s
sugges ha WIP and WASP play an impo an ole in hom-
ing o T cells o lymphoid o gans.
Te he ing o selec in-coa ed su aces is no mal in
WASP
ÿ/ÿ
, WIP
ÿ/ÿ
and DKO T cells
In e ac ion be ween selec in ligands on ci cula ing cells and
endo helial selec ins media es T cell e he ing and olling o e
endo helial su aces (34–36). Figu e 2(A and B) shows ha
exp ession o L-selec in (CD62L) and E-selec in ligand is
WIP and WASP in T cell homing and chemo axis o SDF-1a223
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no mal on WASP
ÿ/ÿ
, WIP
ÿ/ÿ
and DKO T cells. In he case o
E-selec in ligand, only a ac ion o he T cells s ained wi h
E-selec in Fc and s aining was sligh ly lowe in all h ee KO
s ains compa ed wi h WT. Figu e 2(C) shows ha WASP
ÿ/ÿ
,
WIP
ÿ/ÿ
and DKO T cells e he ed no mally o e E-selec in-
coa ed and P-selec in-coa ed su aces. These esul s sugges
ha WIP and WASP a e no essen ial o selec in-media ed
e he ing o T cells on endo helial cells.
Spon aneous and SDF-1-a-induced adhesion o FN a e
no mal in WASP
ÿ/ÿ
, WIP
ÿ/ÿ
and DKO T cells
T cells exp ess he b1 in eg ins VLA-4 (a4b1) and VLA-5 (a5
b1) which media e adhesion o FN (37, 38). VLA-4 and VLA-5
exp essions we e no mal in WASP
ÿ/ÿ
, WIP
ÿ/ÿ
and DKO Tcells
(Fig. 3A). Figu e 3(B) shows ha he adhesion o uns imula ed
T cells o FN is no mal in T cells om all h ee KO mice s ains.
The chemokine SDF-1aup- egula es he a ini y o b1 in eg ins
o hei ligand (39–41). SDF-1aup- egula ed no mally he
adhe ence o T cells o FN in all h ee KOs mice s ains
(Fig. 3B). These esul s sugges ha WIP and WASP a e no
essen ial o baseline o chemokine-s imula ed T cell adhesion
media ed by b1 in eg ins.
In i o chemo axis o SDF-1ain i o is de ec i e in
WASP
ÿ/ÿ
, WIP
ÿ/ÿ
and DKO T cells
The chemokine SDF-1ahas been implica ed in he mig a ion
o T lymphocy es ac oss LN high endo helial cells (42) and
o lymphoid o gans in i o (43). To de e mine whe he WIP
and WASP play a ole in his esponse, we examined he
chemo ac ic esponse o T cells om WASP
ÿ/ÿ
, WIP
ÿ/ÿ
and
Fig. 1. Homing o T cells o pe iphe al lymphoid issues. A mix u e o equal numbe s o Alexa-488-labeled WT cells and TRITC-labeled WT o KO
(WASP
ÿ/ÿ
, WIP
ÿ/ÿ
and DKO) pu i ied splenic T lymphocy es was injec ed in o gene ically ma ched WT ecipien s. (A) FACS analysis o ecipien
spleen, mesen e ic LNs and inguinal LNs. (B) Homing index de i ed om da a o h ee independen expe imen s. The index ep esen s he a io o
TRITC-labeled Tcells ( om WTand KO mice) o Alexa-488-labeled WT cells. Columns and e o ba s ep esen mean 6SD. *P<0.05 compa ed
wi h WT cells.
224 WIP and WASP in T cell homing and chemo axis o SDF-1a
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DKO mice o SDF-1a. FACS analysis using mAb o he SDF-1a
ecep o CXCR4 e ealed ha Tcells om all h ee (KO) s ains
exp essed CXCR4 a a le el simila o ha o T cells om WT
con ols (Fig. 4A). Figu e 4(B) shows ha he chemo ac ic
esponse o WASP
ÿ/ÿ
T cells o SDF-1awas sligh ly, bu no
signi ican ly, dec eased. The chemo ac ic esponse o WIP
ÿ/ÿ
-
de icien T cells was signi ican ly educed o ~50% o con ol.
The chemo ac ic esponse o DKO T cells was d ama ically
educed o ~10% o he esponse o con ol. These esul s
sugges ha WIP and WASP play pa ially edundan oles in
in i o T cell chemo axis o SDF-1a.
