Accelerated chemokine receptor 7-mediated dendritic cell migration in Runx3 knockout mice and the spontaneous development of asthma-like disease.

Abstract

The Runx3 transcription factor is a key regulator of lineage-specific gene expression in several developmental pathways and could also be involved in autoimmunity. We report that, in dendritic cells (DC), Runx3 regulates TGFbeta-mediated transcriptional attenuation of the chemokine receptor CCR7. When Runx3 is lost, i.e., in Runx3 knockout mice, expression of CCR7 is enhanced, resulting in increased migration of alveolar DC to the lung-draining lymph nodes. This increased DC migration and the consequent accumulation of activated DC in draining lymph nodes is associated with the development of asthma-like features, including increased serum IgE, hypersensitivity to inhaled bacterial lipopolysaccharide, and methacholine-induced airway hyperresponsiveness. The enhanced migration of DC in the knockout mice could be blocked in vivo by anti-CCR7 antibodies and by the drug Ciglitazone, known to inhibit CCR7 expression. The data indicate that Runx3 transcriptionally regulates CCR7 and that, when absent, the dysregulated expression of CCR7 in DC plays a role in the etiology of asthmatic conditions that recapitulate clinical symptoms of the human disease. Interestingly, human RUNX3 resides in a region of chromosome 1p36 that contains susceptibility genes for asthma and hypersensitivity against environmental antigens. Thus, mutations in RUNX3 may be associated with increased sensitivity to asthma development.

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Accelerated chemokine receptor 7-mediated dendritic
cell migration in Runx3 knockout mice and the
spontaneous development of asthma-like disease
Ofer Fainaru*, David Shseyov†, Shay Hantisteanu*, and Yoram Groner*‡
*Department of Molecular Genetics, The Weizmann Institute of Science, Rehovot 76100, Israel; and†Department of Pediatrics, Hadassah Mount Scopus
University Hospital, Jerusalem 91120, Israel
Communicated by Leo Sachs, The Weizmann Institute of Science, Rehovot, Israel, June 13, 2005 (received for review May 9, 2005)
The Runx3 transcription factor is a key regulator of lineage-specific
geneexpressioninseveraldevelopmentalpathwaysandcouldalso
beinvolvedinautoimmunity.Wereportthat,indendriticcells(DC),
Runx3 regulates TGF?-mediated transcriptional attenuation of the
chemokine receptor CCR7. When Runx3 is lost, i.e., in Runx3
knockout mice, expression of CCR7 is enhanced, resulting in in-
creasedmigrationofalveolarDCtothelung-draininglymphnodes.
This increased DC migration and the consequent accumulation of
activated DC in draining lymph nodes is associated with the
development of asthma-like features, including increased serum
IgE, hypersensitivity to inhaled bacterial lipopolysaccharide, and
methacholine-induced airway hyperresponsiveness. The enhanced
migration of DC in the knockout mice could be blocked in vivo by
anti-CCR7 antibodies and by the drug Ciglitazone, known to inhibit
CCR7 expression. The data indicate that Runx3 transcriptionally
regulates CCR7 and that, when absent, the dysregulated expres-
sion of CCR7 in DC plays a role in the etiology of asthmatic
conditions that recapitulate clinical symptoms of the human dis-
ease. Interestingly, human RUNX3 resides in a region of chromo-
some 1p36 that contains susceptibility genes for asthma and
hypersensitivity against environmental antigens. Thus, mutations
in RUNX3 may be associated with increased sensitivity to asthma
development.
transcription regulation ? autoimmunity ? Ciglitazone ? human
chromosome 1p36
O
torespondaccordinglybydevelopingactiveimmunityortolerance,
respectively (1). Allergic asthma is a multifactorial disease charac-
terized by a T cell-mediated immune reaction and chronic airway
inflammation in response to inhaled allergen (2). The disease is
oftenaccompaniedbyanincreaseinserumIgEandinvolvesairway
hyperresponsiveness (AHR) (3, 4). Recent studies point at a
primary role of dendritic cells (DC) in regulating in vivo allergic
responses (5, 6). The respiratory tract DC are responsible for
orchestratingtheimmunereactionagainstinhaledantigensthrough
initiation of a T helper 2 (Th2) response and by stimulating
cytokine-producingTcellsduringtheongoingairwayinflammation
(7, 8). Additionally, DC play an important role in the maintenance
of self-tolerance in the periphery (9–11). How do these two
functions converge?
