The Journal of Experimental Medicine
JEM © The Rockefeller University Press $8.00
Vol. 203, No. 12, November 27, 2006 2763–2777 www.jem.org/cgi/doi/10.1084/jem.20051759
The hallmarks of skin infl ammation are an in-
fi ltration of the dermis by monocytes and acti-
vated T cells and an increase in draining lymph
node cellularity primed by the large-scale im-
migration of dermal DCs via aff erent lymphat-
ics. These events are coordinated by cytokines,
including TNF-α and IL-1, that induce matu-
ration and emigration of skin-derived DCs
(1), leading to exacerbation of infl ammation
or eventual fi brosis through pleiotropic eff ects
within aff ected dermis. To date, much research
has focused on the mechanisms by which T
cells are recruited to infl amed tissues by trans-
migration of blood vessels (for review see 2).
However, the equally important question of
how DCs and other leukocytes are recruited
to lymph nodes by transmigration of lym-
phatic vessels has received far less attention (3).
Knowledge about the mechanisms for the en-
try of DCs into lymphatics is mostly restricted
to chemotaxis and the eff ects of chemokines
on the process. For example, CC chemokine
receptor 7 (CCR7), which binds the lymph
node/lymphatic endothelial–derived CC che-
mokine ligand 21 (CCL21) and CCL19, is
expressed in mature DCs and mediates their
traffi cking from tissue to lymph nodes (4–6).
Recent evidence indicates the same receptor
also mediates exit of CD4+ eff ector memory
T cells from tissue to lymph (7, 8). Moreover,
deletion of the gene for CCR7 in knockout
mice abolishes migration of Langerhans cells
into dermal lymphatics, whereas the absence
of lymph node CCL21 (as in the plt mouse,
paucity of lymph node T cells) suppresses re-
cruitment of DCs to draining lymph nodes and
subsequent T cell–mediated immunity (9, 10).
Nonetheless, it remains unclear whether these
An infl ammation-induced mechanism
for leukocyte transmigration across
lymphatic vessel endothelium
Louise A. Johnson,1 Steven Clasper,1 Andrew P. Holt,2 Patricia F. Lalor,2
Dilair Baban,3 and David G. Jackson1
1Medical Research Council (MRC) Human Immunology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe
Hospital, Headington, Oxford OX3 9DS, England, UK
2Liver Research Group, Institute of Biomedical Research, MRC Centre for Immune Regulation, University of Birmingham
Medical School, Edgbaston, Birmingham B15 2TT, England, UK
3MRC Functional Genetics Unit, Department of Human Anatomy and Genetics, University of Oxford, Oxford OX1 3QX,
The exit of antigen-presenting cells and lymphocytes from infl amed skin to afferent lymph
is vital for the initiation and maintenance of dermal immune responses. How such an exit
is achieved and how cells transmigrate the distinct endothelium of lymphatic vessels are
unknown. We show that infl ammatory cytokines trigger activation of dermal lymphatic
endothelial cells (LECs), leading to expression of the key leukocyte adhesion receptors
intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1),
and E-selectin, as well as a discrete panel of chemokines and other potential regulators
of leukocyte transmigration. Furthermore, we show that both ICAM-1 and VCAM-1 are
induced in the dermal lymphatic vessels of mice exposed to skin contact hypersensitivity
where they mediate lymph node traffi cking of dendritic cells (DCs) via afferent lymphatics.
Lastly, we show that tumor necrosis factor α stimulates both DC adhesion and transmigra-
tion of dermal LEC monolayers in vitro and that the process is effi ciently inhibited by
ICAM-1 and VCAM-1 adhesion-blocking monoclonal antibodies. These results reveal a
CAM-mediated mechanism for recruiting leukocytes to the lymph nodes in infl ammation
and highlight the process of lymphatic transmigration as a potential new target for
antiinfl ammatory therapy.
David G. Jackson:
Abbreviations used: CAM, cell
adhesion molecule; CCL, CC
chemokine ligand; CCR, CC
chemokine receptor; CMFDA,
5-chloromethylfl uorescein diac-
etate; CXCL, CXC chemokine
ligand; ENA-78, epithelial neu-
trophil activator 78; GRO,
growth-regulated oncogene β;
HDLEC, human dermal LEC;
ICAM, intercellular adhesion
molecule; JAM, junctional
adhesion molecule; LEC, lym-
phatic endothelial cell; LYVE-1,
lymph vessel endothelial
hyaluronan receptor 1; MCP-1,
protein 1; MDDC, monocyte-
derived DC; MDLEC, mouse
dermal LEC; MIP-3α, macro-
phage infl ammatory protein 3α;
RANTES, regulated on activa-
tion, normal T cell expressed
The online version of this article contains supplemental material.
2764 TRANSMIGRATION MECHANISMS IN INFLAMED LYMPHATIC ENDOTHELIUM | Johnson et al.
important lymphatic traffi cking events are regulated solely by
chemokines or whether there is an additional requirement
for adhesion between leukocytes and lymphatic endothelium
in the process.
In the case of the blood vasculature, it is well documented
that adhesion and migration across infl amed vessel endothe-
lium typically involves cytokine-induced expression of key
leukocyte–endothelial cell adhesion molecules (CAMs)—
intercellular adhesion molecule 1 (ICAM-1), vascular CAM
(VCAM-1), and E-selectin (in endothelium)—that promote
adherence and diapedesis of lymphocytes and monocytes
through ligation of the counterreceptors αLβ2/αΜβ2 integ-
rin (CD11a/CD18, CD11b/CD18, and LFA-1/Mac I) and
α4β1 integrin (very late antigen 4) Sialyl LewisX-modifi ed
mucoproteins, respectively (for review see 11; 12–14). In
contrast, cytokine-induced expression of these and other leu-
kocyte–endothelial CAMs on lymphatic vessels has not been
adequately explored, despite indications that ICAM-1 knock-
out mice have defects in lymph node recruitment of DCs
(12, 13) and that expression of VCAM-1 in medullary sinus
coincides with lymphocyte retention in infl amed lymph
The cellular route by which leukocytes transmigrate lym-
phatic endothelium is equally uncertain. The ultrastructure
of lymphatic capillaries is distinct from that of small blood
vessels; most notably, they are not invested with mural cells
and do not have a conventional basement membrane (3, 15,
16). Moreover, the initial lymphatics culminate in blind-
ended termini that display occasional interendothelial gaps
(17, 18) through which plasma and dissolved macromolecules
are drawn in response to the negative pressure evoked by the
innate pumping activity of larger lymphatic collectors (19).
Whether these gaps can also serve as entry points for leuko-
cytes or whether entry involves transmigration at interendo-
thelial tight junctions (paracellular route) or across the
endothelial cell body (transcellular route) as in the blood vas-
culature (20, 21) is open to conjecture. Likewise, which, if
any, of these routes contributes to the acceleration in leuko-
cyte transmigration that is triggered by infl ammation or how
accelerated entry might be regulated is unclear. In the few
cases where transmigration has been observed in detail (e.g.,
the entry of DCs to lymph vessels in infl amed mouse skin
explants), leukocytes were seen to undergo massive shape
changes during passage into the vessel lumen (22, 23). Thus,
whatever the route of entry, it is likely that intimate contacts
form between leukocytes and infl amed lymphatic endothe-
lium that could well regulate cell transit.
