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Journal ArticleDOI

Interstitial dendritic cell guidance by haptotactic chemokine gradients.

18 Jan 2013-Science (American Association for the Advancement of Science)-Vol. 339, Iss: 6117, pp 328-332

TL;DR: These findings functionally establish the concept of haptotaxis, directed migration along immobilized gradients, in tissues.
Abstract: Directional guidance of cells via gradients of chemokines is considered crucial for embryonic development, cancer dissemination, and immune responses. Nevertheless, the concept still lacks direct experimental confirmation in vivo. Here, we identify endogenous gradients of the chemokine CCL21 within mouse skin and show that they guide dendritic cells toward lymphatic vessels. Quantitative imaging reveals depots of CCL21 within lymphatic endothelial cells and steeply decaying gradients within the perilymphatic interstitium. These gradients match the migratory patterns of the dendritic cells, which directionally approach vessels from a distance of up to 90-micrometers. Interstitial CCL21 is immobilized to heparan sulfates, and its experimental delocalization or swamping the endogenous gradients abolishes directed migration. These findings functionally establish the concept of haptotaxis, directed migration along immobilized gradients, in tissues.
Topics: Haptotaxis (57%), CCL21 (53%), Dendritic cell (50%)

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Interstitial Dendritic Cell Guidance by
Haptotactic Chemokine Gradients
Michele Weber,
1
Robert Hauschild,
1
Jan Schwarz,
1
Christine Moussion,
1
Ingrid de Vries,
1
Daniel F. Legler,
2
Sanjiv A. Luther,
3
Tobias Bollenbach,
1
Michael Sixt
1
*
Directional guidance of cells via gradients of chemokines is considered crucial for embryonic
development, cancer dissemination, and immune responses. Nevertheless, the concept still lacks
direct experimental confirmation in vivo. Here, we identify endogenous gradients of the chemokine
CCL21 within mouse skin and show that they guide dendritic cells toward lymphatic vessels.
Quantitative imaging reveals depots of CCL21 within lymphatic endothelial cells and steeply
decaying gradients within the perilymphatic interstitium. These gradients match the migratory patterns
of the dendritic cells, which directionally approach vessels from a distance of up to 90-micrometers.
Interstitial CCL21 is immobilized to heparan sulfates, and its experimental delocalization or swamping
the endogenous gradients abolishes directed migration. These findings functionally establish the
concept of haptotaxis, directed migration along immobilized gradients, in tissues.
S
everal guidance cues operate in verte
brates, with the most prominent group be
ing chemokines. In vitro, many chemokines
induce directional cell migration when offered
as gradients. However, the best established in vivo
example of chemokine function does not rely
on gradients: During extravasation from the blood
stream, chemokines immobilized on the luminal
surface of blood vessels (13) trigger the local
arrest of leukocytes, which precedes their exit
into the tissue (4). Less is known about how
chemokines act beyond the endothelium (5), and,
especially within lymphatic organs, chemokines
seem to rather cause random motility than di
rectional responses (5). The sparse body of exist
ing evidence for directional guidance is largely
inferred from the migratory trajectories of cells
without information on actual chemokine dis
tribution (68). Only two studies visualized che
mokine gradients in parenchymal organs (9, 10),
328
Konstanzer Online-Publikations-System (KOPS)
URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-263418
Erschienen in: Science ; 339 (2013), 6117. - S. 328-332
https://dx.doi.org/10.1126/science.1228456

and
the
CODcept
that grndients trigger directional
migration
bas
not been addressed with manip
ulative approaches.
We used mature dendritic
cells
(OCs) that
migrate from
the
dermal interstitium into affer
ent lymphatic vessels (LVs)
(11)
as
a model sys
tern
to
study chernokine function
in
situ.
To
track
DCs
en route
to
LVs,
we
used
tissue explants
of
split
mouse
ears (12). Within minutes after
additioo
of
exogenous
DCs
onto exposed der
mal tissue, the cells entered the interstitium and
approached nearby LVs in a directed manner
(Fig. l,
A to D, and movie
Sl
). Single cell track
ing within the almost planar anatomy
of
the
mouse ear (fig.