Ac in polyme iza ion in esponse o SDF-1ais de ec i e in
T cells double de icien in WASP and WIP
Ac in polyme iza ion is equi ed o chemo axis (13, 18). One o
he i s e en s in ol ed in he mig a ion o Tcells in esponse o
chemokines is an inc ease in cellula F-ac in con en . Pu i ied
splenic T cells om WASP
ÿ/ÿ
, WIP
ÿ/ÿ
and DKO mice we e
s imula ed wi h SDF-1a, hen s ained wi h TRITC–phalloidin
and analyzed by FACS o F-ac in con en . As expec ed (16),
SDF-1as imula ion o WT T cells esul ed in an inc ease in
F-ac in con en wi hin 15–30 min o s imula ion. The le el o
F-ac in hen p og essi ely dec eased and e u ned o baseline
Fig. 2. Selec in ligand exp ession and unc ion in Tcells om WASP
ÿ/ÿ
, WIP
ÿ/ÿ
and DKO mice (A and B). FACS analysis o L-selec in (CD62L) and
E-selec in ligand exp ession on pu i ied splenic T cells. Splenocy es we e double s ained wi h an i-CD3–FITC and an i-CD62L–PE (A) o wi h
bio inyla ed E-selec in : IgG chime ic p o ein (E-selec in : Fc) ollowed by s ep a idin–PE. As con ol, incuba ion wi h he chime ic p o ein was
omi ed (B). Resul s a e ep esen a i e o wo independen expe imen s. (C) A achmen o T cells o E-selec in- and P-selec in-coa ed low
chambe s. Resul s ep esen he mean 6SD o h ee expe imen s.
WIP and WASP in T cell homing and chemo axis o SDF-1a225
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60 min a e s imula ion (Fig. 5). SDF-1ainduced a no mal
F-ac in inc ease in WASP
ÿ/ÿ
and WIP
ÿ/ÿ
T cells. In con as ,
Tcells om DKO mice we e se e ely impai ed in hei abili y o
inc ease hei con en o F-ac in ollowing SDF-1as imula ion.
ANOVA e ealed a signi ican di e ence be ween he ime
cu es o F-ac in con en o DKO and con ols. These esul s
sugges ha WIP and WASP play edundan oles in ac in
polyme iza ion igge ed by SDF-1a.
Re-o ganiza ion o he ac in cy oskele on in T cells in
esponse o SDF-1a
SDF-1ainduces ac in e-o ganiza ion and pola iza ion in cells
(44). To in es iga e ac in cy oskele on ea angemen , pu i ied
Tcells we e s imula ed wi h SDF-1a o 0, 1 and 5 min, hen we e
ixed, s ained wi h TRITC–phalloidin and examined by luo es-
cence mic oscopy. Uns imula ed cells om WT, WASP
ÿ/ÿ
,
WIP
ÿ/ÿ
and DKO mice exhibi ed a ing o pe iphe al ac in.
Small and compa able ac ions o hese cells (~10%)
exhibi ed pola ized ac in (Fig. 6A and B). A e 1 min o
s imula ion wi h SDF-1a, T cells om WT mice s a ed o lose
hei ound shape, and ~50% o hem pola ized ac in o one
pole. A 5 min, he e was a u he inc ease in he ac ion o
pola ized cells and a dis inc u opod was o med in he
majo i y o WT cells (Fig. 6A and B). Signi ican ly mo e cells
om WASP
ÿ/ÿ
, WIP
ÿ/ÿ
and DKO mice e ained hei ounded
shape and signi ican ly less o hem accumula ed F-ac in a
one pole and o med u opods 1 min a e SDF-1as imula ion
(Fig. 6A and B). This end pe sis ed 5 min a e s imula ion.
A ac ion (>15%) o T cells om all h ee KO mice s ains,
exhibi ed abno mal pola iza ion wi h mul iple pa ches ( wo o
mo e) o F-ac in a e SDF-1as imula ion. Only a negligible
ac ion o WT T cells (<3%) exhibi ed mo e han one pole o
F-ac in a 5 min pos -s imula ion. These esul s sugges ha
WIP and WASP a e impo an o no mal ac in cy oskele on
e-o ganiza ion a e SDF-1as imula ion.
