Plasmacytoid dendritic cells employ multiple cell adhesion molecules sequentially to interact with high endothelial venule cells--molecular basis of their trafficking to lymph nodes.
ABSTRACT Plasmacytoid dendritic cells (pDCs) are natural type I IFN-producing cells found in lymphoid tissues, where they support both innate and adaptive immune responses. They emigrate from the blood to lymph nodes, apparently through high endothelial venules (HEVs), but little is known about the mechanism. We have investigated the molecular mechanisms of pDC migration using freshly isolated DCs and HEV cells. We found that pDCs bound avidly to HEV cells and then transmigrated underneath them. Two observations suggested that these binding and migration steps are differentially regulated. First, treatment of pDCs with pertussis toxin blocked transmigration but not binding. Second, pDCs were able to bind but not to transmigrate under non-HEV endothelial cells, although the binding was observed to both HEV and non-HEV endothelial cells. Antibody inhibition studies indicated that the binding process was mediated by alphaL and alpha4 integrins on pDCs and by intercellular adhesion molecule (ICAM)-1, ICAM-2 and vascular cell adhesion molecule-1 on HEVs. The transmigration process was also mediated by alphaL and alpha4 integrins on pDCs, with junctional adhesion molecule-A on HEV cells apparently serving as an additional ligand for alphaL integrin. These data show for the first time that pDCs employ multiple adhesion molecules sequentially in the processes of adhesion to and transmigration through HEVs.
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ABSTRACT: Dendritic cells (DCs) serve a critical role both in promoting and inhibiting adaptive immunity. The goal of this study was to investigate the effect of natalizumab (NTZ) treatment on DC numbers, phenotype, and function in patients with multiple sclerosis (MS).PLoS ONE 01/2014; 9(7):e103716. · 3.53 Impact Factor
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ABSTRACT: Plausible representatives of plasmacytoid dendritic cells (pDCs) in pigs have been characterized as being CD4(hi)CD172(lo). Due to their paucity in blood, we utilized novel fluorescent-activated cell sorting procedures to isolate them from PBMC. The resultant subset was greater than 98% homogeneous in regards to the selected phenotype and contained the preponderance of individuals secreting IFN-alpha after exposure to a known stimulant, transmissible gastroenteritis virus (TGEV). In addition to being a potent source of IFN-alpha, other properties of these porcine CD4(hi)CD172(lo) cells including their morphological transition from a plasma cell-like shape during quiescence to one resembling a dendritic cell (DC) after activation by TGEV and their relatively strong constitutive expression of interferon regulatory factor-7 (IRF-7) conformed to the expectations of genuine pDCs. While a substantial IFN-alpha response was also elicited from the porcine pDCs by pseudorabies virus (PrV), swine influenza virus (SIV), and TLR7 and 9 agonists, there was an agent-dependent induction of varying amounts of IL-2, IL-6, IL-8, IL-12, IFN-gamma, and TNF-alpha. Notably, porcine reproductive and respiratory syndrome virus (PRRSV) failed to provoke the pDCs to secrete any of the measured cytokines except IL-2. Moreover, whereas pDCs exposed to TGEV or the TLR9 agonist rapidly increased IRF-7 production and morphed into DCs with enhanced CD80/86 expression, similar alterations were not observed during incubation with PRRSV. This atypical response of pDCs to PRRSV may contribute to its pathogenesis, which unlike that associated with PrV, SIV or TGEV includes persistent infection and limited development of protective immunity.Veterinary Immunology and Immunopathology 10/2009; 135(1-2):20-33. · 1.88 Impact Factor
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ABSTRACT: In response to bacterial stimuli, multiple dendritic cell (DC) populations accumulate within the draining lymph node, thus enhancing opportunities for effective T cell-DC interaction. DC subpopulations, such as plasmacytoid, CD8(+), and CD11b(+) subsets, have distinct roles in determining the nature of the immune response. The mechanisms whereby individual DC subpopulations are mobilized and the extent to which these processes are linked to increases in overall lymph node cellularity have not been determined. In the current study, the mechanisms of DC subset mobilization to the draining auricular lymph node were examined after intradermal injection of Staphylococcus aureus-derived peptidoglycan. Using mast cell-deficient mice and local mast cell reconstitution, plasmacytoid and CD8(+) DC responses were shown to be mast cell dependent, whereas the CD11b(+) DC response was not. A histamine H2 receptor-dependent, CXCL9-independent pathway controlled the selective influx of both plasmacytoid and CD11b(+) DC into the lymph node, but not lymph node cellularity. In contrast, IL-6 was important for the mobilization of CD8(+) and CD11b(+) DC. TNF and IL-1 receptor were dispensable for plasmacytoid, CD11b(+), and CD8(+) DC responses. These findings provide novel opportunities for the selective mobilization of specific DC subsets to lymph nodes and demonstrate critical roles for both histamine and IL-6 in this process.The Journal of Immunology 02/2010; 184(4):2116-23. · 5.52 Impact Factor
International Immunology 2007; 1 of 7
ª The Author 2007. Published by Oxford University Press on behalf of
The Japanese Society for Immunology. All rights reserved.