LungalveolarDC,whichconstantlysampleinhaledantigens,are
normally maintained at an immature state by immunosuppressive
cytokines, such as TGF?, secreted by the surrounding cellular
environment, including lung macrophages and epithelial cells (12–
15). At this immature state, alveolar DC have a poor capacity to
present antigens and?or to migrate to the draining lymph nodes
(LN) and function in maintenance of peripheral tolerance (9, 11,
16). Detection of pathogen-associated molecular patterns
(PAMPs) by the immature tissue-resident DC initiates their mat-
uration and migration to the regional LN where they prime naive
T cells. This maturation process involves up-regulation of specific
ne of the challenges encountered by the immune system is to
distinguish between pathogenic and innocuous antigens and
chemokine receptors (5) that mediate DC migration to the T cell
zones of the draining LN. This DC trafficking is directed by the
secondary lymphoid tissue chemokine (SLC)?CCL21 through
binding to its cognate receptor, the chemokine receptor 7 (CCR7)
(5).SLC?CCL21isconstitutivelyproducedinthehighendothelium
venules (HEVs) of LN and Peyer’s patches (PP) and in the
lymphaticendotheliumofmultipletissues(17).Uponup-regulation
of CCR7 expression, DC respond to these homing signals and
migrate to draining LN (18–20). Because the migratory capacity of
DC is important for their function in both immunity and tolerance,
the regulation of CCR7 expression constitutes a crucial checkpoint
(20), tightly regulated by the antiinflammatory cytokine TGF?
(21).
Runx3belongstotheruntdomainfamilyoftranscriptionfactors,
which are key regulators of lineage-specific gene expression, and
when mutated are associated with human diseases (22). Interest-
ingly, recent findings raised the possibility of RUNX involvement
in autoimmunity (23). In the developing mouse embryo, Runx3
displays a distinct tissue-specific expression pattern. It is expressed
in hematopoietic organs, developing bones, peripheral nervous
system, and skin appendages (24). Studies in knockout (KO) mice
have delineated several cell-autonomous functions of Runx3. In
neurogenesis, Runx3 is required for the development and survival
of dorsal root ganglia TrkC neurons (25, 26). In thymopoiesis,
Runx3 is required for silencing of CD4 during T cell lineage
decisions (27–29), and, in DC, Runx3 functions as a component of
theTGF?-signalingcascade(30).IntheabsenceofRunx3,KODC
do not respond to TGF?; their maturation is accelerated and
accompanied by an increased efficacy to stimulate T cells (30).
Runx3 KO mice also develop lesions in the gastrointestinal tract
(GIT). Li et al. (31) have reported that Runx3 KO newborn mice
exhibit hyperplasia of gastric epithelium (31, 32). They have attrib-
utedthisdefecttothelossofRunx3functioninGITepitheliumand
suggested that Runx3 is a novel tumor suppressor involved in
stomach cancer (31). Brenner et al. (33), on the other hand, have
reported that, at ?4 weeks of age, Runx3 KO mice develop colitis
and only at a much older age of 8 months also develop gastric
mucosal hyperplasia (33). Because Runx3 could not be detected in
GIT epithelium (24, 33, 34), but is readily detected in the resident
leukocytic population, the current conclusion is that the GIT
ailments of the KO mice are due to the loss of an intrinsic cell
function of Runx3 in leukocytes (33).
