Lymph node chemokines promote sustained T lymphocyte motility without triggering stable integrin adhesiveness in the absence of shear forces.

Abstract

Lymphocyte motility in lymph nodes is regulated by chemokines, but the contribution of integrins to this motility remains obscure. Here we examined lymphocyte migration over CCR7-binding chemokines that 'decorate' lymph node stroma. In a shear-free environment, surface-bound lymph node chemokines but not their soluble counterparts promoted robust and sustained T lymphocyte motility. The chemokine CCL21 induced compartmentalized clustering of the integrins LFA-1 and VLA-4 in motile lymphocytes, but both integrins remained nonadhesive to ligands on lymphocytes, dendritic cells and stroma. The application of shear stress to lymphocytes interacting with CCL21 and integrin ligands promoted robust integrin-mediated adhesion. Thus, lymph node chemokines that promote motility and strongly activate lymphocyte integrins under shear forces fail to stimulate stable integrin adhesiveness in extravascular shear-free environments.

Full text

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Lymph node chemokines promote sustained
T lymphocyte motility without triggering stable integrin
adhesiveness in the absence of shear forces
Eilon Woolf1, Irina Grigorova2, Adi Sagiv1, Valentin Grabovsky1, Sara W Feigelson1, Ziv Shulman1,
Tanja Hartmann1, Michael Sixt3, Jason G Cyster2& Ronen Alon1
Lymphocyte motility in lymph nodes is regulated by chemokines, but the contribution of integrins to this motility remains
obscure. Here we examined lymphocyte migration over CCR7-binding chemokines that ‘decorate’ lymph node stroma. In
a shear-free environment, surface-bound lymph node chemokines but not their soluble counterparts promoted robust and
sustained T lymphocyte motility. The chemokine CCL21 induced compartmentalized clustering of the integrins LFA-1 and
VLA-4 in motile lymphocytes, but both integrins remained nonadhesive to ligands on lymphocytes, dendritic cells and stroma.
The application of shear stress to lymphocytes interacting with CCL21 and integrin ligands promoted robust integrin-mediated
adhesion. Thus, lymph node chemokines that promote motility and strongly activate lymphocyte integrins under shear forces fail
to stimulate stable integrin adhesiveness in extravascular shear-free environments.
Naive and memory T cells emigrate into specialized T cell zones in
lymph nodes, where they scan for cognate antigen presented by major
histocompatibility complex molecules on dendritic cells (DCs)1. The
arrest of T cells on high endothelial venules is regulated by the integrin
LFA-1 and the chemokines CCL21 and CCL19, which bind to the
receptor CCR7 on T cells2,3. In vivo imaging with confocal and
multiphoton intravital microscopy suggests that lymphocytes entering
the T cell zones follow a ‘random walk’ over densely packed DC
networks4and specialized fibroblastic reticular cells (FRCs)5. Pub-
lished findings suggest that CCR7-binding chemokines are involved in
supporting this motility. Inhibition of chemokine signaling in
T lymphocytes after they enter the lymph node considerably attenu-
ates their interstitial locomotion6. In addition, large quantities of the
CCR7 ligand CCL21 are expressed in the T cell zone7and attract
activated CCR7+B cells8. Finally, suppression of CCR7 expression on
T cells decreases their entry into lymph nodes and attenuates the
random locomotion of residual T cells that enter the T cell zone6,9.
Additional cues for lymphocyte motility in the T cell zone could be
provided by integrin ligands expressed on FRCs and DCs10. The LFA-1
ligand ICAM-1 is ubiquitously expressed by DCs11, and histological
studies suggest that 60% of T cells in lymph nodes are in contact with
DCs at any given moment12. FRCs express both ICAM-1 and VCAM-1
(ref. 13), the main ligands of the integrins LFA-1 and VLA-4,
respectively. FRCs also display CCL21 and CXCL12, ligands of
CCR7 and CXCR4, respectively13. FRCs could therefore be ideally
suited to present both chemokine and integrin ligand cues to
T lymphocytes5. In addition to their functions promoting motility,
CCL19, CCL21 and CXCL12 activate LFA-1 adhesiveness on naive and
memory T lymphocytes14,15. As ICAM-1 and a second LFA-1 ligand,
ICAM-3, are also expressed on T cells16, exposure of lymphocytes to
lymph node chemokines, if not tightly regulated, could cause non-
productive interlymphocyte clumping or premature lymphocyte arrest
on DCs and FRCs independently of arrest signals triggered by contact
of lymphocytes with cognate antigen. To optimize their antigen-
scanning ability, T cells must escape such antigen-independent,
integrin-mediated arrest cues.