Plasmacytoid dendritic cells employ multiple cell
adhesion molecules sequentially to interact with
high endothelial venule cells – molecular basis of
their trafficking to lymph nodes
Takahiro Matsutani1,2,*, Toshiyuki Tanaka1,3,*, Kazuo Tohya4, Kazuhiro Otani1,2, Myoung Ho Jang1,
Eiji Umemoto1, Kanako Taniguchi1, Haruko Hayasaka1, Koichi Ueda2and Masayuki Miyasaka1
1Laboratory of Immunodynamics, Department of Microbiology and Immunology, Osaka University Graduate School of Medicine,
Suita, 565-0871, Japan
2Department of Plastic and Reconstructive Surgery, The Postgraduate Course of Osaka Medical College, Takatsuki, 569-8686,
3Department of Pharmacy, Laboratory of Immunobiology, School of Pharmacy, Hyogo University of Health Sciences, Kobe,
4Department of Anatomy, Kansai College of Oriental Medicine, Sennan, 590-0482, Japan
Keywords: adhesion molecules, cell trafficking, high endothelial venule cells, plasmacytoid dendritic cells, transmigration
Plasmacytoid dendritic cells (pDCs) are natural type I IFN-producing cells found in lymphoid tissues,
where they support both innate and adaptive immune responses. They emigrate from the blood to
lymph nodes, apparently through high endothelial venules (HEVs), but little is known about the
mechanism. We have investigated the molecular mechanisms of pDC migration using freshly isolated
DCs and HEV cells. We found that pDCs bound avidly to HEV cells and then transmigrated underneath
them. Two observations suggested that these binding and migration steps are differentially regulated.
First, treatment of pDCs with pertussis toxin blocked transmigration but not binding. Second, pDCs
were able to bind but not to transmigrate under non-HEV endothelial cells, although the binding was
observed to both HEV and non-HEV endothelial cells. Antibody inhibition studies indicated that the
binding process was mediated by aL and a4 integrins on pDCs and by intercellular adhesion
molecule (ICAM)-1, ICAM-2 and vascular cell adhesion molecule-1 on HEVs. The transmigration
process was also mediated by aL and a4 integrins on pDCs, with junctional adhesion molecule-A on
HEV cells apparently serving as an additional ligand for aL integrin. These data show for the first time
that pDCs employ multiple adhesion molecules sequentially in the processes of adhesion to and
transmigration through HEVs.
In anti-viral immune responses, plasmacytoid dendritic cells
(pDCs) directly inhibit viral replication and contribute to the
activation of NK cells, T cells, B cells and conventional DCs
by producing type 1 IFNs (1, 2). pDCs are constitutively
present in lymph nodes (LNs) under steady-state conditions
(3) but are substantially reduced in the LNs of L-selectin-
deficient mice (4), indicating that their trafficking to LNs is at
least partially dependent on L-selectin under physiological
conditions. However, detailed studies on adhesion mole-
cules involved in pDC trafficking to lymphoid tissues are
scarce and have often depended on in vitro- (5) or in vivo-
expanded counterpart (6), because it is difficult to obtain
sufficient quantities of unmanipulated pDCs from intact ani-
mals. As a result, little information is currently available
about the molecular mechanisms regulating the steady-state
trafficking of pDCs.
Here we investigated the interactions between pDCs and
high endothelial venules (HEVs) directly using purified pDCs
and HEV endothelial cells. We show for the first time that
pDCs have the full capacity to adhere to and transmigrate
*These authors contributed equally to this work.