We have focused on the molecular mechanisms of lym-
phatic transmigration in the belief that an understanding of
the process is important not only from the standpoint of
fundamental biology but also for the development of more
eff ective therapies to block infl ammation and enhance vac-
cine delivery. We have found that lymphatic endothelium
undergoes a program of activation during infl ammation to
support increased leukocyte transmigration in which the
adhesion molecules ICAM-1 and VCAM-1 play key roles.
Figure 1. Characteristics of primary HDLECs isolated by LYVE-1
immunoselection. HDLECs isolated from human dermis by LYVE-1
immunomagnetic bead selection are shown after dual immunofl uore-
scence staining for lymphatic and blood vascular-specific markers.
(A and B) Confl uent monolayers stained for the panendothelial marker
CD31 and the lymphatic endothelial markers podoplanin or LYVE-1,
respectively. In contrast to podoplanin, which is expressed by all HDLECs,
LYVE-1 shows considerable variation, refl ecting the heterogeneity seen in
normal tissue lymphatics (see Results). (C) Triple staining for CD31, the
lymphatic endothelial transcription factor PROX-1, and the nuclear stain
DAPI. Note that all cells contain PROX-1–positive nuclei. (D) HDLEC mono-
layers stained for podoplanin and the blood vascular marker pal-E. Note
the complete absence of pal-E–positive cells. Blood vascular endothelial
cells positive for pal-E in the mixed endothelial culture preceding LYVE-1
immunoselection are shown for comparison (inset). (E) FACS histograms
of HDLECs stained for CD31, podoplanin, or LYVE-1 (red). Isotype-matched
controls are shown (black).
JEM VOL. 203, November 27, 2006
These important fi ndings reveal a functional similarity be-
tween two otherwise distinct vasculatures and point to a
much more active role for the lymphatics in infl ammation
than previously anticipated.
Cytokine-induced expression of key leukocyte adhesion
molecules and chemokines in primary human dermal
lymphatic endothelial cells (HDLECs)
To generate cells for analysis of the infl ammatory response
and its eff ects on DC transmigration, we isolated primary
HDLECs from the dermis of freshly resected breast tissue or
abdominoplasty by enzymatic digestion and magnetic bead
immunoselection using antibody to lymph vessel endothelial
hyaluronan receptor 1 (LYVE-1), a molecule whose pref-
erential expression in lymphatic endothelium has been well
documented (see Materials and methods) (24–26). The re-
sulting cells comprised at least 99% LECs, as assessed by im-
munostaining for the sialomucin marker podoplanin (27) and
the lymphatic lineage-associated transcription factor Prox-1
(28, 29), either alone or in combination with the panen-
dothelial marker CD31 (Fig. 1, A, C, and E) (30). Parallel
immunostaining for LYVE-1 (both alone and in combina-
tion with CD31) showed that the majority of the population
was positive for the receptor even though there was clear
heterogeneity in the level of expression among individual
LECs (Fig. 1, B and E). This is in line with the observations
of other researchers that indicate the receptor is expressed
to variable extents in individual lymphatic vessels (19, 31)
and primary LEC lines (32, 33, 34, 35), as well as with our
own fi ndings that LYVE-1 can be specifi cally downmod-
ulated in cultured LECs, e.g., after exposure to cytokines
Figure 2. Infl ammatory cytokines up-regulate surface expression
of ICAM-1, VCAM-1, and E-selectin in cultured HDLECs. Cells were
cultured for 24 h in the presence of individual proinfl ammatory cytokines
or chemokines before immunostaining for either (A) VCAM-1 or (B) ICAM-1
and quantitation by FACS analysis. Data represent the mean ± SEM
(n = 3). (C and D) Dot plots show VCAM-1 or ICAM-1 expression in HDLECs
cultured in the presence or absence (control) of TNF-α and assessed by
dual staining for podoplanin and CAMs. Note that all cells expressing
CAMs are positive for podoplanin (top right quadrants). (E) Representative
double immunofl uorescence micrographs showing induction of CAMs and
E-selectin (green) in podoplanin-positive (red) HDLECs, as indicated with
nuclei counterstained for DAPI. Bar, 50 μm.
2766 TRANSMIGRATION MECHANISMS IN INFLAMED LYMPHATIC ENDOTHELIUM | Johnson et al.
(Fig. S1 E, available at http://www.jem.org/cgi/content/
full/jem.20051759/DC1; see Fig. S3 B; and not depicted).
Importantly, no cells expressing the blood vascular endo-
thelial-specifi c, vesicle-associated pal-E antigen (36, 37)
were detected in our LEC cultures, these having been effi -
ciently depleted in two successive rounds of LYVE-1 immuno-
selection (Fig. 1 D).
We next investigated the eff ects of infl ammatory cyto-
kines, focusing in particular on the leukocyte adhesion mole-
cules ICAM-1, VCAM-1, and E-selectin, whose surface
expression is known to be induced on activated blood vascu-
lar endothelium (38–40). As shown by FACS analysis and
immunofl uorescence microscopy (Fig. 2, A–E), a minor pro-
portion of resting HDLECs expressed low levels of ICAM-1,
whereas VCAM-1 and E-selectin were virtually absent.
However, treatment with the cytokine TNF-α led to a dra-
matic up-regulation in surface expression (40–80-fold) of all
three CAMs. These eff ects of TNF-α were both potent
(requiring levels below 1 ng/ml for half-maximal induction;
Fig. S1) and rapid (T1/2 of 6–12 h for ICAM-1 and VCAM-1;
T1/2 of ?4 h for E-selectin; Fig. 3, A–C). The characteristics
of the response, its magnitude, and the transient nature of
E-selectin expression (Fig. 3 C) were rather similar to those
previously described for blood vascular endothelium (i.e., the
human umbilical vein epithelial cell line; references 41, 42).
Moreover, the induction of CAM expression by TNF-α ap-
peared to be independent of mitogenesis or apoptosis within
the range used in our experiments, involved no loss of
cell integrity, and was completely reversible within 24 h of
cytokine withdrawal (Fig. S2, A–C, available at http://www.
jem.org/cgi/content/full/jem.20051759/DC1). Among other
cytokines, TNF-β (lymphotoxin-α), IL-1α, and to a marginal
extent IFN-γ also induced expression of ICAM-1, whereas
IL-2, IL-6, IL-8, and the infl ammatory chemokine macrophage
infl ammatory protein 3α (MIP-3α; CCL20) had no such
eff ect (Fig. 2, A and B).
As induction of CAMs had not been reported previously
in LECs, we considered the possibility that the CAM-positive
cells in our experiments represented a subpopulation of con-
taminating blood vascular endothelial cells. However, two-
color fl ow cytometry and immunofl uorescence microscopy
(Fig. 2, C–E) clearly demonstrated that all VCAM-1– and
Figure 3. Kinetics of TNF-induced CAM and E-selectin expression
in cultured primary HDLECs. (A–C) Respective time courses for induc-
tion of VCAM-1, ICAM-1, and E-selectin in HDLECs cultured for 0–48 h in
the presence or absence of 1 ng/ml TNF-α, as assessed by FACS analysis.