To delineate the individual functions of integrin and chemokine
cues in mediating T cell arrest, we used a series of molecularly defined
model surfaces ‘decorated’ with the chief T cell zone chemokine
CCL21 as well as the integrin ligands ICAM-1 and VCAM-1. We
found that surface-bound CCL21 but not its soluble counterpart
triggered robust and sustained T cell locomotion in the absence of
any additional adhesive ligands. Signals from surface-bound CCL21
were insufficient to trigger LFA-1 and VLA-4 adhesiveness and thereby
minimized lymphocyte clumping and arrest on DCs and stroma. This
‘silenced’ integrin adhesiveness was immediately reversed by the
application of shear forces. Our data suggest that chemokine-primed
lymphocyte integrins need shear forces to optimally adhere to their
ligands. This mechanism may allow lymph node chemokines to
maximize lymphocyte motility and minimize integrin-mediated
Received 14 May; accepted 6 July; published online 26 August 2007; doi:10.1038/ni1499
1Department of Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel.2Department of Microbiology and Immunology and Howard Hughes Medical
Institute, University of California, San Francisco, California 94143, USA.3Max Planck Institute of Biochemistry, Munich 82152, Germany. Correspondence should be
addressed to R.A. (ronen.alon@weizmann.ac.il).
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adhesions to ligands in lymph node interstitial spaces devoid of shear
flow. At the same time, identical chemokines robustly stimulate firm
integrin-mediated adhesion of lymphocytes to blood vessel ligands
under shear flow.
RESULTS
Sustained lymphocyte motility triggered by immobilized CCL21
To determine whether prominent lymph node chemokines can
promote T cell motility in vitro, we initially tracked by video micro-
scopy the motility of resting human CD3+peripheral blood lympho-
cytes (PBLs) on an adhesive matrix coated with immobilized CCL21
(Fig. 1a). Although the spontaneous autonomous motility of resting
lymphocytes was negligible, surface-bound CCL21 triggered robust
and random T cell motility (Fig. 1a,b and Supplementary Video 1
online); this motility was eliminated by pertussis toxin, an irreversible
inhibitor of Gaiprotein signaling (Fig. 1b). Unexpectedly, soluble
CCL21, even in quantities sufficient to trigger phosphorylation of the
Erk1 and Erk2 mitogen-activated protein kinases (Supplementary
Fig. 1 online) and transient lymphocyte polarization (Supplementary
Fig. 2 online), failed to promote any T cell locomotion (Fig. 1a and
Supplementary Video 2 online). T cell locomotion on immobilized
CCL21 was rapid, with velocities within the ranges reported before
in vivo5,17(Fig. 1a). A critical density of 100 sites of immobilized
CCL21 per mm2was needed to support motility, but above this
threshold, locomotion velocity was not altered by increasing chemo-
kine concentration (data not shown). Motile T cells were mainly naive
CD45RA+T cells (Supplementary Fig. 3 online) and about 60–70% of
the memory T cells present among PBLs (data not shown).
Notably, lymphocyte locomotion triggered by CCL21, CCL19 and
CXCL12 did not require the presence of any additional adhesive
ligands (Supplementary Video 3a,b online and data not shown).
Furthermore, blockade of b1integrin did not alter the motility of
T cells exposed to CCL21 immobilized together with the b1ligands
collagen and fibronectin (Supplementary Fig. 4 online and data not
shown). Mouse T cells also moved on immobilized CCL21 regardless
of the presence of an integrin ligand (Sup-
plementary Fig. 5 online). The failure of
soluble CCL21 to promote motility was not
due to insufficient ligand density, as a single T cell encountered a
similar number of chemokine molecules when presented with either
30 nM soluble CCL21 or 100 sites of immobilized CCL21 per mm2
(data not shown). Notably, although soluble CCL21 failed to promote
motility, it also did not antagonize locomotion on immobilized
CCL21 (Fig. 1a). Consistent with the potent activity of immobilized
CCL21 and CXCL12, T cells often simultaneously extended two
pseudopodia throughout their migration period (Supplementary
Video 3a,b).
T cell motility persisted over immobilized CCL21 for at least 3 h
without noticeable changes in velocity (Fig. 1a and data not shown),
suggesting minimal global desensitization of CCR7 by immobilized
CCL21. As CCL21 is a poor desensitizing chemokine18, we compared
two additional lymph node chemokines, the CCR7 ligand CCL19 and
the CXCR4 ligand CXCL12, both known to induce strong desensitiza-
tion of their cognate G protein–coupled receptors19. T cell locomotion
on these chemokines was sustained to a similar degree (Fig. 1c). In
contrast, the small fraction of effector memory PBLs capable of
locomotion on immobilized inflammatory chemokines such as
CXCL9 (Fig. 1c) or CXCL10 (data not shown) rapidly slowed after
initial chemokine exposure, presumably because of desensitization of
CXCR3. Thus, three important homeostatic chemokines but not
inflammatory chemokines act in their surface-bound state as
triggers of robust and sustained integrin-independent locomotion
of resting T lymphocytes.