WASP and WIP a e no essen ial o ea ly signaling e en s
ollowing CXCR4 liga ion in T cells
SDF-1aliga ion o i s G-p o ein-coupled ecep o CXCR4
esul s in he ac i a ion o mul iple signaling pa hways. They
include he phospho yla ion and ac i a ion o PAK1. PAK1
is ups eam o wo pa hways ha a e in ol ed in ac in
Fig. 3. Exp ession o b1 in eg ins and adhesion o FN o T cells om WASP
ÿ/ÿ
, WIP
ÿ/ÿ
and DKO mice. (A) FACS analysis o VLA-4 and VLA-5
exp ession. Pu i ied splenic T cells we e s ained wi h an i-CD3–FITC and an i-CD49d–PE o an i-CD49e–PE. (B) Adhesion o uns imula ed and
SDF-1a-s imula ed pu i ied splenic T cells o BSA- e sus FN-coa ed pla es. Resul s ep esen he mean 6SD o wo expe imen s.
226 WIP and WASP in T cell homing and chemo axis o SDF-1a
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e-o ganiza ion and chemo axis ollowing SDF-1as imula ion:
ac i a ion o he small GTPases Rac-1 and Cdc42 wi h sub-
sequen WASP-media ed, A p2/3 complex-dependen ac in
polyme iza ion (32, 45) and ac i a ion o LIM kinase-media ed
co ilin phospho yla ion and inac i a ion (46, 47). CXCR4
liga ion also causes phospho yla ion and ac i a ion o phos-
pholipase Cb, which ini ia es a biochemical cascade ha
esul s in he phospho yla ion and ac i a ion o he mi ogen-
ac i a ed p o ein kinase E k, which may play a ole in
chemo axis o SDF-1a(48, 49). Finally, SDF-1acauses ZAP-
70 phospho yla ion and ac i a ion o s c kinases, including
Lck, which also ha e been implica ed in chemo axis (50, 51).
Figu e 7 shows ha phospho yla ion o PAK1, co ilin, E k and
Lck a e SDF-1aliga ion was no mal in Tcells om DKO mice.
Figu e 7 also shows ha GTP loading o Rac-1 a e SDF-1a
s imula ion was no mal in DKO T cells. These da a sugges
Fig. 4. CXCR4 exp ession and chemo axis o SDF-1ao T cells om WASP
ÿ/ÿ
,WIP
ÿ/ÿ
and DKO mice. (A) Pu i ied splenic T cells we e double
s ained wi h an i-CD3–FITC and an i-CXCR4–PE. CXCR4 exp ession on ga ed CD3
+
cell is shown. Resul s shown a e ep esen a i e o wo
independen expe imen s. (B) Pe cen o splenic T lymphocy es om WASP
ÿ/ÿ
, WIP
ÿ/ÿ
and DKO mice and WTcon ols ha mig a ed h ough he
il e o he SDF-1a-con aining lowe compa men o a answell chambe . Resul s ep esen he mean 6SD o ou expe imen s. Columns and
e o ba s ep esen mean 6SD. *P<0.05.
WIP and WASP in T cell homing and chemo axis o SDF-1a227
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ha WASP and WIP unc ion downs eam o small GTPases in
SDF-1achemo axis.
Discussion
The esul s o his s udy show ha bo h WASP and i s pa ne
WIP play impo an and pa ially edundan oles in T cell
homing and in chemo axis o T cells o SDF-1a.
T cells om WASP
ÿ/ÿ
and WIP
ÿ/ÿ
mice exhibi ed de ec i e
homing in i o o spleen and LNs o compa able ex en . T cell
homing was educed o 44% o no mal in WASP
ÿ/ÿ
T cells and
o 38% o no mal in WIP
ÿ/ÿ
T cells. The homing de ec was
mo e se e e in Tcells om WASP
ÿ/ÿ
/WIP
ÿ/ÿ
DKO mice, being
educed o 13% o no mal. The mo e se e e educ ion in
homing o DKO T cells compa ed wi h single KO cells
sugges s ha WIP and WASP play complemen a y oles in
he homing o T cells o pe iphe al lymphoid o gans. The T cell
homing de ec in WASP
ÿ/ÿ
mice obse ed is consis en wi h
he obse a ion ha T cell a eas a e educed in spleens and
LNs om WAS pa ien s (52). The obse a ion ha he o al
numbe o CD3
+
Tcells in spleen and pe iphe al LNs is no mal
in WASP
ÿ/ÿ
, WIP
ÿ/ÿ
and DKO mice (21, 29; da a no shown)
sugges s he p esence o compensa o y mechanisms in i o.