Representative histograms are shown for cells stained with isotype-
matched control Ig (light gray) or mAbs to the appropriate adhesion mol-
ecules in unstimulated cells (black) or cells treated with TNF-α for 3 h
(dark gray), 6 h (blue), 12 h (green), and 24 h (red).
JEM VOL. 203, November 27, 2006
ICAM-1–expressing cells were positive for podoplanin, thus
confi rming their identity as LECs. Moreover, we consistently
observed induction of CAMs in a total of fi ve independent cell
preparations isolated by the LYVE-1 immunoselection proce-
dure, in addition to preparations of HDLECs immunoisolated
by either CD34/podoplanin selection (43) or outgrowth from
commercial human dermal microvascular cell cultures (35)
and in mouse dermal LECs (MDLECs) immunoisolated by
LYVE-1 selection (Fig. S3, available at http://www.jem.
org/cgi/content/full/jem.20051759/DC1; and not depicted).
Thus, we are confi dent that the capacity to express leukocyte
CAMs is a genuine property of the bulk HDLEC population
rather than the result of contamination with biliary epithelial
cells or a subpopulation undergoing dediff erentiation.
Finally, we investigated the eff ects of TNF-α on the pro-
duction of leukocyte chemokines using appropriate ELISAs.
The results (Fig. 4) show that resting, unstimulated HDLEC
cultures secrete only trace levels of the major T cell and
monocyte chemokines regulated on activation, normal T cell
expressed and secreted (RANTES; CCL5), monocyte che-
moattractant protein 1 (MCP-1; CCL2), and (MIP-3α;
CCL20), confi rming and extending upon previous fi ndings
(43). Importantly, however, stimulation with TNF-α trig-
gered a considerable (>100-fold) rise in secretion of all three
chemokines over a period of 24–48 h, reaching fi nal levels of
7–55 ng/ml (Fig. 4). In contrast, secretion of both Epstein-
Barr virus–induced molecule 1 ligand chemokine (CCL19)
and secondary lymphoid tissue (CCL21)—chemokines asso-
ciated with lymph node traffi cking (4)—was below detect-
able levels (80 and 15 pg/ml, respectively; unpublished data)
and was evidently not induced by treatment of LECs with
infl ammatory cytokines.
Extent of the infl ammatory cytokine–induced expression
program in LECs revealed by microarray analysis
To defi ne infl ammation-induced changes in HDLEC gene
expression more comprehensively, we examined the RNA
profi les of control and TNF-α–treated cells by gene chip ar-
ray analysis using Aff ymetrix human U-133 arrays. The re-
sults (summarized in Table I and completely presented in
Table S1, available at http://www.jem.org/cgi/content/full/
jem.20051759/DC1) revealed a total of 424 genes that were
up-regulated and 171 that were down-regulated by a factor
of twofold or greater (P ≤ 0.1). Notably, VCAM-1 (214-
fold), ICAM-1 (7.7-fold), and E-selectin (160-fold) were all
highly up-regulated, as were the levels of the leukocyte che-
mokines MCP-1, RANTES, and MIP-3α, confi rming our
fi ndings at the protein level using FACS analysis and ELISA
(see previous section). Moreover, RNA levels were also
up-regulated for the neutrophil chemoattractants epithelial
neutrophil activator 78 (ENA-78; CXC chemokine ligand 5
[CXCL5]) and growth-regulated oncogene β (GROβ;
CXCL2), the monocyte/macrophage chemokines fractalkine
(CX3CL1) and CSF-1, and CCR10, a receptor for the
chemokine CCL27 (cutaneous T cell–attracting chemokine)
Figure 4. TNF-훂 induces proinfl ammatory chemokine production
in cultured primary HDLECs. Cells were stimulated with 1 ng/ml TNF-α
over a time course of 48 h, and concentrations of MCP-1, RANTES, and
MIP-3α secreted into the culture supernatant were measured by ELISA.
Data represent the mean ± SEM (n = 3).
Table I. Up-regulation of representative genes
Transcript Fold change
(P < 0.05)
Toll-like receptor 2
Toll-like receptor 1
Representative genes were up-regulated more than twofold by TNF-α stimulation
of primary HDLECs at P < 0.05 (or P < 0.1, as indicated by *).
2768 TRANSMIGRATION MECHANISMS IN INFLAMED LYMPHATIC ENDOTHELIUM | Johnson et al.
produced by infl amed epidermal keratinocytes (44), indicat-
ing a broad chemoattractant response within infl amed lym-
phatic endothelium. Interestingly, the array data also showed
signifi cant up-regulation of Toll-like receptor 2, the tight
junctional component claudin-1, and the metalloproteinases
MMP19 and ADAMTS3 (Table I). However, there was
no change in abundance of transcripts for either CCL21 or
CCL19, both of which are implicated in the exit of DCs
from tissues to aff erent lymphatics and traffi cking to lymph
nodes (9, 10).
Comparable changes in the transcriptional profi le of
MDLECs were induced by treatment with TNF-α, as revealed
in microarray analyses using the Aff ymetrix mouse 430 array
(Table S2, available at http://www.jem.org/cgi/content/
full/jem.20051759/DC1), indicating the broad similarity in
LEC response to infl ammation between species.
In vivo evidence of a functional role for VCAM-1 and
ICAM-1 in infl ammation-induced lymphatic transmigration
Having established infl ammation-induced expression of leu-
kocyte CAMs in primary LECs in vitro, we next asked
whether a similar response occurs during infl ammation in
vivo, using the well-characterized oxazolone-induced skin
contact hypersensitivity model in BALB/c mice (45, 46). To
achieve this, we prepared sections of dermis from infl amed
and contralateral uninfl amed ears (see Materials and methods)
at various time points after oxazolone challenge and immuno-
stained for CAMs in both whole-mount and thin sections.
The results (Fig. 5, A and B) show that VCAM-1 and ICAM-1
are indeed expressed on infl amed lymphatic vessels—distin-
guished by their expression of podoplanin and comparatively
large vessel diameters—in addition to small blood capillaries,
whereas both CAMs were absent from the lymphatics of un-
challenged tissue. Interestingly, expression in infl amed lym-
phatics was more focal than in infl amed blood vessels (Fig. 5,
A and B; and not depicted), possibly indicative of diff erent
kinetics of induction within the two vasculatures. Neverthe-
less, quantitative estimates revealed that ?50% of podo-
planin-positive lymphatics stained for ICAM-1 and 60% for
VCAM-1 within 18–24 h of allergen administration (Fig. 5,
C and D). Similar levels of CAM expression were also seen
among podoplanin-positive lymphatics (60% ICAM-1+ ves-
sels vs. 70% VCAM-1+ vessels) 24 h after direct stimulation
with TNF-α when ex vivo mouse skin explants were incu-
bated with the cytokine (unpublished data). Moreover, visu-
alization of APCs in the dermis of oxazolone-treated mice by
immunostaining for MHC class II, ICAM-1, and podoplanin
revealed many such cells in close association with CAM-pos-
itive lymphatic vessels (Fig. S4, available at http://www.jem.
org/cgi/content/full/jem.20051759/DC1), suggesting but
not proving that ICAM-1 might indeed play a role in lym-
phatic transmigration in an infl ammatory context. The diam-
eter of these superfi cial vessels (average ?50 μm) is also
consistent with their identity as initial lymphatics rather than
the larger smooth muscle–invested lymphatic collectors
(100–200 μm) that are found in the deeper dermis.