Sl
) revealed that cells switched
from noodirectional
to
increasingly directional
persistent movement toward the vessel at a
distance
of
about
90
J.Ull
from the vessel wall
(Fig. 1, B to D, and fig. 82). When located
be
tween
two
LVs,
DCs
occasionally extended
protrusions toward both vessels, indicating
the
simultaneous presence
of
two
conflicting
sig
nals.
There
the cells often spanned distances
of
more than
50
J.UI1
before they retracted ooe
protrusion and finally entered
one
of
the vessels
(Fig.
IE
and
movie 82).
In
line with previous
~ST
Austria
(Institute
ol
Sdence
and
Technology
Austria)
,
Am
Campus
1, A
3400
Klosterneuburg,
Austria
.
2
Biotechnol
ogy
lnstitul!!
Thurgau
(BITg)
at
the
University
of
Konstanz,
Unterseestrasse
4 7,
CH
8280
KreU2tingen
,
Switzerland.
3
De
partment
ol
Biochemistry
,
University
of
lausanne
,
Chemin
des
Boveresses
155,
CH
1066
Epatinges
,
Switzerland.
"To
whom
correspondence
should
be
addressed.
Email:
Sixt@ist.ac.at
A
Tracks
8
studies (13), DCs deficient for the chemokine
receptor
CCR7
did
not
approach LVs.
CCR7
dependency was cell autonomous, because
the
presence
of
comigrating
wild
type
DCs
could
not
rescue CCR7 deficient cells
(Fig.
IF). Hence,
the involvement
of
secoodarily
induced
paracrine
guidance cues that might relay the directional
information was excluded
CCR7 has two
Jig
ands, CCL19 and CCL21.
By
using tissues and
DCs
from
CCL19 deficient
animals
(14)
, we found,
in
agreement with an earlier report (15), that
CCL19
was
not
required for intravasation into
afferent LVs (fig. 83).
We therefore measured the localizatioo
of
the other CCR7 ligand, CCL21, within the der
mis.
Whole mount immunostainings
of
ear
sheets
that were previously fixed and penneabilized
re
vealed a punctuate CCL21 staining that was
exclusively associated with LVs
as
revealed
by
lymphatic vessel endothelial hyalurooan receptor
l (LYVE
1)
and basement membrane stainings
(Fig. 2, A
and B). Disrupting the trans Golgi
network
by
tissue treatment with brefeldin A led
to loss
of
the punctuate pattern and to scattering
of
CCL21 within individual LYVE I bordered
(16) lymphatic endothelial cells (Fig. 2B).
This
suggests that
the
main source
of
CCL21 produc
tioo
is
lymphatic endothelium, which harbors
intracellular
depots
of
this chemokine. Because
intracellular
CCL21
is
not
available for cells, es
pecially when located
at
a distance from the ves
se~
we
used unfixed, nonpenneabilized tissues
to
exclusively detect extracellular chemokine.
The
punctuate pattern was
not
detected. Although the
E
75
100
E
~230
8
~380
!11
0
c
530
75 230
380
530
Distance
(Jlm)
Distance
(J.1m)
D
'[
50
Ql
g 0
as
~
50
0
100
F
WT
/Ccr7 -1- DCs
50
100
Distance
{l.tm)
signals were
CODsiderably
lower than
in
the per
meabilized tissue, the interstitial
distn"bution
of
CCL21 became apparent CCL21 peaked oo the
vessel
wall and appeared
to
grndually fade with
increasing distance
froo1
the vessel (Fig. 2C).
To
quantitatively analyze these interstitial
gradients,
we
first ensured that our detection
method amplified the chemokine signal linearly
(fig.
84)
and then
measured
the averaged
in
tensity
of
interstitial CCL21 staining as a
time
tioo
of
distance from the
LV
margin. Integration
of
data from multiple
ear
sheets revealed that
the
mean CCL2I signal steeply decayed around
the vessel and flattened
in
the
intervessel
area
(Fig.
2D
and
fig.