Correspondence to: M. Miyasaka; E-mail: firstname.lastname@example.orgReceived 24 May 2007, accepted 27 June 2007
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International Immunology Advance Access published September 5, 2007
by guest on June 3, 2013
beneath HEV endothelial cells under steady-state conditions
and that they interact with HEVs through a precise sequence
of events using specific pairs of adhesion molecules. pDCs
show robust transmigration underneath HEV endothelial cells
but not non-HEV endothelial cells, indicating that pDC trans-
migration is at least in part regulated differently from their
adhesion to these cells. We also demonstrate the involve-
ment of leukocyte function-associated antigen-1 (LFA-1)/
junctional adhesion molecule (JAM)-A adhesion pathway in
BALB/c mice were purchased from Japan SLC (Hamamatsu,
Japan). All animal experiments used experimental protocols
that were approved by the Ethics Review Committee for Ani-
mal Experimentation of Osaka University Graduate School of
Pertussis toxin (PTX) and polymyxin B were obtained from
Calbiochem (La Jolla, CA, USA). Hybridomas producing
mAbs against peripheral node addressin (PNAd) (MECA-79)
(7) and mucosal addressin cell adhesion molecule-1 (MAd-
CAM-1) (MECA-89 and MECA-367) (8) were kindly provided
by E. C. Butcher (Stanford University, Stanford, CA, USA). Hy-
bridomas for anti-vascular cell adhesion molecule-1 (VCAM-1)
(M/K-1) (9) and CD49d (PS/2) (10) were generous gifts of
K. Miyake (University of Tokyo, Tokyo, Japan). mAbs to JAM-A
(H202.106) (11) and JAM-C (H33) (12) were kind gifts from
Beat A. Imhof and Michel Aurrand-Lions (the University Medi-
cal Center, Geneva, Switzerland). mAbs to CD11a (KBA) and
CD54 (KAT-1) were described previously (13). Biotin-conjugated
(Bergisch Gladbach, Germany). Purified rat IgG and FITC-
conjugated goat anti-rat IgG were from Cappel (Durham, NC,
USA). Allophycocyanin-conjugated anti-CD11c (HL3), Alexa
647-conjugated anti-CD34 (RAM34) and FITC-conjugated
anti-CD45R/B220 (RA3-6B2) or CD49d (R1-2) were from eBio-
science (San Diego, CA, USA). FITC-conjugated anti-CD11a
(M17/4), CD18 (C71/16), CD29 (Ha2/5), b7 (M293) or CD62L
(MEL-14), PE-conjugated anti-B220 (RA3-6B2) or CD11c
(HL3), Fc block CD16/CD32 (2.4G2) and mAb to CD62L
(MEL-14) were from BD PharMingen (San Diego, CA, USA).
Purification of HEV cells
MAdCAM-1+endothelial cells of HEVs were purified from
mouse mesenteric LNs by MACS (Miltenyi Biotec), using
biotin-conjugated anti-MAdCAM-1 mAb (MECA-89), as previ-
ously described (14). Some experiments used CD34+PNAd+
HEV endothelial cells and CD34+
MAdCAM-1?non-HEV endothelial cells that had been sorted
by FACSAria (BD Biosciences). The purity of the isolated
HEV cells was routinely >90%.
Preparation of pDCs
Spleens removed from mice were digested with 400 Mandl
U/ml collagenaseD (Roche Diagnostics, Mannheim,
Germany) and 10 lg/ml DNase I (Roche Diagnostics) in RPMI
1640/10% FCS, with continuous stirring at 37?C for 30 min.
After the addition of 10 mM EDTA, the cell suspension was
incubated at 37?C for an additional 5 min. The cells obtained
were then spun through a 17.5% Accudenz solution (Accu-
rate Chemical & Scientific, Westbury, NY, USA) to enrich for
pDCs. These cells were then incubated with anti-CD16/
CD32 for FcR blocking followed by an incubation with
biotin-conjugated anti-mPDCA-1 (10 lg/ml) for 15 min. After
being washed with PBS containing 10% FCS, 20 mM
HEPES, 100 U/ml penicillin, 100 lg/ml streptomycin, 1 mM
sodium pyruvate, 10 mM EDTA and 10 lg/ml polymyxin B,
the cells were incubated with a mixture of 10-fold-diluted
streptavidin-conjugated microbeads (Miltenyi Biotec) and
subjected to two rounds of MACS (Miltenyi Biotec). The
purity of the sorted pDCs was routinely >95%.
For phenotypic analysis, cells were incubated with a combi-
nation of allophycocyanin-conjugated anti-CD11c, PE-conju-
gated anti-B220 and FITC-conjugated anti-CD11a, -CD18,
-CD29, -CD49d, -CD62L or -integrin b7 mAbs, and analysed
on a flow cytometer (FACSCalibur, BD Biosciences). In some
experiments, the cells were first incubated with anti-JAM-A
or JAM-C mAb, followed by FITC-conjugated goat anti-rat
IgG (Cappel). After blocking with rat IgG, cells were further
incubated with a combination of allophycocyanin-conjugated
anti-CD11c and PE-conjugated anti-B220 mAbs. To analyse
L-selectin expression, pDCs in unseparated cell populations
were used. Bone marrow (BM)-derived pDCs prepared as
described (15) and CD11chighB220?DCs from the spleen
were also analysed as above.