Figure 5. In vivo expression of ICAM-1 and VCAM-1 in mouse
dermal lymphatics induced by skin contact hypersensitivity. Skin
infl ammation was induced in mouse ear by sensitization and subsequent
challenge with oxazolone before analysis of lymphatic vessel CAM ex-
pression by immunofl uorescence microscopy. (A and B) Whole-mount
sections of oxazolone-challenged and contralateral-unchallenged
(control) ears dual-stained for podoplanin (green) and ICAM-1 or VCAM-1,
respectively (red). Note the weak expression of ICAM-1 confined to
podoplanin-negative (blood) vessels in uninfl amed skin (A) and the focal
up-regulation of both ICAM-1 and VCAM-1 on podoplanin-positive
(lymphatic) vessels in infl amed skin (A and B). Images were captured by
confocal microscopy. Bars, 100 μm. (C and D) Quantitative estimates for
the numbers of ICAM-1+/podoplanin+ and VCAM-1+/podoplanin+ ves-
sels determined by counting 21 separate fi elds of view (7 fi elds/mouse)
in control and oxazolone-treated ear sections. Data represent the mean
JEM VOL. 203, November 27, 2006
To assess the functional role of CAMs in lymphatic trans-
migration in vivo, we imposed blockade by the injection
of oxazolone-treated mice with neutralizing antibodies to
ICAM-1 (mAb YN1/1.7.4 ) or VCAM-1 (mAb 6C7.1
) and tracked migration of cutaneous DCs to lymph
nodes detected by FITC skin painting. The results of these
experiments (Fig. 6 A) show that blocking antibodies to ei-
ther VCAM-1 or ICAM-1 consistently suppressed lymph
node traffi cking of FITC+/CD11c+ by >60% compared
with isotype-matched Ig controls. Given that cutaneous DCs
migrate to lymph nodes almost exclusively via aff erent lym-
phatics rather than entering the blood circulation (49), these
fi ndings argue for a role of ICAM-1 and VCAM-1 in either
proximal vessel entry, migration, or egress from the drain-
ing lymph node. To further distinguish between these pos-
sibilities, we traced the fate of emigrating DCs during CAM
adhesion blockade in a separate set of experiments in which
we adoptively transferred mature 5-chloromethylfl uorescein
diacetate (CMFDA)–labeled bone marrow–derived DCs into
the dermis of oxazolone-treated mice. As shown in Fig. 6 B,
virtually all CMFDA-labeled DCs had already exited the
dermis by 24 h after adoptive transfer in mice treated with
isotype-matched control Ig and few, if any, were detected
in the vicinity of lymphatics. In contrast, large numbers
of CMFDA-labeled DCs could be seen in the dermis of
ICAM-1 mAb YN1-1–treated mice, and these were associated
Figure 6. In vivo traffi cking of skin DCs via afferent lymphatics is
dependent on ICAM-1 and VCAM-1 adhesion. The involvement of
ICAM-1 and VCAM-1 in the traffi cking of DCs via afferent lymphatics
was investigated in mice with oxazolone-induced skin hypersensitivity.
(A) Recoveries of FITC+/CD11c+ skin DCs in the draining lymph nodes 24 h
after FITC skin painting of oxazolone-sensitized mice that received prior
injection of neutralizing mAbs to VCAM-1, ICAM-1, or control rat Ig. Data
represent the mean recoveries ± SEM (obtained from three separate
experiments). (B) To show retention of DCs within the skin, CMFDA-labeled
bone marrow–derived DCs from a littermate were intradermally injected
into the ear tissue of sensitized mice that received prior injection of a
neutralizing mAb to ICAM-1 (YN1-1) or control rat Ig. After 24 h, ears
were removed, and whole-mount staining was performed using anti-
podoplanin with Alexa Fluor 568 (red) and Cy5-conjugated goat anti–rat
Cy5 (blue) to detect binding of neutralizing antibody within the tissue.
Bars, 100 μm.
2770 TRANSMIGRATION MECHANISMS IN INFLAMED LYMPHATIC ENDOTHELIUM | Johnson et al.
with podoplanin-positive initial lymphatics that could be
seen to express high levels of ICAM-1 through YN1-1 stain-
ing (Fig. 6 B, bottom). There was no obvious association
of DCs with ICAM-1-positive blood vessels. Although the
ICAM-1 molecule is expressed at low levels by mature DCs
themselves, this was previously shown not to be required
for traffi cking to lymph nodes (13). Hence, our results
provide new evidence that CAMs expressed on lymphatic
endothelium are required for effi cient DC transmigration of
infl amed lymphatics.
Analysis of the molecular mechanisms for ICAM-1–
and VCAM-1–mediated translymphatic migration
Finally, we investigated the mechanistic basis for CAM-me-
diated lymphatic transmigration using a quantitative in vitro
assay that measured the passage of in vitro LPS- stimulated
CD14+ human blood monocyte-derived DCs (MDDCs) across
monolayers of primary HDLECs in Boyden chambers. The
mature promigratory phenotype of these DCs (CD80+, CD83+,
CD86+, and MHC class IIhigh) is shown in Fig. S5 (available
Figure 7. MDDC transmigration of TNF-α–stimulated HDLEC
monolayers is dependent on ICAM-1 and VCAM-1. Transmigration
of Cell Tracker Green fl uorescently labeled MDDCs across either unstimu-
lated or TNF-α–stimulated HDLEC monolayers plated on the undersurface
of Fluoroblok fi lters was monitored in the presence or absence of selected
adhesion blocking antibodies over a 12-h period. Progress curves are
shown for MDDC transmigration across (A) control unstimulated versus
TNF-α–stimulated HDLECs, (B) TNF-α–stimulated HDLECs treated with
control rat IgG versus VCAM-1–neutralizing mAb P8B1, and (C) TNF-α–
stimulated HDLECs treated with control rat IgG versus ICAM-1–
neutralizing mAb P2A4. Data represent the mean ± SEM (n = 4).
(D) Comparative effects of individual ICAM-1 mAbs 15.2 and P2A4,
VCAM-1 mAbs 51-10C9 and P8B1, ICAM-1 mAb 15.2 and VCAM-1 mAb
P8B1 together, the LFA-1 mAb 24, and control mouse IgG on MDDC
transmigration of TNF-α–stimulated HDLECs. The level of transmigration
across unstimulated HDLECs is shown for comparison. Data from three
independent experiments are normalized to the measured levels of trans-
migration in the presence of control IgG (100% maximal transmigration)
in each case and represent the mean ± SEM (n = 4). (E) Permeability of
confl uent HDLEC monolayers to unconjugated Alexa Fluor 488 measured
as dye recovered in the lower chamber of Fluoroblok fi lter wells after a 6-h
incubation at 37°C. Data represent the mean ± SEM.