85
, A and B). Stainings for
CCL19 with identical
secondary reagents revealed
a
uniform
pattern
of
background signal in both
CCL19 deficient and oootrol tissues. This back
ground signal invariably approached the leveled
out CCL21 signal remote from the vessel (Fig.
2D
and
fig.
85
, C
to
G).
The
slopes
of
CCL21
gradients appeared smooth upon signal integra
tion over
l
ar&>e
areas or multiple samples.
How
ever, migrating single cells have only
access
to
local informatioo, which exhibited considerably
more noise, with local concentratioo
peaks
that
could potentially trap the cells
oo their path
to
the
ves.~el.
The widely accepted spatial para
digm
of
euk:aryotic (as opposed
to
prokaryotic)
gradient sensing assumes that cells quantita
tively detect concentration differences over their
entire
surfuce
and
polarize toward higher
COD
centrations (17). Hence,
we
calculated. vector
maps
of
the
local gradients as they would
be
75
E
.2; 230
~
c
~
380
i5
530
WT
/Ccr7 -1- DCs
75
230
380
530
Distance
{l.tm)
Fig
.
1.
D
Cs
move
directionally
toward
CU21-expressing
L
Vs
in
the
dermal
interstitium.
(A
to
D)
Tracks
of
D
Cs
migrating
in
ear
explants.
(A)
Migratory
paths
(tines
il
gray)
t
racked
from
a
representative
60-min
movie,
o~.Erlaid
onto
the
LV
mask
(dark
gray).
(B)
Selected
tracks
(gray)
from
five
movies
(n =
200,
three
independent
experiments)
reorientated
to
nearest LV (LV
margin
at
distance
= 0
indicated
by
the
black
horizontal
line).
(C
and
D)
Direc-
tionality
and
velocity
toward
LV
as
a
function
of
distance
to
the nearest L
V.
M
ean
± S
EM
is
shown.
n = 200,
three
independent
experiments.
a.u.,
arbitrary
units.
(E) W
ild-type
DC
(highlighted
by
the white
arrow)
migrating
be
tw
een
tw
o
adjacent
vessels
(indicated
by
yello
w
dotted
lines).
Scale
ba
r
indicates
50
Jlm.
(F) (L
eft)
Z
-stack
projection
showing
wild-type
(red)
and
Ccrr'
- D
Cs
(green)
after 120.min
incubation
with
ear
sheets
stained
for
LYVE
-1
(blue).
Scale
bar,
100
Jlm.
(Right)
DC
migration
paths
tr
acked
from
a
representative
60-min
movie,
overlaid
onto
t
he
LV
mask
(dark
gray).
329

330
sensed by a cell
of
a given diameter. For cell
diameters below 9
IJ-01,
no
coherent vector field
emerged (Fig.
2E
and movie S3). When
as
suming a cell size
of
15
to 50
JlDl
, which reflects
the fluctuating span
of
a migrating
OC
(Fig.
IE
),
we
retrieved vector fields pointing
from
the
interstitiwn toward
the
next vessel for CCL21
but
not for control stainings (Fig. 2, E and F,
and movie S2). We next computed average local
concentration
deltas
fur
given cell sizes as a time
tion
of
distance
from
the vessel. For
an
assumed
cell size
of
36 1!01, this "functional gradient''
A
B
c
E
F
-
9~tm
-
18~tm
-
36~tm
Fi
g.
2. V
isuali
zation
and
quantification
of
an
interstitial
CCL21
gradient
(A
)
Z-stack
projection
of
permeabilized
ear
dermis
stained
for
CCL21
(red)
and
laminin
(gr
een
).
Scale
bar,
100
~J.m.
(B)
CCL21
(green)
LYVE
-1
+
(red)
co-
staining
of
permeabilized
ear
derrnis
after
treatment
with
25
~J.g/ml
br
efeldin
A.
Scale
bar,
25
~J.m.
(C)
Z-stack
projection
of
nonpermeabilized
ear
dermis
stained
for
CCL21.
Gray
scale
shows
maximum
intensity
projection
(left).