Cell adhesion and transmigration assay
Purified HEV cells (1–2 3 104cells per well) were allowed to
adhere to collagen I-coated wells of 8-well chamber slides
(BD Biosciences) at 37?C for 2 h. pDCs (1–2 3 105cells per
well) were then added, and the cultures were incubated at
37?C for an additional 3 h. The cells were washed 3 times
with PBS, fixed with 4% PFA and stained with May–Grunwald–
Giemsa solution. Under the light microscope, we observed
pDCs bound to the surface of HEV cells (stained dark blue),
and other pDCs that transmigrated underneath the HEV cells
(stained pale blue). These cells were scored separately for
at least 100 HEV cells. For antibody inhibition studies, purified
mAbs were used at a final concentration of 50 lg/ml. Anti-
bodies to VCAM-1 or CD49d were used as hybridoma culture
supernatants. In some experiments, pDCs were pre-treated
with various concentrations of PTX at 37?C for 1 h.
For scanning electron microscopy, purified HEV cells (2 3
104cells per well) and pDCs (4 3 105cells per well) in the
wells of collagen I-coated 8-well culture slides (BD Bioscien-
ces) were allowed to interact for 3 h as above. The cell
layers were fixed in 1% glutaraldehyde in 0.1 M phosphate
buffer, then post-fixed in 1% OsO4for 30 min at room tem-
perature. The samples were then dehydrated in graded con-
centrations of ethanol, and critical-point dried in CO2. After
2Multiple adhesion pathways in pDC-HEV interactions
by guest on June 3, 2013
sputter coating with gold, the specimens were examined un-
der a Hitachi S-3000H electron microscope operated at 15 kV.
For transmission electron microscopy, purified HEV cells
(7 3 104cells per well) and pDCs (5 3 105cells per well)
were cultured in collagen I-coated 48-well culture slides (BD
Biosciences) as above. The cell layers were fixed as above,
then dehydrated in graded ethanol and embedded in Quetol
812. Vertically cut ultrathin sections of the layer were stained
with uranyl acetate/lead citrate and examined using a JEOL
JEM-1230 electron microscope operated at 60 kV.
A Student’s t-test was applied to compare the statistical dif-
ference within two groups.
Results and discussions
Splenic pDCs express an array of adhesion molecules that
can interact with HEVs
To study the mechanisms by which pDCs are recruited to
LNs under steady-state conditions, we wished to use natu-
rally circulating pDCs rather than in vitro- or in vivo-
expanded pDCs, because any cell amplification process
may result in changes in the cell phenotype and trafficking
patterns. A phenotypic comparison of the pDCs from differ-
ent lymphoid organs of unperturbed mice showed that pDCs
expressed readily detectable levels of L-selectin and aL, a4,
b1 and b2 integrins, low levels of b7 integrin and were nega-
tive for JAM-A and JAM-C (Fig. 1). This pattern corresponds
to the phenotype of circulating pDCs in the peripheral blood
of mice (5). Myeloid (CD11chighB220?) DCs from the spleen
showed a similar pattern of adhesion molecule expression
except that they showed higher expression of b7-integrin
and JAM-A than pDCs. L-selectin expression is apparently
susceptible to isolation procedures, because the prolonged
exposure of pDCs to digestion enzymes and/or density gra-
dient solutions during their isolation rapidly diminished the
L-selectin expression (data not shown). This might at least
partly account for the previous observation that pDCs iso-
lated from the secondary lymphoid organs showed negative
or very low L-selectin expression in the mouse (4) and hu-
man (16). Because the expression pattern of these mole-
cules was almost identical to that seen with pDCs obtained
from other lymphoid tissues and peripheral blood, we
judged our pDCs to be representative of blood-borne circu-
lating pDCs and used them as such throughout the present
investigation. Their localization in the trafficking area of the
spleen, i.e. in the periarteriolar lymphoid sheath or at the
marginal zone (3) also supports the contention that splenic
pDCs represent circulating pDCs.
pDCs bind readily to purified HEV cells and subsequently
transmigrate beneath them
To establish whether pDCs can interact with HEV endothelial
cells, we purified pDCs and HEV endothelial cells and exam-
ined their interactions in vitro. After being seeded on collagen
I-coated slides, most HEV endothelial cells spread well, but
some retained a plump morphology and adhered to the sub-
stratum by extending pseudopods from the cell surface.