JEM VOL. 203, November 27, 2006
To mimic the tissue exit of DCs via initial lymphatics in vivo
(i.e., basolateral to luminal migration), we plated HDLECs
on the lower side of UV-opaque Fluoroblok fi lters and
monitored transit of CMFDA (Cell Tracker Green)-labeled
MDDCs from the upper to the lower compartment. The result-
ing monolayers showed some permeability to a small-molecule
probe (fl uorescent-labeled goat IgG; see Materials and methods)
even when fully confl uent (Fig. 7, E and F; and Fig. S6),
perhaps refl ecting some retention of the native overlapping
junction architecture. As shown by the time course in Fig.
7 A, LPS-matured DCs displayed substantial migration across
resting unstimulated HDLECs (?10% of input cells trans-
migrated after 8 h). However, earlier stimulation of HDLEC
monolayers with 1 ng/ml TNF-α for 24 h led to a substan-
tial increase in the rate and extent of MDDC transmigration
after a lag period of ?3 h (range = 1–3 h) after MDDC
Figure 8. VCAM-1 and ICAM-1 are expressed on both luminal
and basolateral surfaces of HDLECs. Cells were cultured on clear-
membrane inserts and stimulated with TNF-α for 24 h before staining
with antipodoplanin (red) and either anti–ICAM-1 or anti–VCAM-1 (blue)
and analysis by confocal microscopy. Staining was performed either on
HDLECs cultured (A) in the absence of MDDCs or (B) at 10 h after addition
of Cell Tracker Green fl uorescently labeled MDDCs. (A, top) Asterisks de-
pict the axis through which individual LECs were imaged in z-section
(bottom). Bars, 10 μm.
2772 TRANSMIGRATION MECHANISMS IN INFLAMED LYMPHATIC ENDOTHELIUM | Johnson et al.
addition (Fig. 7 A). This TNF-α–induced component of
transmigration was not caused by a simple increase in mono-
layer permeability because it was blocked in dose-dependent
fashion by the addition of neutralizing antibodies specifi c
to either ICAM-1 (mAbs P2A4 or 15.2) or VCAM-1 (mAb
51-10C9), added either singly or in combination (Fig. 7, B–D;
and Fig. S7). No such inhibition was seen with unstimulated
LECs (unpublished data). In addition to these CAM-blocking
mAbs, considerable inhibition of transmigration was also
observed after the addition of the β2 integrin–neutralizing
antibody mAb 24, consistent with a role for this integrin as
an LEC CAM counterreceptor on DCs (Fig. 7 D). However,
in the case of the ICAM-1 mAbs, inhibition of transmigra-
tion could be assigned to the blockade of antigen on LECs
rather than DCs (which express lower but substantial levels of
ICAM-1; Fig. S5) because preincubation with the latter had
no eff ect (not depicted).
Interestingly, MDDCs were also found to transmigrate
TNF-α–activated LECs in the luminal to basolateral direc-
tion (assessed by plating LECs on the upper surface of fi lters)
with a similar time course and CAM dependence to basolateral-
luminal transmigration (Fig. S8, available at http://www.jem.
org/cgi/content/full/jem.20051759/DC1; and not depicted).
To determine whether these properties are refl ected in the
polarity of CAM distribution in TNF-activated LECs, we
performed dual fl uorescence staining of monolayers for
ICAM/VCAM and podoplanin and analyzed the resulting
sections by confocal microscopy. As shown in Fig. 8, both
ICAM-1 and VCAM-1 could be seen on the apical as well
as the basolateral face of the endothelial plasma membrane,
indicating no obvious polarity in distribution. Moreover,
ICAM-1 appeared to be distributed in a ring beneath indi-
vidual MDDCs contacting the LEC monolayer as visualized
by CMFDA labeling (Fig. 8 A), whereas VCAM-1 showed a
more patchy distribution (Fig. 8 B).
The clearest interpretation of our fi ndings is that ICAM-1
and VCAM-1 in LECs mediate a leukocyte adhesion step
that is prerequisite for activation-induced transmigration,
analogous to the CAM-mediated fi rm adhesion that precedes
leukocyte diapedesis in blood vascular endothelium. To ex-
plore this issue further, we measured adhesion of CMFDA-
labeled MDDCs to TNF-activated HDLECs cultured in
24-well plates. As shown in Fig. 9, MDDCs displayed con-
siderable binding to activated LECs within 3 h of incubation,
increasing twofold after 10 h in a time course that resembled
MDDC transmigration (Fig. 7). Moreover, when the eff ects
of antibody blockade were examined (ICAM-1 mAbs P2A4
and 15.2 and VCAM-1 mAbs Ig11 and P8B1), it was clear
that only binding at the later (10 h) and not the earlier (3 h)
time point was reduced (Fig. 9). Binding during this early lag
period is therefore CAM-independent and may represent a
period during which chemokine-induced activation of DC
In summary, we conclude that MDDC transmigration of
activated LECs in vitro involves an initial CAM-independent
interaction between the two cell types that is followed by sec-
ondary ICAM/VCAM-mediated adhesion and transmigration.
D I S C U S S I O N
The mechanisms by which leukocytes exit the tissues via
lymph and migrate to draining lymph nodes have remained
unclear despite their central importance for effi cient genera-
tion of the immune response. The aff erent lymphatics must
cater for both low-level traffi cking of APCs during nor-
mal immune surveillance and the increase in DC, eff ector
memory T cell, and neutrophil traffi cking that is triggered
by tissue infl ammation (for review see reference 3). In this
manuscript, we set out to defi ne the mechanisms underly-
ing these processes using a combination of in vitro studies
with primary dermal LECs and in vivo studies with a mouse
model of oxazolone-induced skin infl ammation. Specifi cally,
we showed that cultured primary HDLECs and MDLECs
respond to the cytokines TNF-α and TNF-β (lymphotoxin-α),
and to a lesser extent IL-1, by rapidly and reversibly up-
regulating expression of the leukocyte adhesion molecules
ICAM-1, VCAM-1, and E-selectin, together with synthesis
and release of chemotactic agents, including the key infl am-
matory CC chemokines CCL5 (RANTES), CCL2 (MCP-
1/JE), and CCL20 (MIP-3α). In addition, we demonstrated
the induction of ICAM-1 and VCAM-1 expression in aff er-
ent lymphatic vessels draining the skin of oxazolone-treated
mice in vivo and presented evidence that administration of
Figure 9. MDDC adhesion to TNF-α–stimulated HDLECs is de-
pendent on ICAM-1 and VCAM-1. Cell Tracker Green fl uorescently
labeled MDDCs were applied to TNF-α–stimulated HDLEC monolayers
plated in 24-well plates, preincubated with either IgG control or ICAM-1
mAbs (15.2 or P2A4) or VCAM-1 mAbs (P8B1 or IGII), in triplicate. At 3 and
10 h, the numbers of adherent MDDCs were measured. Representative
data from three independent experiments are shown and represent
the mean ± SEM (n = 3).
JEM VOL. 203, November 27, 2006
ICAM-1– and VCAM-1–neutralizing antibodies blocked
exit of CD11c-positive skin DCs via aff erent lymphatics.
Finally, we presented evidence from in vitro lymphatic trans-
migration assays with MDDCs that ICAM-1 and VCAM-1
mediate infl ammation-induced transmigration by promoting
The induction of CAM expression in activated lymphatic
vessel endothelium reveals an unexpected similarity with the
blood vasculature, where up-regulation of these same molecules
in infl amed postcapillary venules promotes leukocyte transmi-
gration by mediating fi rm adhesion (for review see reference 2).