Right
image
sho\IIS
same
staining
as
color-coded
average
projection.
LV
bound-
aries
are
indicated
by
the
blue
dotted
line
based
on
L
YVE-1
staining
(as
shown
in
fig.
52).
Scale
bars,
100
~J.m.
A
representative
image
from
n = 9
out
of
four
independent
experiments
is
shown.
(D
)
Quantification
r:J
interstitial
CCL21
and
CCU9
staining
as
function
of
distance
from
the
nearest
LV margil.
Mean
signal
intensities
relative
to
average
maximum
CCL21
signal
±
SEM
are
shO\WI
(red,
D
50 100
Distance from
LV
(~tm)
G
-
9~tm
-
18~tm
-
36~tm
<l
-0.1
CCL21;
green,
CCL19
in
Ccl19+1
+
ear;
n =
9;
four
independent
experiments;
black.
CCL19
in
Cc/1~
1
-
ear;
n =
5;
two
experiments).
(E)
Vector
maps
of
local
chemokine
gradients.
Arrow
length
and
direction
indicate
concentration
rise
in
CCL21
after
averaging
over
circular
surface
area
with
indicated
diameter
(virtual
cell
size).
Gray
scale
indicates
averaged
intensities.
Scale
bar,
15
~J.m.
(F
) D
irectionality
of
CC
L21
gradients
{1),)
for
indicated
virtual
cell
size
as
cosine
of
angle
between
direction
toward
dosest
point
of
LV
and
direction
of
increasing
chemokine
concentration.
Means
±
SEM
for
n = 6
fr
om
three
independent
experiments
are
shown.
(G)
Average
local
CCL21
concentration
delta~)
as
a
function
of
distance
to
nearest
LV
,
calculated
for
indicated
virtual
cell
si
ze.
Means
±
SEM
for
n = 7
from
four
independent
experiments
are
shown.

fuded
at a distance
of
75
to 90 IJlil
from
the
vessel
wall
(Fig.
20)
and
thus matched the
mi&rratory
behavior
of
the cells that increased directionality
in
a similar perimeter around
the
vessel as we
have shown above (Fig. 1
C).
To estimate bow
well
the
detected gradients comply with the dis
tnbution
of
LVs
within
the
dermis, we calcu
lated distance maps
of
afferent LVs over large
areas.
We
found that a cell that is randomly lo
caliz.ed
within the interstitium would need to
travel an average distance
of
47 ± 3
J.im
to reach
the nearest
LV
(fig. 86, A and B) and that 92%
of
the
interstitial space lies within a
90
IJlil
perimeter around
the
LV
netwmk (fig. S6C).
In
accordance with this, endpoint analysis
of
in
travasation assays only occasionally revealed few
cells that were left "stranded"
in
the
middle be
tween vessels
with
large
spacing
(fig.
860). These
data demonstrate a relative invariability
of
the
intervessel distance and imply that the range
of
the CCL21 gradient is very
well
adapted to the
distribution
ofLVs
in
the
skin.
We
next challenged our correlative evidence
for
DC
guidance by a
CCL21
gradient using
experimental manipulations. To test to what de
gree a continuous release
of
CCL21 is required
to
maintain
the
gradient, we extensively washed
the ear explants
and
performed migration assays
in
the
presence
of
brefeldin A
to
terminate pos
Sible
chemokine secretion tium
LVs.
Because
we
previously
found
that
CCL21
can be
proteolytical
ly cleaved at the C terminus by a
DC
associated
protease
(18),
we also
pharmacol!Jbrically
blocked
this putative cleavage event, which might cause
solubilization
of
CCL21. In both
cases,
we found
that cells still approached LVs (fig.
S7
, A and B)
and conclude that a continuous release
ofCCL21
is
dispensable for directed DC migration within
the
approximate 2 hour time window
of
our as
says. This suggests that the functionally active
CCL21 gradient is stored within
the
homeostatic
dermis.
In contrast to CCL19, CCL21 has a highly
charged
C terminal extension that binds glyco
saminoglycans
(GAGs)
and
is
thought to
im
mobilize the chemolcine to extracellular matrix
or
cell surfaces (18, 19).