Scanning electron microscopy showed that purified pDCs
bound avidly to both the flat and plump types of HEV endo-
thelial cells, which occurs within 30 min of co-incubation
(Fig. 2A and B), and the pDCs occasionally induced distinct
morphological changes, such as pseudopod protrusion, in the
endothelial cells in the vicinity of their adhesion (Fig. 2A). As
shown in Fig. 2(C and D), pDCs often transmigrated beneath
HEV endothelial cells from the site where the morphological
Fig. 1. pDCs obtained from various lymphoid tissues express a variety of adhesion molecules that can interact with HEVs. pDCs were obtained
from the spleen, mesenteric LNs (MLN), peripheral LNs (PLN), BM and blood, and were examined for the expression of L-selectin and integrin
aL, a4, b1, b2, b7 chains, JAM-A and JAM-C. Myeloid (CD11bhighB220?) DCs from the spleen were also analysed in the same way. Note that the
pDCs from various tissues showed comparable expression profiles of the trafficking-associated adhesion molecules.
Multiple adhesion pathways in pDC-HEV interactions3
by guest on June 3, 2013
changes were observed. These findings are consistent with
the process by which leukocyte adhesion is thought to trigger
signals in the endothelial cells that promote subsequent cellu-
lar interactions such as transmigration (17). Fig. 2(E) illus-
trates numerous pDCs, identified by their unique cytoplasmic
tail, binding to and transmigrating underneath HEV endothe-
lial cells. The transmigration was readily observed soon after
pDC adhesion, reaching a plateau at 2 h after adhesion.
pDCs transmigrate along the periphery of an endothelial
cell or between adjacent endothelial cells in a PTX-sensitive
After adhesion, pDCs appeared to swiftly insinuate their cell
bodies beneath the HEV endothelial cells. As seen in the set
of transmission electron micrographs in Fig. 3(A–E), pDCs
appeared to crawl underneath the HEV endothelial cells
along the cell periphery. Once more than half of the pDC cell
body was underneath the endothelial cell, a thin protrusion
from the endothelial cell appeared that was tightly apposed
to the transmigrating pDCs (Fig. 3D and E). Eventually, this
protrusion surrounded the entire apical surface of the pDCs,
completing the process of pseudoemperipolesis (18) by the
HEV endothelial cells. As shown in Fig. 3(F), pDCs some-
times appeared to make their way to endothelial junctions
and then transmigrate between tightly apposed HEV endo-
appeared to lift in a way that would facilitate the pDC’s pas-
sage through the junction, indicating that pDC adhesion
may initiate the opening of endothelial cell junctions to expe-
dite the pDC transmigration process. In the scanning of
>1000 HEV endothelial cells interacting with pDCs, there
was no indication that pDCs penetrated the HEV endothelial
cell body or passed through the cytoplasm of the endothelial
cell during the course of diapedesis. This is consistent with
the idea that pDCs use mainly the paracellular and not the
transcellular route to extravasate through HEVs, as do naive
lymphocytes (19, 20).
The transmigration by pDCs was apparently regulated by
Gai-protein-mediated signaling, because PTX treatment of
the pDCs almost completely abrogated the transmigration
underneath HEV endothelial cells (Fig. 3G). It is also of note
that no transmigration was observed when non-HEV endo-
thelial cells from LNs were used instead of HEV endothelial
cells, although appreciable pDC binding to the non-HEV en-
dothelial cells was observed (Fig. 3H). These findings indi-
cate that pDC transmigration occurs with a specific type of
endothelial cell and that pDC transmigration is, at least in
part, regulated differently from pDC adhesion to endothelial
aL and a4 integrins on pDCs and ICAM-1, ICAM-2, VCAM-1
and JAM-A on HEVs were involved in pDC–HEV interactions
We next performed antibody inhibition studies to identify the
adhesion molecules involved in the interaction between
pDCs and HEV endothelial cells. As shown in Fig. 4(A),
mAbs to aL or a4 integrin inhibited the pDC adhesion to
HEV endothelial cells by about 30–40% compared with con-
trol rat IgG, and a mixture of the two mAbs inhibited the ad-
hesion by ;70%. Correspondingly, mAbs to the ligands for
these integrins, intercellular adhesion molecule (ICAM)-1,
ICAM-2 or VCAM-1 inhibited pDC adhesion to HEV endothe-
lial cells by 30–50% individually and by ;60% when applied
in combination. In contrast, mAbs to L-selectin and MAd-
CAM-1 did not inhibit the adhesion. These results indicate
that pDC adhesion to HEV endothelial cells is regulated
mainly by the aLb2 integrin/ICAM-1, -2 and a4b1 integrin/
VCAM-1 pathways, although another adhesion pathway
is also involved. As shown in Fig. 4(B), the transmigration
process also appears to be regulated by the aLb2 integrin/
ICAM-1, -2 and a4b1 integrin/VCAM-1 pathways, because
individual mAbs to these molecules inhibited transmigration
by >50%: a mixture of mAbs to aL and a4 integrins almost
completely abolished the transmigration of pDCs, while mAbs
to ICAM-1, -2 plus VCAM-1 inhibited pDC transmigration by
;80%. The effect of these mAbs on transmigration might in
Fig. 2. Scanning electron micrographs showing pDCs adhere to HEV
endothelial cells and induce morphological changes in the endothelial
cells. Splenic pDCs were incubated with purified HEV endothelial
cells. pDCs bound to both the plump (A) and flat (B) types of HEV
endothelial cells. After adhesion, HEVendothelial cells often extended
pseudopods (shown by an arrow in A) or a fold-like structure near the
site of pDC adhesion (shown by an arrow in C and D). Panel (E)
shows numerous pDCs adhering to and transmigrating underneath
HEV endothelial cells. The unique cytoplasmic tail extending from the
adherent or transmigrating cells verifies that the cells interacting with
the endothelial cells were pDCs. Scale bars, 2 lm.