In hemovascular transmigration, ICAM-1 and VCAM-1 pro-
mote stable adhesion of leukocytes to the apical endothelial
membrane surface after their initial capture from blood fl ow and
tethering on E- and P-selectin (50). Binding via ICAM-1 and
VCAM-1 then allows the adherent leukocytes to crawl toward
intercellular junctions (51), where additional interactions with
the homophilic adhesion molecules CD31 (platelet/endothelial
cell adhesion molecule 1) and CD99, together with junctional
molecules such as junctional adhesion molecule 1 (JAM-1)
and VE-cadherin, promote diapedesis (52–54). The predic-
tion from our own experiments that ICAM-1 and VCAM-1
would mediate lymphatic vascular transmigration through a
similar mechanism of leukocyte adhesion was confi rmed by
our fi nding that LPS-treated MDDCs bind to TNF-activated
LECs in static assays and that the interaction was blocked by
CAM-neutralizing mAbs using similar concentrations to those
that blocked transmigration. There is also the likelihood that
events downstream of this CAM-mediated adhesion might be
similar to those in the hemovasculature, given that primary
HDLECs express similar junctional molecules, including VE-
cadherin and JAMs, and that LEC monolayers can form both
tight and adherens junctions in vitro (43). Furthermore, mice
with targeted deletion of the gene for JAM-1 (JAM-A) dis-
play abnormalities in DC traffi cking to lymph nodes, consistent
with a role for the molecule in lymphatic vessel diapedesis (55).
Detailed functional studies of these molecules in lymphatics are
therefore clearly warranted.
A key question regarding entry of leukocytes to the aff er-
ent lymphatics is whether transmigration occurs at the dis-
tinctive overlapping junctions found within some initial
lymphatics, at conventional interendothelial junctions, or
through the endothelial cell body. It is interesting to note
that docking structures containing ICAM-1 and VCAM-1
that were shown to mediate leukocyte transcellular/paracel-
lular migration in hemovascular endothelial cells have also
been observed in cells resembling a lymphatic phenotype
(56). Hence, it may be that leukocytes can transmigrate lym-
phatic endothelium using more than one mechanism, and it
will be of major interest to determine the preferred route for
individual leukocyte populations and the factors infl uencing
such choice in future experiments.
It is generally assumed that most leukocyte traffi c through
aff erent lymphatics involves transmigration in the basolateral
to luminal direction. Nevertheless, it is also possible that re-
verse migration in a luminal to basolateral direction might
occur. Such a notion is supported by our fi nding that ICAM-1
and VCAM-1 are expressed on both the upper and lower
faces of the endothelial membrane in primary HDLECs in
vitro and that activated HDLECs promote CAM-dependent
migration of MDDCs equally in both directions. If this phe-
nomenon occurs in vivo, it raises the intriguing possibility
that some leukocytes might exit the aff erent lymphatics and
reenter at diff erent points within infl amed tissue, a process
that might increase the effi ciency of immune surveillance.
Bidirectional migration across activated lymphatic sinus en-
dothelium could also be envisaged to play roles in traffi cking
within infl amed lymph nodes.
Finally, besides identifying mechanisms by which adhe-
sion molecules facilitate transmigration of activated lym-
phatics, we also identifi ed several chemokines that could
potentially direct the process. Although existing evidence
indicates that the major chemokine for directing lymphatic
entry of mature DC and memory T cell entry is CCL21
(also known as secondary lymphoid chemokine, or slc),
which binds the G protein–coupled receptor CCR7 (5, 9,
10), it seemed likely to us that other chemoattractants might
also be involved. As indicated by the results of gene chip
microarray analyses and chemokine ELISAs presented in this
manuscript, it is now clear that activated HDLECs synthe-
size a large number of diff erent chemoattractants, including
the T cell/monocyte chemokines CCL20 (MIP-3α), CCL5
(RANTES), CCL2 (MCP-1), and CX3CL1 (fractalkine).
Release of these chemokines in vivo might be envisaged to
promote lymph node traffi cking of those newly extravasated
monocytes and immature DCs bearing cognate CCR2,
CCR5, CCR6, and CX3CR that enter skin and other
tissues in response to infl ammation and that subsequently
mature into professional APCs (57, 58). Moreover, secretion
into lymph could have long-range eff ects on cell traffi cking
in the blood vasculature (59). Thus, “activated” LECs may
well be the source of CCL2 (MCP-1) in infl amed skin that
was shown recently to be rapidly transported via aff erent
lymph to the luminal surface of draining lymph node high
endothelial venues, where it triggered integrin-mediated
arrest and recruitment of monocytes from the blood cir-
culation (60). Overall, the broad range of chemoattractants
that we observed in cytokine-stimulated LECs, including
both monocyte/T cell chemokines and neutrophil chemo-
kines such as CXCL2 (GROβ) and CXCL5 (ENA-78),
suggests a far greater role for lymphatics in coordinating
infl ammatory leukocyte recruitment than has previously
In conclusion, we have shown for the fi rst time that in-
fl amed lymphatic endothelium promotes the exit of leuko-
cytes from tissue to aff erent lymph through newly induced
expression of the adhesion molecules ICAM-1 and VCAM-1,
which were previously thought to be specifi c for blood vessel
transmigration. These fi ndings reveal an overlap between the
traffi c signals within the blood and lymphatic circulations and
identify the process of lymphatic transmigration as a potential
target for antiinfl ammatory drug therapy.
2774 TRANSMIGRATION MECHANISMS IN INFLAMED LYMPHATIC ENDOTHELIUM | Johnson et al.
MATERIALS AND METHODS
Human and animal studies. All studies using human tissue were approved
by the Oxford Regional Ethics Committee. All animal studies were per-
formed under the appropriate Home Offi ce licenses and institute guidelines.
Isolation of primary HDLECs and MDLECs. HDLECs were prepared
from healthy adults undergoing elective surgery (breast reduction and ab-
dominoplasty). MDLECs were prepared from the skin of BALB/c pups. In
both cases, skin was digested overnight at 4°C with 2 mg/ml Dispase (Invitrogen)
in PBS, pH 7.5, to remove the epidermis. Dermal cells were released
from human tissue by scraping, passed through a 70-μm cell strainer (BD
Biosciences), and expanded in 0.1% gelatine-coated fl asks in complete me-
dium (EGM-2 MV; Cambrex Bio Science) at 37°C/5% CO2 in a humidi-
fi ed atmosphere. Dermal cells were released from mouse skin by digestion
for 30 min at 37°C with a mixture of 2 mg/ml collagenase A, 0.2 mg/ml
ovine testicular hyaluronidase, 0.05 mg/ml DNase I, and 0.05 mg/ml
elastase (all obtained from Roche). Digests were fi ltered through a 70-μm
cell strainer, and cells were cultured overnight in complete EGM-2 MV
medium. HDLECs and MDLECs were lifted with Accutase (PAA Laboratories)
and immunoselected with mouse anti–human LYVE-1 mAb and rat anti–
mouse LYVE-1 mAb, respectively, followed by magnetic retrieval with the
appropriate MACS bead preparations (Miltenyi Biotec). The resulting cells
were cultured in EGM-2 MV using plastic tissue culture fl asks that had been
precoated with 0.1% gelatin (Invitrogen). All experiments were performed
on confl uent cells.