Our
previous findings
together with the fact that we could detect
CCL21
gradients by histological methods, which
necessarily include washing steps where unbound
proteins
are
removed, were consistent with the
assumption that
CCL21
in
the
dermis is not sol
B
Distance from
LV
{J.tm)
D
200 -
1
00
200 300 400 500
Distance
{J.tm)
.t::.
'i
:2
Q)
~
':
100
"'l!t
as
"'
~:8
G>
as
O.t::.
-a.
o E
0~
z
uble.
We
therefore performed a series
of
experi
ments
in which we perturbed the distribution
of
endogenous CCL21 by addition
of
exogenous
chemokine.
To
this end, we pre incubated
ear
explants with an excess
of
exogenous recombi
nant
CCL21
, washed them to remove the solu
ble
CCL21
fraction, and quantified the impact
on
gradient shapes and
DC
migration.
We
found
that exogenous
CCL21
diffusely localized with
in
the dermal interstitium
and
thereby masked
and flattened
the
endogenous
CCL21
gradients
(Fig.
3A
and
fig.
S8A).
In
the
pretreated ex
plants, a
laJb>e
fiaction
of
DCs was misguided
within
the
interstitium and ultimately remained
scattered remote
from
the vessels (Fig. 3, B to
D;
fig.
S8B; and movie
S4).
Additionally, pre
treatment
of
the
explants with a C terminally
truncated version ofCCL21, which is incapable
of
binding GAGs (19), as
well
as
with a GAG
binding but nonsignaling mutant did not change
the migratory pattern
of
the cells (Fig.
3D
and
fig.
S8C). To exclude
that
exogenous
CCL21
af
fects
DC
migration nonspecifically,
for
example,
by
masking electrostatic interactions, we pre
treated
ear
explants with exogenous CXCL13
and CXCLI2, which also bind GAGs
(20)
yet
- 1.0
~
'd
"5l
0.5
N
~
0
z
0
+-----~-----.--
0
50
100
Distance from
LV
().l.m)
Fig. 3.
The
CQ.21
gradient is functional and
immobiUzed
to heparan sulfates. (A)
(Left)
Z-stack
projection
of
CCL21
F
immunostaining (false
colo
r-coded)
of
nonpermeabilized ear
dermis
after incubation
with
0.6
j.l.g/ml
CQ.21.
L
YV
E
-1-
positive L
Vs
are indicated
by
the blue dotted line.
Scale
bar, 100
j.l.m.
(Right)
cCCL21
after incubation
with
exogenous
CCL21.
Gray
line indicates endogenous
CCL21
as
shown
in
Fig.
2.
n = 3,
two
independent experiments. (B)
Z-stack
projections
of
D
Cs
(r
ed)
and L
YV
E-
1 immunostaining (green) after a 120-min co-incubation of D
Cs
with
ear sheets.
Tissue
was
either vehicle-treated or incubated
with
CC
L21 before addition
of
D
Cs.
n = 3,
two
independent
ex-
periments. (C)
DC
tracks
(light gray
lines)
from
representative 60-min movie
with
CQ.21
pre-incubation,
overlaid
onto
LV
mask
(dark
gray).
(D)
Number
of
D
Cs
aS$odated
with
LVs
after
120-min
crav-A-in.
I
ndicated
chemokine
Y.as
pre-
incubated
at 0.6
j.l.g/ml
before
addition
ci
D
Cs.
Control
versus
CQ.21,
**P
= 0.006;
control
versus
CQ.21trunc,
P = 0382;
control
versus
CCL21mut.
P = 0.8163,
control
versus
CXG.l3,
P = 0.458;
control
versus
CXCU2,
P = 0.0642. n = 3, at
least
two
independent
experiments.
(E)
Z-stack
projection
of
nonpermeabiized ear
dermis
stailed
for
CG.21
(false
color-
coded,
left)
and
quantification
of
cCCl21
(right)
in
nonpermeabilized
ear
dermis
after treatment
with
50
mlU
heparitinase.