4 Multiple adhesion pathways in pDC-HEV interactions
by guest on June 3, 2013
part reflect their inhibitory effects on adhesion and/or cell
movement: firm adhesion is a prerequisite for transmigration,
so blocking adhesion necessarily blocks transmigration (21).
In addition, blocking a4b1 integrin inhibits leukocyte chemo-
taxis (Y. Srinoulprasert and M. Miyasaka, unpublished obser-
vation). Nevertheless, the observation that these mAbs
invariably inhibited transmigration far more efficiently than
adhesion argues for a role of these molecules in the transmi-
gration process, and the involvement of other molecules is
not ruled out.
Previous studies by Ostermann et al. (22) showed that
JAM-A could serve as a functional ligand for LFA-1 and me-
diate transendothelial migration of certain leukocytes. We
therefore sought to test the functional contribution of JAM-A
expressed in HEV endothelial cells (23) in pDC–HEV interac-
tions. As shown in Fig. 4(C), mAb to JAM-A inhibited trans-
migration of pDCs underneath HEV endothelial cells without
significantly affecting the pDC binding to HEV cells, whereas
mAb to JAM-C affected neither adhesion nor transmigration.
In addition, mAbs to JAM-A and ICAM-1 additively inhibited
transmigration of pDCs to a similar extent that mAb to aL
integrin did (Fig. 4D). These observations are consistent with
previous findings that JAM-A can function as an additional
ligand to LFA-1 mediating transendothelial migration of leu-
kocytes and suggest an important role of JAM-A in the trans-
migration of pDCs. The possible involvement of CD31 (24),
CD99 (25) and other JAM family members (26), such as
JAM-B and ESAM, in pDC transmigration needs to be inves-
tigated in future studies.
Although mAbs to MAdCAM-1 or L-selectin had little effect
on adhesion and the anti-MAdCAM-1 did not affect transmi-
gration in our analysis (Fig. 4A and B), it does not rule out
the possible involvement of these molecules in pDC–HEV
interactions in vivo. Our in vitro static assay system may
bypass the initial shear-dependent tethering processes medi-
ated by L-selectin and/or MAdCAM-1 that may be necessary
for firm adhesion. In support of this idea, our preliminary
analysis indicated that pDCs could adhere to and transmi-
grate underneath HEV endothelial cells independently of
shear stress (data not shown). Although we could not formally
address the role of L-selectin under shear stress conditions
in the present investigation, a previous study demonstrated
the critical role of L-selectin as a tethering receptor in pDC–
HEV interactions in vivo (5).
Finally, it has been generally thought that trafficking of cir-
culating pDCs from the blood to LNs is primarily driven by
inflammatory stimuli (1, 5, 27). However, our study clearly
demonstrates that circulating pDCs are fully capable of inter-
acting with HEV endothelial cells under physiological condi-
tions and are likely to use a mechanism very similar to that
described for T and B naive lymphocytes (28). Our study
also indicates that pDCs interact with HEVs via a specific
Fig. 3. pDCs transmigrate under HEV endothelial cells in a PTX-
sensitive manner. After their adhesion to HEV endothelial cells, pDCs
appeared to crawl along the cell periphery and transmigrate beneath
the HEV cells. Panels (A–E) show the presumed sequence of events
in the pDC–HEV endothelial cell interactions. Toward the end of this
process, HEVendothelial cells provide a cellular protrusion in the form
of a thin flap in close apposition to the pDCs, as seen in (D and E).