Here,wereportthat,inadditiontotheirlunginflammation(30),
Runx3 KO mice spontaneously develop physiological conditions
characteristic of asthma, including AHR, increased levels of serum
IgE, and hypersensitivity to inhaled LPS. In the KO mice, the
traffickingofDCfromairwaystotheregionalLNisaccelerateddue
Abbreviations: AHR, airway hyper-responsiveness; DC, dendritic cell; CCR7, chemokine
receptor 7; LN, lymph node; BAL, bronchoalveolar lavage; KO, knockout; CFSE, carboxy-
fluorescein diacetate-succinimidyl ester; BMDC, bone marrow-derived DC; SLC, secondary
lymphoid-tissue chemokine.
‡To whom correspondence should be addressed. E-mail: yoram.groner@weizmann.ac.il.
© 2005 by The National Academy of Sciences of the USA
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to dysregulated expression of the chemokine receptor CCR7. We
show that Runx3 regulates the TGF?-mediated transcription at-
tenuation of CCR7, and, when it is lost, CCR7 expression on KO
DC is enhanced. The enhanced CCR7 expression results in in-
creased migration of alveolar DC and accumulation of mature
activated DC in lung-draining LN. The accelerated DC migration
could be blocked by anti-CCR7 antibodies and by the drug Cigli-
tazone, known to inhibit DC CCR7 expression (35). The data
delineate Runx3 as a transcriptional regulator of CCR7 expression
and indicate that dysregulation of CCR7 in DC could lead to
development of asthmatic conditions recapitulating clinical symp-
toms of the human disease. Interestingly, human RUNX3 resides in
aregionofchromosome1p36(36)thatcontainssusceptibilitygenes
for asthma and hypersensitivity against environmental antigens
(37, 38).
Materials and Methods
Mice and Treatments. Runx3-KO mice were generated as described
(25) and bred on ICR and MF1 backgrounds. KO mice and WT
littermates of both backgrounds were used for the experiments.
Mice were maintained in individually ventilated cages in a specific
pathogen-free (SPF) facility free of known viral and bacterial
pathogens. For AHR measurements, Runx3 KO and WT litter-
mates were placed in a whole-body plethysmograph (Buxco, Win-
chester,U.K.)afteranesthesiawithi.p.injectionof10?l?gsolution
containing ketamine (10 mg?ml) and xylazine (2 mg?ml). Record-
ing of baseline respiratory patterns was followed by measurements
after 1 min inhalation of the muscarinic agonist methacholine (40
mg?ml) (Sigma), by using an ultrasonic nebulizer. For the analysis
of serum and bronchoalveolar lavage (BAL) IgE, mice were bled
from the retroorbital plexus and serum was prepared and kept at
?70°C until further analyzed. BAL was prepared as described (30)
and kept at ?70°C. In vivo cell labeling with carboxyfluorescein
diacetate-succinimidylester(CFSE,MolecularProbes)wascarried
out by intranasal instillation of 50 ?l per mouse of 8 mM CFSE in
RPMI medium 1640 to isoflurane-anesthetized mice. For analysis
of BAL and LN, mice were killed by CO2asphyxiation 16 h after
CFSE labeling, tracheae were cannulated, and lungs washed by
gentle infusion of 2–4 aliquots of 1 ml PBS. Subsequently, medi-
astinal and axillary LN were removed, incubated with 1 mg?ml
collagenase type VIII (C-2139, Sigma) for 45 min at 37°C, minced,
and pressed through an 80-?m nylon mesh to obtain a single cell
suspension. The administration of reagents affecting DC migratory
capacity was carried out by intranasal instillation of 50 ?g of LPS
(Sigma) per mouse 5 h after CFSE treatment, or anti-CCR7
antibodies (purified goat anti-mouse CIO131, Capralogics, Hard-
wick, MA) 8 ?g per mouse, 1 h after CFSE treatment. For the
Ciglitazone treatment, mice were subjected to inhalation of 50 ?g
per mouse of Ciglitazone (71730, Cayman Chemical, Ann Arbor,
MI) for 20 min before CFSE treatment.