Cytokines and chemokines. Recombinant cytokines and chemokines
(R&D Systems) were used at the following concentrations: IL-1α, 1 ng/ml;
IL-2, 2 ng/ml; IL-6, 20 ng/ml; IL-8, 50 ng/ml; TNF-α, 0.1–10 ng/ml (see
Results); TNF-β, 100 ng/ml; IFN-γ, 100 ng/ml; and MIP3-α, 100 ng/ml.
Recombinant mouse TNF-α was used at 100 ng/ml.
Antibodies. mAb to soluble mouse LYVE–1 Fc (25) was generated in the
rat, and polyclonal antisera to mouse or human LYVE-1 were generated
and used as described previously after purifi cation of Ig using Protein A/G–
Sepharose (25, 61). Other antibodies were rat anti–mouse CD31 (Cymbus
Biotechnology); goat anti–mouse ICAM-1, SLC, and JE (R&D Systems);
mouse anti–human ICAM-1 and ICAM-2 (Serotec); mouse anti–human
E-selectin (R&D Systems); and mouse anti–human VCAM-1, CD31,
and CD34 (BD Biosciences). Rabbit anti–human Prox-1 and anti–human
podoplanin were purchased from Fitzgerald Industries International, Inc.
Specifi city of rabbit antisera was confi rmed using podoplanin Fc. Hamster
anti–mouse podoplanin (clone 8.1.1) was from the Developmental Studies
Hybridoma Bank (University of Iowa, Iowa City, IA). Rabbit polyclonal
sera against mouse and human podoplanin were donated by D. Kerjaschki
(Medical University of Vienna, Vienna, Austria). Function-blocking
mAbs P2A4 and P8B1 (anti–ICAM-1 and anti–VCAM-1, respectively)
(62) were from the Developmental Studies Hybridoma Bank; 51-10C9
(anti–VCAM-1) was from BD Biosciences; and both 15.2 (anti–ICAM-1)
and 24 (anti–LFA-1) were gifts from N. Hogg (Cancer Research UK
London Research Institute, London, United Kingdom). Rat anti–mouse
function-blocking mAbs against VCAM-1 (clone 6C7.1) (48) and ICAM-1
(YN1/1.7.4) (47) were gifts from D. Vestweber (University of Münster,
Münster, Germany). Isotype-matched antibodies (mouse, rat, rabbit, and
goat IgG) were purchased from Sigma-Aldrich. All other primary anti-
bodies were obtained from Cancer Research UK. Secondary antibody Alexa
Fluor 488 (green) or Alexa Fluor 568/594 (red) conjugates were obtained
Flow cytometry. Cells were lifted with Accutase (PAA Laboratories), sus-
pended in incubation buff er (PBS–5% FCS, 0.1% azide) and incubated for
30 min at 5°C with primary antibody, followed by washing and reincubation
for 30 min at 5°C with the appropriate Alexa Fluor 488 goat conjugate be-
fore analysis on a fl ow cytometer (FACSCalibur; BD Biosciences) using
Immunofl uorescent antibody staining of cells and tissues. For single/
double immunofl uorescent staining of cultured HDLECs and MDLECs in
plastic dishes, appropriate primary antibodies were applied in PBS–5% FCS,
and cells were incubated at 25°C for 30 min, followed by washing and rein-
cubating with Alexa Fluor secondary antibodies in PBS–5% FCS. Samples
were fi xed in 2% formaldehyde-PBS (vol/vol) for 10 min and mounted in
Vectashield-DAPI (Vector Laboratories) before viewing under a fl uores-
cence microscope (Axiovert; Carl Zeiss MicroImaging, Inc.).
For whole-mount staining, tissues were fi xed overnight at 5°C in para-
formaldehyde (4% wt/vol in PBS, pH 7.4), blocked in PBS–Triton X-100
(0.3% vol/vol supplemented with dried milk, 3% wt/vol), and incubated
with the appropriate primary antibodies overnight at 5°C and fl uorescent-
conjugated secondary antibodies for 2 h at 25°C before mounting in Vecta-
shield and viewing on a confocal microscope (Radiance 2000; Bio-Rad
Laboratories) with sequential scanning.
For preparation of thin frozen sections, tissues were frozen in OCT
Embedding Medium (purchased from R.A Lamb Laboratory Supplies) be-
fore cutting 8-μm or thinner sections by cryostat. Primary antibodies were
applied, followed by Alexa Fluor conjugates. Sections were mounted in
Vectashield, and images captured using a confocal microscope with sequen-
To visualize DC migration across HDLEC monolayers, primary
HDLECs were seeded onto the underside of gelatin-coated clear cell culture
inserts (3-μm pore size; BD Biosciences), as described for a transmigration
assay (see MDDC-HDLEC transmigration assay). CMFDA-labeled MDDCs
were applied, and after 10 h cells were fi xed in paraformaldehyde and stained
with rabbit antipodoplanin and mouse anti–VCAM-1 or anti–ICAM-1, and
with goat anti–rabbit–conjugated Alexa Fluor 568 and goat anti–mouse Cy5
(Chemicon). Images were captured using a confocal microscope with xy
and xz scanning.
Endothelial cell proliferation assay. The proliferation rate of HDLECs
was determined using a colorimetric assay that measured reduction of MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) to the
insoluble formazan product (63). In brief, 40 μl MTT solution (7.5 mg/ml in
PBS, pH 7.5) was mixed with 200 μl culture medium and added to mono-
layers of endothelial cells cultured in 24-well dishes. After incubation for 1 h
at 37°C, supernatants were discarded, and 200 μl of 0.04 M HCL in isopro-
panol was applied to the monolayer. Cell lysates were centrifuged for 4 min
at 900 g, and the resulting supernatants were analyzed in a plate reader
(model 680; Bio-Rad Laboratories) at 590 nm.
Chemokine ELISA. Supernatants from triplicate wells of confl uent
primary HDLECs and MDLECs were assayed for the chemokines RAN-
TES, MCP-1 (JE), and MIP-3α using a commercial antigen capture ELISA
method (Quantikine; R&D Systems), according to the manufacturer’s
instructions. In brief, appropriately diluted supernatants were applied in tripli-
cate to precoated ELISA wells, alongside negative controls of medium alone
and chemokine standards, applied in duplicate. Bound chemokines were
detected using a secondary horseradish peroxidase– conjugated antibody and
substrate for measurement in a microplate reader at 490 nm.
Oxazolone-induced contact hypersensitivity. BALB/c male mice aged
8–10 wk were sensitized by topical application of 3% (wt/vol) oxazolone
(4-ethoxymethylene-2 phenyl-2-oxazoline-5-one; Sigma-Aldrich) in 95%
aqueous. ethanol to the shaved abdomen (50 μl per mouse). The next day,
a further 100 μl of 2.5% (wt/vol) oxazolone was applied to the same site. On
day 5, the dorsal surface of the left ear was challenged in each case by topical
application of 0.5% oxazolone solution (50 μl per ear), while the right ear
(control) was treated with vehicle alone.