In ac , he size and weigh o he spleen a e signi ican ly
inc eased in WASP
ÿ/ÿ
, WIP
ÿ/ÿ
and DKO mice (M.-D.G.,
unpublished esul s).
WASP is exp essed p edominan ly in hema opoie ic cells
while WIP is exp essed in all issues (23). I is he e o e
possible ha hema opoie ic non-T cells, e.g. dend i ic cells
which a e known o sec e e T cell chemoa ac an s (53) may
con ibu e o he Tcell homing de ec in WASP
ÿ/ÿ
and ha bo h
hema opie ic non-T cells and non-hema opoie ic cells, e.g.
endo helial cells and s omal cells, con ibu e o he T cell
homing de ec in WIP
ÿ/ÿ
and DKO mice. Howe e , we ha e
obse ed ha WT T cells home no mally o he pe iphe al
lymphoid o gans o WASP
ÿ/ÿ
, WIP
ÿ/ÿ
and DKO mice (ou
unpublished esul s). This sugges s ha non-T cells do no
subs an ially con ibu e o de ec i e T cell homing in hese
mice.
In i o s udies e ealed ha wo o he p ocesses in ol ed
in T cell homing o pe iphe al issues, namely selec in ligand-
media ed a achmen and b1 in eg in-media ed adhesion,
we e no mal in T cells om WASP
ÿ/ÿ
, WIP
ÿ/ÿ
and DKO mice.
The pe cen ages o Tcells om all h ee s ains ha exp essed
L-selec in and E-selec in ligands on hei su ace we e
compa able o hose obse ed in WT T cells (Fig. 2A and B).
In he case o E-selec in, he e was a sligh educ ion in
he in ensi y o exp ession in all h ee KO s ains. T cells om
all h ee KO s ains e he ed no mally o E-selec in- and
P-selec in-coa ed su aces (Fig. 2C), al hough e he ing was
es ed a only a single concen a ion o ligands coa ing he
pla e. The appa en ly no mal e he ing o P-selec in sugges s
ha , al hough i could no be measu ed di ec ly due o lack o
sui able eagen , P-selec in ligand exp ession was in ac in all
h ee s ains o KO mice. Recen ly, e he ing o WASP
ÿ/ÿ
lymphocy es o pe iphe al node add essin was epo ed o be
educed by ~20%, sugges ing ha lack o WASP may
di e en ially a ec e he ing o a ious ligands (54).
T cells om all h ee s ains exp essed no mal amoun s o
he b1 in eg ins VLA-4 and VLA-5 (Fig. 3A). Mo e impo an ly,
hey adhe ed no mally o FN and up- egula ed no mally hei
adhesion o FN a e SDF-1as imula ion (Fig. 3B). P e ious
wo k has shown ha WASP
ÿ/ÿ
T cells adhe ed no mally o
ICAM-1 and FN a e s imula ion wi h an i-CD3 (55). Thus,
WASP is no impo an o ei he he up- egula ion o b1 in eg in
adhesi e ac i i y by chemokine o an igen ecep o s. Taken
oge he , hese esul s sugges ha nei he WIP no WASP is
essen ial o Tcell e he ing and olling o o in eg in-media ed
adhesion o FN in esponse o SDF-1a.
In i o chemo axis o SDF-1awas de ec i e in WIP
ÿ/ÿ
T cells, being educed o ~50% o no mal, and se e ely
de ec i e in DKO T cells, being educed o ~10% o no mal.
Despi e hei in i o homing de ec , WASP
ÿ/ÿ
T cells did no
1.0
2.0
1.5
Fold inc ease in F-ac in
WT
WASP-/-
1.0
2.0
1.5
Fold inc ease in F-ac in
WIP-/-
WT
1.0
2.0
1.5
015 6030 45
Fold inc ease in F-ac in
WT
DKO
min.
015 6030 45 min.
015 6030 45 min.
*
A
B
C
Fig. 5. Changes in F-ac in con en in T cells s imula ed wi h SDF-1a.