RT-PCR Analysis. RT-PCR products were generated by using the
following primers: 5?-CATCAGCATTGACCGCTACGT-3? and
5?-GGTACGGATGATAATGAGGTAGCA-3? for murine
CCR7; 5?-GTGTTCATCATTGGAGTGGTG-3? and 5?-GGT-
TGAACAGGTAGATGCTGGTC-3? for murine CCR1; 5?-GG-
GACATCATCAAACCAGACC-3? and 5?-GCCAACCAAGCA-
GAAGACAGC-3? for murine IL-12.
Culturing Bone Marrow DC. Bone marrow-derived DC (BMDC)
werepreparedasdescribed(39).Briefly,micewerekilled,andbone
marrow was extracted from femurs and tibias by flushing the shaft
with5mlofRPMImedium1640.RBCwerelyzedin1.66%NH4Cl,
and cells were seeded into non-tissue culture plates at a density of
1 ? 106cells per ml in medium (RPMI medium 1640?5% FCS?50
?M 2-mercaptoethanol?penicillin?streptomycin) containing 10
ng?ml murine recombinant granulocyte?macrophage colony-
stimulating factor (GM-CSF, PeproTech, Rehovot, Israel). Me-
dium was replenished every three days, and the loosely adherent
DC were collected and used for further studies at designated time
points. To induce DC maturation, day 7–12 cultures were treated
with LPS (1 ?g?ml) and analyzed 1 day later.
Flow Cytometry. Single cells were suspended in FACS buffer
(PBS?1mMEDTA?1%BSA?0.05%sodiumazide).Immunostain-
ing (1–2 ? 106cells) was performed in the presence of rat
anti-mouseFc?RIII?IIreceptor(CD16?32;clone2.4G2,American
Type Culture Collection), by incubating the cells with monoclonal
antibodies for 30 min on ice (100 ?l per 1 ? 106cells). Flow
cytometrywasperformedwithaFACSCalibur(BectonDickinson)
and CELLQUEST software (Becton Dickinson). Staining reagents
included CD11c APC?PE and IA?IE PE. For analysis of IgE
Receptors (Fc?R), cells were treated by acid elution before specific
staining. Specifically, cells were suspended in an acidic buffer (0.05
M sodium acetate?acetic acid, pH3.5?0.085 M NaCl?0.005 M
KCl?1% FCS) and incubated at room temperature for 3 min. After
neutralizing with PBS?BSA 1%, cells were washed twice with
FACS buffer and subjected to further staining with IgE-FITC
antibodies.
DC Migration Assays.BMDCweregrowninthepresenceorabsence
of TGF? (10 ng?ml). On day 6, cells were treated overnight with
LPS(1?g?ml)toinducematuration.BMDC(5?105)wereplaced
in a Transwell migration chamber (Costar 3421, Corning) and
allowed to migrate through a polycarbonate mesh (pore size 5 ?m)
at 37°C. SLC (100 ng?ml) (457-6C, R & D Systems) or control
bufferwasplacedinthelowerchambertoinduceCCR7-dependent
chemotaxis. After 3 h, cells that migrated to the lower chamber
were counted, and SLC-dependent migration was calculated as the
ratio of the number of cells migrating with?without SLC. For the
dermal DC migration assay, mice were killed, and dermal sheaths
were prepared by splitting the ears. The sheaths were incubated
dermal side down at 37°C, in medium (RPMI medium 1640?10%
FCS?20 ?M 2-mercaptoethanol?penicillin?streptomycin), with or
without TGF? (10 ng?ml). Twenty-four hours later, SLC (100
ng?ml)orbufferwasadded.Afteranadditional48h,migratedcells
werecollectedandstained,andthenumberofCD11c?MHCIIbright
DC were determined by FACS analysis.