CCL21 polarizes integrins without activating ligand binding
LFA-1 on lymphocytes is rapidly activated by CCL21 displayed on
endothelial cells14,15. We therefore next analyzed the distribution
and adhesiveness of LFA-1 as well as VLA-4, a second main
integrin expressed on T lymphocytes, on lymphocytes moving over
surface-bound CCL21. The a4integrins VLA-4 and a4b7clustered at
the uropods of most lymphocytes exposed to immobilized CCL21,
whereas LFA-1 clustered mainly in the highly dynamic pseudopodia of
these migrating cells (Fig. 2a and Supplementary Videos 4 and 5
a
bc
80
None
Immob CCL21
Sol CCL21
0–20 min
100
80
60
40
20
0
70–90 min
100
80
60
40
20
0
Vmean = 11 ± 1.5
–40
–60
–80
–100
–100–50 –20
50
100
–40
–60
–80
–100
–100–50–200100
Vmean = 10 ± 2.3
Immob + sol CCL21
Immob CCL21
Immob CCL21 + PTX
Immob CXCL9
Immob CXCL12
Immob CCL19
60
40
20
0
0 1020 80
Time (min)
5
0
20
40
60
80
10 15 2025
Time (min)
Motile lymphocytes
(% of total cells)
0
0 10 20
Time (min)
8090
20
40
60
**
80
100
Motile lymphocytes
(% of total cells)
Motile lymphocytes
(% of total cells)
90
Immob CCL21
Sol CCL21
50
0
Figure 1 Immobilized but not soluble CCL21
promotes sustained locomotion of resting
peripheral blood T lymphocytes without apparent
desensitization. (a) Left, fraction of human PBLs
moving over a matrix coated with collagen I and
fibronectin alone (None) or in the presence of
soluble (Sol) CCL21 (30 nM; saturating CCR7)
or immobilized (Immob) CCL21 (600 sites/mm2).
Middle, T cells interacting with immobilized or
soluble CCL21 (from Supplementary Videos 1
and 2). Original magnification, ?40. Right, tracks
of individual T cells (different color for each)
normalized to their starting coordinates; Vmean,
average instantaneous velocity of total T cells
analyzed. (b) Fraction of moving T cells among
PBLs pretreated for 12 h with pertussis toxin
(+ PTX) or carrier and plated without washing on
a matrix coated with CCL21, collagen I and
fibronectin. (c) Fraction of primary PBLs moving
over CCL19, CXCL12 or CXCL9 (1 mg/ml each)
immobilized together with collagen I and
fibronectin. **, P o 0.01 (Student’s t-test).
Data are representative of ten (a; left, means
of three fields of view), three (b) or four (c)
independent experiments.
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online). The key integrin linker talin20was distributed among
both edges of motile T cells (Fig. 2b), but phophatidylinositol-4,5-
bisphosphate, which enhances the binding of talin to integrins, was
enriched only in the leading edge (Fig. 2b), along with the actin-
remodeling GTPase Rac1 (Supplementary Fig. 6 online).
To directly assess the adhesive state of LFA-1 at the leading and
trailing edges of motile T lymphocytes, we monitored both the
frequency and strength of productive encounters between motile
T cells and ICAM-1-coated beads (Fig. 3a and Supplementary
Video 6 online). Neither the number of ICAM-1 bead encounters
nor the duration of each encounter was lower after LFA-1 blockade
(Fig. 3a). Thus, LFA-1 is not adhesive at either the leading or trailing
edges of lymphocytes migrating over immobilized CCL21. A similar
analysis with VCAM-1-coated beads indicated a lack of VLA-4
adhesiveness in motile lymphocytes (data not shown). Whereas
T cells express two LFA-1 ligands, ICAM-1 and ICAM-3, and both
ligands clustered at the uropod of migrating lymphocytes (Fig. 3b),
LFA-1 on motile T cells failed to bind to neighboring T cells (Fig. 3b,c
and Supplementary Video 7 online) unless their LFA-1 was artificially
activated (Fig. 3c and Supplementary Video 8 online). Lymphocytes
moving over immobilized CCL19 and CXCL12 showed a similar lack
of LFA-1 adhesiveness to ligand-coated beads and to other passing
T cells (data not shown).
Shear forces are critical for integrin activation by chemokines
Rapid activation of LFA-1 triggered by CCL21 displayed on the
vascular endothelium involves
ICAM-1 and juxtaposed chemokines15. Thus, the failure of immobi-
lized CCL21 to induce adhesiveness of LFA-1 on motile T cells could
have been due to physical separation of the chemokine and ICAM-1,
each of which were presented on a different surface in these assays.