Pu i ied splenic T lymphocy es om (A) WASP
ÿ/ÿ
, (B) WIP
ÿ/ÿ
and
(C) DKO mice and li e ma e WT con ols we e s imula ed wi h SDF-1a
o indica ed imes. Cells we e hen ixed, pe meabilized, s ained
wi h phalloidin–TRITC and analyzed by FACS. Resul s a e exp essed
as old inc ease in mean luo escence in ensi y o e baseline (0 ime).
The esul s shown a e ep esen a i e o ou independen expe imen s.
*P<0.05 (by ANOVA).
228 WIP and WASP in T cell homing and chemo axis o SDF-1a
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exhibi a signi ican dec ease in chemo axis o SDF-1ain i o.
This is no en i ely su p ising because homing in i o in ol es
a mul i ude o signals and complex equi emen s compa ed
wi h in i o chemo axis o a single chemokine. In his ega d, i
was ecen ly shown ha lymphocy es om WASP
ÿ/ÿ
mice
ha e de ec i e chemo axis o CCL19. (21). The i ually no mal
chemo axis o mu ine WASP-de icien T cells o SDF-1ais in
con as o he de ec i e chemo axis exhibi ed by T cells om
WAS pa ien s in esponse o his chemokine (32). I is possible
ha di e ences in he exp ession o N-WASP may explain he
di e ence be ween he esponse o WASP-de icien human
and mouse T cells o SDF-1a. Highe exp ession o N-WASP in
mouse pla ele s han in human pla ele s is hough o accoun
o he p ese a ion o ci cula ing pla ele numbe s in WASP-
de icien mice (21).
Ac in polyme iza ion a e SDF-1as imula ion was no mal in
Tcells om WASP
ÿ/ÿ
and WIP
ÿ/ÿ
mice. This inding in WASP
ÿ/ÿ
T cells is consis en wi h he obse a ion ha o e -exp ession
o he CRIB domain o WASP in a T cell line does no inhibi
SDF-1aac in polyme iza ion, al hough i inhibi s SDF-1a
chemo axis (45). The no mal ac in polyme iza ion in WIP
ÿ/ÿ
T cells in esponse o SDF-1asugges s ha ac in polyme iza-
ion is no su icien o chemo axis. In con as o he esul s in
WASP
ÿ/ÿ
and WIP
ÿ/ÿ
T cells, ac in polyme iza ion in esponse
o SDF-1awas se e ely impai ed in T cells om DKO mice.
This sugges s ha WIP and WASP play edundan oles in
ac in polyme iza ion in esponse o CXCR4 liga ion in T cells.
In con as , WASP and WIP play non- edundan oles in he
ac in polyme iza ion esponse o T cells o TCR liga ion, as
e idenced by he obse a ion ha his esponse is impai ed in
bo h WASP
ÿ/ÿ
and WIP
ÿ/ÿ
T cells (21, 22, 29).
Examina ion o he ac in cy oskele on e ealed de ec i e
ac in cy oskele on e-o ganiza ion in Tcells o WASP
ÿ/ÿ
,WIP
ÿ/ÿ
and DKO mice in esponse o SDF-1a. These esul s sugges
ha WASP and WIP play complemen a y oles in ac in
cy oskele al changes induced by SDF-1a. The impo ance o
bo h WASP and WIP in ac in e-o ganiza ion a e TCR liga ion
is well documen ed (21, 22, 29). The ac ha T cells om
WASP
ÿ/ÿ
mice exhibi ed a signi ican de ec in hei abili y o
e-o ganize hei ac in cy oskele on, despi e hei nea -no mal
chemo ac ic esponse o SDF-1ain i o, is eminiscen o
obse a ions on megaka yocy es om WAS pa ien s, which
exhibi abno mal ilopodia o ma ion, and ac in e-dis ibu ion
in esponse o SDF-1a, bu mig a e no mally in esponse o his
Fig. 6. Ac in cy oskele al e-o ganiza ion a e s imula ion wi h SDF-1a. Pu i ied splenic T lymphocy es om WASP
ÿ/ÿ
, WIP
ÿ/ÿ
and DKO mice and
WT con ols we e s imula ed o 1 and 5 min wi h SDF-1a, ixed, pe meabilized and s ained wi h phalloidin–TRITC and hen examined by
luo escen mic oscopy a 360 magni ica ion. (A) Rep esen a i e images. (B) Quan i a ion o esul s om h ee expe imen s. *P<0.05 compa ed
wi h WT cells.
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