Results
Runx3 KO Mice Display Major Hallmarks of Human Asthma. In an
earlierstudy,wereportedthatRunx3KOmicedevelopeosinophilic
lung inflammation associated with airway remodeling and mucus
hypersecretion (30). However, the question of whether the spon-
taneous development of these features in the KO mice was asso-
ciated with other symptoms characteristic of human asthma re-
mained open. AHR is a cardinal pathophysiological feature of
humanasthma,clinicallydefinedbyanincreaseinairwaysensitivity
to inhaled histamine or methacholine (40). In mouse models of
asthma, AHR could be assessed by using the noninvasive enhanced
pause (Penh) method (41). Examination of 4- to 9-week-old Runx3
KO mice and WT littermates revealed significantly increased
methacholine-induced AHR in the KO mice (Fig. 1A). Of note,
increasing the dosage of methacholine from 40 mg?ml to the
reported 200 mg?ml (42) resulted in suffocation of the KO mice,
whereastheWTmiceremainedunaffected.Interestingly,however,
thisAHRwastransientandnolongerobservedin5-to6-month-old
KO mice.
Human asthma is characterized by a pronounced elevation in
serum IgE (43, 44). In the KO mice, concentrations of IgE in both
BAL and blood serum were 3- and 4-fold higher, respectively, than
in WT littermate mice (Fig. 1B). High surface expression of the
high-affinity receptor for IgE, Fc?R, on LN DC is another impor-
tant feature of human asthma (45, 46). Fc?R expression on DC
increases the efficiency of allergen presentation up to 1,000-fold
through an antigen-focusing mechanism that enhances MHC pre-
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sentation (45). We used FACS analysis to assess surface expression
of Fc?R on DC derived from the lung-draining thoracic LN.
Significantly, as much as 16% of the DC in the thoracic LN of the
KO mice expressed this allergy-mediating receptor, compared with
a mere 1% in WT littermates (Fig. 1C). This latter low abundance
of Fc?R-expressing WT thoracic LN DC was similar to that in
non-lung-drainingLN(i.e.,axillaryLN)ofeitherWTorRunx3KO
mice (Fig. 1C). These data are also consistent with findings that, in
humans, increased serum IgE was associated with elevated Fc?R
expression on LN DC (46).
The severity of asthma in humans is influenced by exposure to
LPS (47). We next assessed whether the KO mice are more
susceptiblethanWTmicetominuteamountsofinhaledLPS.Even
without LPS (i.e., inhalation of PBS), a significant increase in the
proportion of the highly allergenic (48) CD11c??CD11b?alveolar
DC was observed in KO mice compared with WT littermates (Fig.
1D).InhalationofLPS(0.1?g)ledtoafurtheraccumulationofthe
CD11c??CD11b?DC subset and to a pronounced recruitment of
CD11c??CD11b?eosinophils and neutrophils to the lung of KO
mice (Fig. 1D). This influx of eosinophils and neutrophils to the
lungsoftheKOmiceindicatesacuteinflammationandunderscores
the oversensitivity of these mice to exogenous inducers of lung
inflammation.
The results of methacholine-induced AHR, the increased serum
andBALIgE,theelevatedDCsurfaceexpressionofFc?R,andthe
hyperresponsiveness to inhaled LPS demonstrate that the patho-
physiological features found in Runx3 KO mice recapitulate the
clinical symptoms characterizing human asthma.
Runx3IsRequiredforTGF?-MediatedAttenuationofCCR7Expression.
Transcription and surface expression of CCR7 in DC are up-
regulated by maturation inducers (49) and down-regulated by
TGF? (21). Because our previous findings indicate that Runx3
mediates TGF? signaling in DC, we addressed whether Runx3 is
also involved in TGF?-directed inhibition of CCR7 expression. A
marked decrease in CCR7 transcription and surface expression
occurred when WT BMDC were treated with TGF? during mat-
uration (Fig. 2 A and B). However, no such inhibition of CCR7
expression was noted in KO BMDC (Fig. 2 A and B). Of note,
expression of another chemokine receptor, CCR1, known to be
refractory to TGF? inhibition (21), was not reduced in either KO
or WT BMDC, whereas expression of IL-12, known to respond to
TGF?(50),wasreducedinbothKOandWTBMDC(Fig.2A).The
data indicate that Runx3 is specifically involved in TGF?-
dependent transcriptional attenuation of CCR7 expression in
BMDC.