A dotted line in (C–E) demarcates a junction between the endothelial
thin flap and the pDC cell body. (F) pDCs sometimes showed
transmigration through the junctions of HEV endothelial cells. This
panel shows a pDC that was just about to migrate into the junction
formed by two HEV endothelial cells. A cell on the left adhering to the
surface of one HEV cell appears to be a lymphocyte. Note the
prominent flap sticking out from the endothelial cell on the right, which
made close contact with the transmigrating pDC. Scale bars, 1 lm.
(G) Pre-treatment of pDCs with PTX strongly inhibited their trans-
migration underneath HEV endothelial cells. (H) pDCs transmigrated
under HEV endothelial cells but not under non-HEV endothelial cells.
Multiple adhesion pathways in pDC-HEV interactions5
by guest on June 3, 2013
Fig. 4. InvolvementofmultiplecelladhesionpathwaysinpDC–HEVinteractions. (A)pDCadhesiontoHEVendothelial cells wasinhibitedby mAbs
against JAM-A. (D) Similar inhibitory effects on pDC transmigration were observed with anti-aL integrin mAb and anti-ICAM-1 plus anti-JAM-A
mAbs. Data are presented as mean number of pDCs per 100 HEVs 6 SD (n = 3). *P < 0.001, **P < 0.01, ***P < 0.05 compared with controls.
6 Multiple adhesion pathways in pDC-HEV interactions
by guest on June 3, 2013
order of events: they first bind to HEV endothelial cells, then
invoke morphological changes in the endothelial cells and
subsequently transmigrate underneath them. Because it is
technically difficult to monitor continuously the process of
pDC diapedesis/transmigration with the currently available
intravital microscopy technique (5), our assay system may
provide a useful new tool for studying the entire process of
pDC transmigration in detail in vitro. Although the present
study did not address the mechanisms of integrin activation
in pDC–HEV interactions, future studies using our in vitro
system may help define the chemokines and lysophospholi-
pid mediators, such as sphingosine-1-phosphate and lyso-
phosphatidic acid, that might be involved in this process
under physiological and pathological conditions. Such find-
ings would undoubtedly contribute to the development of
techniques for manipulating pDC trafficking in vivo.
Ministry of Education, Culture, Sports, Science and Technol-
ogy of Japan (17047025 and 17590432 to T.T.); Advanced
Research on Cancer from the Ministry of Education, Culture,
Sports, Science and Technology of Japan (17014056 to M.M.).
We thank E. C. Butcher for the hybridomas for MECA-79, MECA-89
and MECA-367 mAbs, K. Miyake for the hybridomas for M/K-1 and
PS/2 mAbs and Beat A. Imhof and Michel Aurrand-Lions for anti-JAM-A
and anti-JAM-C mAbs. We also thank Ms S. Yamashita and M.
Komine for their secretarial assistance.
Funding to pay the Open Access publication charges for this
article was provided by the research funds to Dr. M. Miyasaka.
MAdCAM-1 mucosal addressin cell adhesion molecule-1
pDC plasmacytoid dendritic cell
PNAd peripheral node addressin
PTX pertussis toxin
VCAM-1 vascular cell adhesion molecule-1
high endothelial venule
intercellular adhesion molecule
junctional adhesion molecule
leukocyte function-associated antigen-1
1 Colonna, M., Trinchieri, G. and Liu, Y. J. 2004. Plasmacytoid
dendritic cells in immunity. Nat. Immunol. 5:1219.
2 Liu, Y. J. 2005. IPC: professional type 1 interferon-producing cells
and plasmacytoid dendritic cell precursors. Annu. Rev. Immunol.
3 Asselin-Paturel, C., Brizard, G., Pin, J. J., Brie `re, F. and Trinchieri,
G. 2003. Mouse strain differences in plasmacytoid dendritic cell
frequency and function revealed by a novel monoclonal antibody.
J. Immunol. 171:6466.
4 Nakano, H., Yanagita, M. and Gunn, M. D. 2001. CD11c+B220+Gr-
1+cells in mouse lymph nodes and spleen display characteristics
of plasmacytoid dendritic cells. J. Exp. Med. 194:1171.
5 Diacovo, T. G., Blasius, A. L., Mak, T. W., Cella, M. and Colonna,
M. 2005. Adhesive mechanisms governing interferon-producing
cell recruitment into lymph nodes. J. Exp. Med. 202:687.
6 Bonasio, R., Scimone, M. L., Schaerli, P., Grabie, N., Lichtman, A. H.
and von Andrian, U. H. 2006. Clonal deletion of thymocytes by
circulating dendritic cells homing to the thymus. Nat. Immunol
7 Streeter, P. R., Rouse, B. T.N. and Butcher, E. C. 1988.
Immunohistologic and functional characterization of a vascular
addressin involved in lymphocyte homing into peripheral lymph
nodes. J. Cell Biol. 107:1853.