Csirf
line
in
graph ildicates
endogenous
CQ.21
as
il
Fig.
2.
(F
)
Numbers
of
D
Cs
assodated
with
L
Vs
in
ear
dermis
after 120
min
of
crawl-in.
n =
5,
three independent
experiments
(**P
=
0.004).
All
scale
bars,
100
j.l.m.
Error
bars
indicate S
fM
.
33
1

do not trigger CCR7, and saw no significant ef
fects on DC migration (Fig. 3D and fig. S8C).
Together, these findings establish the functional
activity of the immobilized CCL21 gradients that
we identified before and show that, although DCs
carry the receptor for and can respond to CXCL12,
CCL21 gradients dominate. In addition, the che
mokine pattern appears not to be determined by
the distribution of CCL21 binding sites but most
likely by the diffusion range of CCL21, which is
trapped by sugar residues once it is released from
the LVs (21).
CCL21wasshowntobindviaitsCterminal
domain sulfated sugars like heparin, heparan,
dermatan, and chondroitin sulfates with low nano
molar affinities (19, 22, 23), potentially explain
ing the observed long retention times of CCL21
within the dermis and the inability of C terminally
truncated CCL21 to outcompete the endogenous
gradients. To test which of the sugar moieties
are involved in the immobilization of CCL21,
we pretreated ear explants with sugar degrading
enzymes and found that heparitinase, which ef
fectively removed heparan sulfates (fig. S9A),
severely changed the CCL21 pattern. Quantifica
tions revealed an almost complete flattening of
the gradient and a drop to signal levels similar to
control stainings, whereas tissue integrity was not
affected (Fig. 3E and figs. S5G, S9B, and S10A).
Consequently, DC migration in heparitinase
treated explants was severely diminished (Fig.
3F and fig. S10B). Heparan sulfate distribution
patterns in untreated dermis did not match these
of CCL21 (fig. S9A), corroborating the concept
that not the tissue binding sites for CCL21 but
rather its distribution range determines the shape
of the CCL21 gradient. These findings demon
strate that, like in the lumen of blood endothe
lium (2), interstitial CCL21 is immobilized to
heparan sulfate residues, which either decorate
cell surfaces or interstitial matrix components.
This immobilized chemokine fraction is sufficient
to guide intravasation of DCs, whereas adhesion
molecules of the integrin family are dispensable
for path finding (24).
The term haptotaxis was originally intro
duced to describe cell migration along adhesive
gradients, a phenomenon that was successfully
constituted in vitro but still lacks direct in vivo
support (25). Interstitial guidance by heparan sul
fate immobilized chemokine gradients, as dem
onstrated here, can be viewed as a second variant
of haptotaxis. The facts that (i) many chemokines
bind GAGs (26), (ii) GAG interaction is impor
tant for the leukocyte recruiting activity of some
chemokines upon instillation into animals (27),
and (iii) leukocytes have the ability to migrate
along immobilized chemokine gradients in vitro
(28, 29) suggest that haptotaxis could be a wide
ly used principle. Because immobilized gradients
are insensitive to mechanical perturbations, they
certainly constitute a robust and stable infrastruc
ture for cellular guidance, whereas attraction
by soluble gradients might be rather transient
in nature.
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Acknowledgments: We thank M. Frank for technical assistance
and S. Cremer, P. Schmalhorst, and E. Kiermaier for critical
reading of the manuscript. This work was supported by a
Humboldt Foundation postdoctoral fellowship (to M.W.), the
German Research Foundation (Si1323 1,2 to M.S.), the Human
Frontier Science Program (HFSP RGP0058/2011 to M.S.), the
European Research Council (ERC StG 281556 to M.S.), and
the Swiss National Science Foundation (31003A 127474 to
D.F.L., 130488 to S.A.L.). The authors declare no conflicts
of interest. The data reported in the manuscript are tabulated
in the main paper and in the supplementary materials.
Supplementary Materials
www.sciencemag.org/cgi/content/full/339/6117/328/DC1
Materials and Methods
Figs. S1 to S10
References (30)
Movies S1 to S4
332
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