Elevated CCR7 Expression on Alveolar and LN DC of Runx3 KO Mice.
The fact that, in the absence of Runx3, transcriptional regulation of
CCR7expressioninBMDCisimpairedledustoexaminetheinvivo
levels of CCR7 in WT and KO DC. Lung DC isolated from BAL
of Runx3 KO mice express significantly higher levels of CCR7 than
the corresponding DC of WT littermates (Fig. 2C). Of note, the
highest level of CCR7 was recorded on the allergenic CD11c??
CD11b?DC subset, which are not present in WT lung but are
abundant in BAL of Runx3 KO mice (30). These results indicate
that alveolar DC of the KO mice, including the CD11c??CD11b?
allergenic subset, overexpress CCR7 and should thus possess an
increased propensity to migrate. Significantly, increased surface
expression of CCR7 was also noted on DC derived from either
thoracicoraxillaryLNofKOmice,comparedwithWTlittermates,
(Fig. 2D).
Runx3 KO DC Display Increased Migration Ability.BecauseCCR7and
its ligands (ELC?CCL19 and SLC?CCL21) are important compo-
nents of the DC migration and navigation system (51), we asked
whether increased CCR7 due to the loss of Runx3 indeed affects
DCmigratoryproperties.WTandKOBMDCtreatedoruntreated
with TGF? were induced to undergo maturation by LPS and then
subjected to an SLC?CCL21-dependent transmigration assay.
TGF? treatment significantly reduced the migration rate of WT
BMDC(Fig.3A),consistentwiththeinhibitionofCCR7expression
by this cytokine (Fig. 2A). No such inhibition of SLC?CCL21-
mediated chemotaxis was noted in TGF?-treated KO BMDC
(Fig. 3A).
DC migration was also assessed by using another in vitro assay.
Fig. 1.
mice.(A)IncreaseofAHRintheKOmice.AHRwas
determined in 4- to 9-week-old KO mice and WT
littermatesasdescribedinMaterialsandMethods.
Resultsarepresentedasfoldincreaseinenhanced
pause (Penh) [a parameter related to pulmonary
resistance (41)] relative to baseline value. Data
represent the mean ? SEM of 15 mice per group
and disclose a significant difference between WT
and KO mice (Student’s two-tailed t test;*, P ?
0.0005).(B)IncreaseinserumandBALIgEintheKO
mice. Total IgE in serum and BAL of 4- to 6-week-
oldKOmice(n?7)andWTlittermates(n?6)was
quantitated by ELISA (Mouse IgE ELISA Quantiza-
tion Kit, E90-115, Bethyl Laboratories, Montgom-
ery, TX) according to the manufacturer’s recom-
mended conditions. Values in ng?ml disclose
significant differences between WT and KO mice
(Student’s two-tailed t test;*, P ? 0.026;**, P ?
0.014). (C) Increased expression of Fc?R receptors
onDCfromKOthoracicLN.Single-cellsuspension
wasobtainedfromLNofWTandKOmice(n?3),
subjected to acid elution of occupied Fc?R recep-
tor, incubated with IgE-FITC conjugate and with
anti CD11c, and subjected to FACS analysis.
CD11c?cells were gated, and Fc?R level was de-
termined(D)IncreasedresponsivenessofKOmice
toinhaledLPS.Runx3KOmiceandWTlittermates
(n?4)wereanesthetizedandsubjectedtointranasalinhalationof25?lofPBScontaining0.1?gofLPSor25?lofPBSalone.Sixteenhourslatermicewerekilledand
BALwasobtainedandanalyzedbyFACSand,aftercytospin,byGiemsastaining.Circledarespecificpopulations:CD11c?DC(circles1and2),neutrophils(circle3),and
eosinophils (circle 4), as was also confirmed by Giemsa staining (data not shown).