In vivo blockade of DC migration. BALB/c male mice aged 8–10 wk
were sensitized by topical application of 3% (wt/vol) oxazolone in 95%
aqueous ethanol to both ears (50 μl per ear). The next day, a further 50 μl
of 3% (wt/vol) oxazolone was applied to the same site. On day 5, an
JEM VOL. 203, November 27, 2006
intraperitoneal injection of 0.5mg of antibody in PBS—6C7.1 (anti–VCAM-1)
(48), YN1/1.7.4 (anti–ICAM-1) (47), or rat IgG—was administered. On
day 6, the shaved abdomen was challenged by topical application of 0.8%
oxazolone solution and 1.5mg/ml FITC at 150 μl per animal. 24 h after
challenge, mice were killed, and the axillary and inguinal lymph nodes were
removed. Tissue was disrupted by 0.5 mg/ml collagenase D (Roche) for 30
min at 37°C, passed through a 70-μm cell strainer (BD Biosciences), and
stained with PE anti–mouse CD11c (BD Biosciences) for FACS analysis.
To observe blocking of DC migration from the dermis, contact hypersen-
sitivity was induced by oxazolone in BALB/c male mice by application to the
abdomen. 5 d after sensitization, either YN1/1.7.4 (anti–ICAM-1) or 0.5 mg rat
IgG was administered by intraperitoneal injection. 24h later, oxazolone was ap-
plied to both ears. 8 h after the challenge, 106 CMFDA-labeled bone marrow–
derived DCs per ear from a littermate (diff erentiated in vitro in 20 ng/ml IL-4
and GM-CSF) were dermally injected. 24 h later, animals were killed, and ear
tissue was fi xed in paraformaldehyde. Whole-mount staining was performed us-
ing hamster anti–mouse podoplanin with Alexa Fluor 568 and Cy5-conjugated
goat anti–rat Cy5 to detect binding of neutralizing antibody within the tissue.
DNA microarray analyses. Total cellular RNA was isolated (RNeasy;
QIAGEN) from freshly isolated primary HDLECs and MDLECs cultured
for 24 h in EGM-2 MV medium alone or supplemented with 1 ng/ml TNF-α
before synthesis and biotin labeling of cRNA probes and fragmentation
according to standard Aff ymetrix protocols at the Cancer Research UK
Microarray Facility (Paterson Institute for Cancer Research, Manchester,
UK). Probes were hybridized for 16 h to Aff ymetrix GeneChip Human
Genome U133 Plus 2.0 or Mouse 430 arrays, as appropriate, and processed
using a GeneChip Fluidics Station 450 according to recommended protocols
(EukGE-WS2v5; Aff ymetrix). Images were captured using the GeneChip
Scanner 3000 (Aff ymetrix). Transcript levels were determined using GeneChip
Operating Software (GCOS1.2; Aff ymetrix), and data were normalized by
global scaling. Robust multi-array average (RMA) expression was measured
by probe sequence information (GCRMA) in BioConductor R statistics and
analyzed using Data Mining Tool (DMT 3.1; Aff ymetrix) and GeneSpring
7.2 (Silicon Genetics). Microarray data are available in the National Center
for Biotechnology Information Gene Expression Omnibus (http://www.
ncbi.nlm.nih.gov/geo/) under accession no. GSE6257.
MDDCs. PBMCs were obtained from healthy donors, and monocytes were
purifi ed by positive selection using anti-CD14–conjugated magnetic micro-
beads (Miltenyi Biotec). MDDCs were generated by culturing monocytes
for 5 d in RPMI–10% FCS supplemented with 50 ng/ml GM-CSF and 10
ng/ml IL-4 (R&D Systems) and matured with LPS from 1μg/ml Salmonella
abortus (Sigma-Aldrich). For fl uorescent labeling, MDDCs were incubated
with 2.5 μM Cell Tracker Green (Invitrogen) in EGM-2 MV media for
40 min, then washed in media and rested for 30 min.
MDDC-HDLEC transmigration assay. Primary HDLECs were seeded
onto the underside of gelatin-coated Fluoroblok cell culture inserts (3-μm
pore size; BD Biosciences) and incubated for 2 h at 37°C before being placed
into a companion plate (BD Biosciences) containing EGM-2 MV medium.
Cells were cultured until confl uent and stimulated with 1 ng/ml TNF-α
(R&D Systems) 24 h before use. To assess the contribution of individual ad-
hesion molecules expressed in HDLECs, cells were incubated in the pres-
ence of blocking antibodies (see Antibodies) for 30 min before the addition
of 0.5 × 106 fl uorescently labeled MDDCs per well. To assess the contribu-
tion of ICAM-1 expressed on MDDCs, fl uorescently labeled MDDCs were
incubated at 37°C for 30 min in the presence of ICAM-1 blocking mAb
15.2 or mouse IgG and washed three times in medium before applying to
the HDLEC monolayer. Numbers of MDDCs transmigrating through the
fi lter and monolayer into the lower chamber were recorded at 30-min inter-
vals on an automated fl uorescent multiprobe plate reader (Synergy HT;
Bio-Tek) at 37°C using KC4 software (Biotech). The fl uorescent signal was
calibrated against a standard curve, and reverse transmigration was expressed
as the number of DCs in the lower chamber.
To assess the permeability of the monolayer, 50 μg/ml Alexa Fluor 488
fl uorescent dye was applied to the upper compartment of the Fluoroblok cell
culture inserts with confl uent HDLECs on the underside. Fluorescence in
the lower chamber was recorded at 30-min intervals at 37°C, calibrated
against a standard curve, and expressed as the concentration of dye in the
MDDC-HDLEC adhesion assay. Primary HDLECs were seeded in gelatin-
coated 24-well dishes and cultured until confl uent, then stimulated with
1 ng/ml TNF-α. Where appropriate, HDLECs were incubated in the pres-
ence of blocking antibodies (see Antibodies) for 30 min before the addition
of fl uorescently labeled 0.5 × 106 MDDCs per well. Plates were incubated
for either 3 or 10 h, medium was removed, and cells were washed with PBS
to remove nonadherent MDDCs. Numbers of MDDCs adhering were mea-
sured by a fl uorescent plate reader, and the fl uorescent signal was calibrated
against a standard curve of fl uorescently labeled DCs.
Online supplemental material. Detailed characterization of the eff ects of
TNF-α on CAM expression by HDLECs and MDLECs (Figs. S1–S3), the
association of DCs with ICAM-1–positive mouse lymphatics in vivo (Fig.
S4), the phenotype of MDDCs (Fig. S5), and various features of the HDLEC
in vitro transmigration assays (Figs. S6–S8) are available online. Tables S1
and S2 show Aff ymetrix GeneChip Human Genome U133 Plus 2.0 and
Mouse 430 array data, respectively. Online supplemental material is available
We thank Professor Nancy Hogg for providing ICAM-1 and LFA-1 blocking
antibodies and Professor Dontscho Kerjaschki for the gift of primary HDLECs.
We acknowledge the fi nancial support of the Medical Research Council and
Cancer Research UK (project grant A3999).
The authors have no confl icting fi nancial interests.
Submitted: 3 October 2005
Accepted: 26 October 2006
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