8 Streeter, P. R., Berg, E. L., Rouse, B. T.N., Bargatze, R. F. and
Butcher, E. C. 1988. A tissue-specific endothelial cell molecule
involved in lymphocyte homing. Nature 331:41.
9 Miyake, K., Medina, K., Ishihara, K., Kimoto, M., Auerbach, R. and
Kincade, P. W. 1991. A VCAM-like adhesion molecule on murine
bone marrow stromal cells mediates binding of lymphocyte
precursors in culture. J. Cell Biol. 114:557.
10 Miyake, K., Weissman, I. L., Greenberger, J. S. and Kincade, P. W.
1991. Evidence for a role of the integrin VLA-4 in lympho-
hemopoiesis. J. Exp. Med 173:599.
11 Malergue, F., Galland, F., Martin, F., Mansuelle, P., Aurrand-Lions,
M. and Naquet, P. 1998. A novel immunoglobulin superfamily
junctional molecule expressed by antigen presenting cells,
endothelial cells and platelets. Mol. Immunol 35:1111.
12 Johnson-Le `ger, C. A., Aurrand-Lions, M., Beltraminelli, N., Fasel,
N. and Imhof, B. A. 2002. Junctional adhesion molecule-2 (JAM-2)
promotes lymphocyte transendothelial migration. Blood 100:2479.
13 Ding, Z., Kawashima, H. and Miyasaka, M. 2000. Sulfatide binding
and activation of leukocytes through an L-selectin-independent
pathway. J. Leukoc. Biol. 68:65.
14 Izawa, D., Tanaka, T., Saito, K. et al. 1999. Expression profile of
active genes in mouse lymph node high endothelial cells. Int.
15 Gilliet, M., Boonstra, A., Paturel, C. et al. 2002. The development
of murine plasmacytoid dendritic cell precursors is differentially
regulated by FLT3-ligand and granulocyte/macrophage colony-
stimulating factor. J. Exp. Med. 195:953.
16 Cella, M., Jarrossay, D., Facchetti, F. et al. 1999. Plasmacytoid
monocytes migrate to inflamed lymph nodes and produce large
amounts of type I interferon. Nat. Med. 5:919.
17 Vestweber, D. 2002. Regulation of endothelial cell contacts during
leukocyte extravasation. Curr. Opin. Cell Biol 14:587.
18 Hiai, H., Shisa, H., Nishi, Y. et al. 1980. Symbiotic culture of mouse
leukaemias: regulation of cell interaction by an activity of serum.
Virchows Arch. 32:261.
19 Schoefl, G. I. 1972. The migration of lymphocytes across the
vascular endothelium in lymphoid tissue. A reexamination. J. Exp.
20 Yamaguchi, K. and Schoefl, G. I. 1983. Blood vessels of the
Peyer’s patch in the mouse: III. High-endothelium venules. Anat.
21 Muller, W. A. 2003. Leukocyte-endothelial-cell interactions in
leukocyte transmigration and the inflammatory response. Trends
22 Ostermann, G., Weber, K. S.C., Zernecke, A., Schro ¨der, A. and
Weber, C. 2002. JAM-1 is a ligand of the b2 integrin LFA-1
involved in transendothelial migration of leukocytes. Nat. Immunol.
23 Aurrand-Lions, M., Johnson-Leger, C., Wong, C., Du Pasquier, L.
and Imhof, B. A. 2001. Heterogeneity of endothelial junctions is
reflected by differential expression and specific subcellular
localization of the three JAM family members. Blood 98:3699.
24 Muller, W. A., Weigl, S. A., Deng, X. and Phillips, D. M. 1993.
PECAM-1 is required for transendothelial migration of leukocytes.
J. Exp. Med. 178:449.
25 Schenkel, A. R., Mamdouh, Z., Chen, X., Liebman, R. M. and
Muller, W. A. 2002. CD99 plays a major role in the migration of
monocytes through endothelial junctions. Nat. Immunol 3:143.
26 Ebnet, K., Suzuki, A., Ohno, S. and Vestweber, D. 2004. Junctional
adhesion molecules (JAMs): more molecules with dual functions?
J. Cell Sci. 117:19.
27 Yoneyama, H., Matsuno, K., Zhang, Y. et al. 2004. Evidence for
recruitment of plasmacytoid dendritic cell precursors to inflamed
lymph nodes through high endothelial venules. Int. Immunol. 16:915.
28 Miyasaka, M. and Tanaka, T. 2004. Lymphocyte trafficking across
high endothelial venules: dogmas and enigmas. Nat. Rev. Immunol.
Multiple adhesion pathways in pDC-HEV interactions7
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