Human asthma hallmarks in Runx3 KO
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SLC is produced at two points along DC migration route from skin
to regional LN, the lymphatic endothelium and the LN T cell zone
(17). We thus examined the migratory capability of dermal DC,
comparing WT with KO mice. Dermal sheaths were prepared and
incubated, dermal side down, with or without TGF? and SLC. In
both WT and Runx3 KO sheaths, a pronounced SLC-dependent
migration of CD11c??MHCIIhighDC was recorded (Fig. 3B).
However, whereas the migration of WT dermal DC was inhibited
by TGF?, migration of the KO DC was not. The results of BMDC
and dermal DC migration assays indicate that loss of Runx3 not
onlyimpairsTGF?-mediatedtranscriptionregulationofCCR7,but
also leads to an impairment of TGF?-dependent migration inhi-
bition of DC toward the CCR7 ligand SLC.
Enhanced CCR7-Dependent Migration of Respiratory DC to Draining
LN in Runx3 KO Mice. Migration of respiratory DC to draining LN
is a key step in the initiation of immune response in the lung (52).
The results of the transmigration in vitro assays led us to examine
theinvivotraffickingofrespiratoryDCinKOandWTmice.Invivo
migration was assessed by monitoring the accumulation of CFSE-
labeledrespiratoryDCintheregionalLN.InWTmice,respiratory
CFSE?CD11c?DC accumulate primarily in the draining thoracic
LN, amounting to 1–3% of the total LN DC (Fig. 4). These data
correspond with previously reported findings (52) and are highly
specific because no CFSE?respiratory DC were detected in the
nondraining axillary LN, used as a reference (Fig. 4A). In com-
parison with WT, significantly more respiratory CFSE?DC were
foundinthoracicLNofKOmice(Fig.4),whichcorrelateswellwith
the higher migratory capacity of KO DC in vitro and the elevated
CCR7 on KO DC. Treatment of WT mice by intranasal instillation
ofLPScausedanincreaseinmigrationofrespiratoryDC,although
toamuchlowerextentcomparedwiththeuntreatedKOmice(Fig.
4B). These results indicate that the increased expression of CCR7
on Runx3 KO DC results in increased CCR7-dependent migration
of respiratory DC to the draining LN. This conclusion is supported
by the demonstration that blocking CCR7 by receptor-specific
antibodiesmarkedlyreducedthemigrationofrespiratoryDCinthe
KO mice (Fig. 4B).
Fig. 3.
Runx3 KO DC is not attenuated by
TGF?.(A)BMDCfromWTandRunx3
KO littermates were cultured as
above with or without TGF? and, at
day 6, were treated overnight with
LPS.BMDC(5?105)wereplacedina
Transwell migration chamber, and
SLC-dependent chemotaxis was
measured as described in Materials
and Methods. The mean ? SEM of
three separate experiments is pre-
sented. Migration inhibition of WT
DC by TGF? was significant (*) by
using the paired Student t test (P ?
0.05). (B) Dermal sheaths of WT and KO mice (n ? 3) were prepared, and SLC-mediated chemotactic migration was assessed. Inhibition of WT DC migration by TGF?
(*) was significant (P ? 0.029) as were the responses of WT and KO DC to SLC and TGF? (**)(P ? 0.019).
SLC-directed chemotaxis of
Fig. 2.
DC. (A) Impaired TGF?-dependent inhibition of CCR7
transcription in KO BMDC. WT and Runx3 KO BMDC
were grown in the presence or absence of TGF? (10
ng?ml). At day 6, cells were induced to undergo mat-
uration by LPS, and, at day 7, RNA was prepared and
analyzed by RT-PCR. (B) Impaired TGF?-dependent in-
hibitionofsurfaceexpressionofCCR7inKOBMDC.WT
and Runx3 KO BMDC were grown and treated as in A.
At day 7, cells were analyzed by FACS by using anti-
CCR7 antibodies (goat anti-mouse CI0131, Capralog-
ics). FSChighCD11c?DC were gated, and their CCR7
expression was determined. Reduction in the level of
surface CCR7 and in the number of cells expressing it
was noted only in WT and not in Runx3 KO BMDC. (C
and D) Increased CCR7 expression on alveolar and LN
DCofRunx3KOmice.BALandperipheralLNcellsofKO
and WT mice (n ? 3) were obtained and analyzed. (C)
Alveolar DC (FSChigh?CD11chigh) were gated and ana-
lyzedforCCR7expression.Ofnote,expressionofCCR7
on the DC subpopulation CD11c??CD11b?present
only in KO lungs is shown along with that of KO
CD11c??CD11b?DC. (D) FSChigh?CD11chighDC of axil-
laryandthoracicLNweregatedandanalyzedforCCR7
expression.
Dysregulated expression of CCR7 in Runx3 KO
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Hammad et al. (35) have reported that activation of the perox-
isome proliferator-activated receptor ? (PPAR?) by selective
PPAR? agonists such as Rosiglitazone and Ciglitazone decreased
CCR7 expression on DC and inhibited their migratory properties.
Consequently, this treatment resulted in reduced DC-induced
eosinophilic lung inflammation (35). We addressed whether
PPAR? activation would inhibit the migration of respiratory KO
DC, as did the anti-CCR7 antibodies. When mice inhaled Ciglita-
zone, before instillation of CFSE, a pronounced inhibition of KO
DC migration to draining LN was observed (Fig. 4B). Of note,
Ciglitazone was almost as effective as the anti-CCR7 antibodies in
inhibitingDCmigrationtoLN,andalsocausedmigrationinhibition
of WT DC in LPS-treated mice (Fig. 4B). Taken together, the
results of elevated CCR7, RNA, and protein, in KO DC and the in
vitro and in vivo DC migration studies, demonstrate that Runx3
functions as a negative regulator of CCR7 transcription in response
to TGF? signaling and thereby regulates the trafficking of alveolar
DC to the draining LN. The data may also suggest that Ciglitazone
could be useful in treatment of DC-induced immunity to self-
antigens.
Discussion
Respiratory tract DC are an important population of regulatory
cells that orchestrate immunity against inhaled antigens (5, 7).
These cells are normally maintained at their immature state
through interaction with antiinflammatory cytokines, such as
TGF?, secreted by the surrounding cellular environment (5). At
this immature state, respiratory DC have a low propensity to
Fig.4.
byintranasaladministrationofCFSEtolabelinvivotherespiratoryDC.Whenindicated,WTmiceweretreatedwithLPS(n?4),andRunx3KOmiceweretreated
with anti-CCR7 antibody (n ? 4) or with buffer only (n ? 4). KO mice (n ? 4) and LPS-treated WT mice (n ? 3) were also treated by inhalation of Ciglitazone.
Eighteen hours later, mice were killed, and single-cell suspensions of BAL, thoracic LN, and axillary LN were prepared and analyzed by FACS. (A) FSChigh?CD11c?
DC were gated (R1 and R2). Shown is representative side scatter (SSC) versus CFSE staining of DC populations in BAL and LN after the various treatments. (B)
Migration index of alveolar DC to thoracic LN represents the ratio between the percentage of CFSE?cells within the CD11c?population in the thoracic LN and
therespectivevalueinBALcells.Resultsarepresentedasmean?SEM.AnalysisofvarianceshowedthatthemigrationindexofuntreatedKODCwassignificantly
higher than that of WT (*, P ? 0.016). Notably, the anti-CCR7-treated KO DC migration index was similar to basal migration of WT, and Ciglitazone significantly
(*, P ? 0.03) reduced the migration of KO DC.
ElevatedCCR7mediatedinvivotraffickingofalveolarDCtothedrainingLNintheKOmice.(AandB)Runx3KO(n?7)andWT(n?5)miceweretreated
10602 ?
www.pnas.org?cgi?doi?10.1073?pnas.0504787102Fainaru et al.