Staining of lymph node sections showed moderate but ubiquitous
ICAM-1 expression on all extravascular components of the T cell zone,
including FRCs associated with laminin-positive conduits (Fig. 4a and
bidirectionalsignaling between
Figure 3 LFA-1 on T cells migrating on CCL21
does not bind ICAM-1. (a) Top row, T cells
moving over immobilized CCL21 and
encountering beads coated with high-density
ICAM-1 (from Supplementary Video 6). The first
image (time 0) was obtained 10 min after the
introduction of T cells to the chemokine-coated
surface. Yellow stars mark the leading edge of a
T cell colliding with an ICAM-1-coated bead.
Below, number and duration of collisions between
T lymphocytes migrating on immobilized
CCL21 and ICAM-1-coated beads. U, uropod;
L, lamelipodium. a-b2, antibody TS1.18
(anti-b2; blocking LFA-1 on T cells); a-ICAM-1,
pretreatment of ICAM-1-coated beads with
polyclonal anti-ICAM-1; Control, isotype-matched
monoclonal antibody. (b) Top row, concentration
of ICAM-1 and ICAM-3 at the uropods of T cells
moving over immobilized CCL21, among T cells
fixed and stained with anti-ICAM-1 or anti-ICAM-
3. Bottom row, collision of a T cell with the
uropod of another T cell (from Supplementary
Video 7), among T cells prelabeled with
nonblocking anti-LFA-1. (c) Frequency and
average duration of contacts between T cells
colliding with each other during locomotion over CCL21 (30 s or less) in the presence of a control antibody, antibody TS1.18 (b2-blocking) or antibody
240Q (b2-activating). Inset (below), two T cells preactivated with 240Q migrating on immobilized CCL21 and colliding into each other; 240Q also increased
by 70% the binding of ICAM-1-coated beads to T cells migrating on CCL21 (data not shown). Original magnification, ?40. Data are one representative of
four (a) or three (b,c) experiments.
a
b
Time (s)
LFA-1
α4
CCL21–+–+
TalinPIP2
01530 45
a
Time (s)
c
b
0
200
180
160
140
120
100
80
60
40
20
T cell–ICAM-1
bead contacts
0
100
T cell contacts (%)
80
60
40
20
0
Contact site:
Duration (min):
LLU
>1
U
>1
ICAM-1
0Time (s)15 3045
ICAM-3
<0.5 <0.5
mAb;None
Time (s)0 45150
β2–
blocking
β2–
activating
*
*
*
*
*
*
*
*
15 30 45
Control
α-β2
α-ICAM-1
Contacts (min)
<0.5
>0.5
ICAM-1-
coated
bead
Figure 2 Immobilized CCL21 triggers polarized redistribution of LFA-1 and
a4integrins. (a) Live images of LFA-1 (top) or of a4integrins (bottom) in
T cells migrating over CCL21 at 5 min after initial T cell exposure to the
immobilized chemokine (from Supplementary Videos 4 and 5); cells are
labeled with Alexa Fluor 568–tagged TS2/4 (nonblocking anti-aL) or
Alexa Fluor 488–tagged B5G10 (nonblocking anti-a4). Top rows, merged
differential interference contrast and fluorescence images obtained
consecutively; bottom rows, spectrum analysis of relative fluorescence
intensity corresponding to the images above. Neither antibody alters
T cell speed. (b) Intracellular staining of talin and phophatidylinositol-4,
5-bisphosphate (PIP2) in resting T cells (–) or in T cells moving for 10 min
over CCL21 (+). Original magnification, ?40. Data are representative of five
(a) or three (b) independent experiments.
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data not shown). Notably, throughout the T cell zone, CCL21 staining
was consistently localized together with FRCs and was therefore
enhanced around high endothelial venules (Fig. 4a). This staining
pattern suggested that stromal and endothelial CCL21 are physio-
logically encountered by T cells in juxtaposition to ICAM-1.
To determine whether immobilized CCL21 juxtaposed with
ICAM-1 can activate LFA-1 on T cells, we reconstituted various
ICAM-1-bearing surfaces with a fixed amount of surface-bound
CCL21. ICAM-1 alone, although shown before to promote motility
of T cell blasts21, failed to support the motility of resting lymphocytes
(Fig. 4b); in contrast, CCL21 triggered robust motility (Supplemen-
tary Video 3a). Notably, increased ICAM-1 densities did not arrest or
decelerate T cells moving on immobilized CCL21 (Fig. 4b). These
results collectively indicate that T cell LFA-1 is not functionally
activated even when T cells encounter CCL21 and ICAM-1 immobi-
lized together. Indeed, blocking LFA-1 on T cells did not affect lym-
phocyte locomotion (Fig. 4b), although on high-density ICAM-1,
blockade of LFA-1 marginally reduced the velocity of motile T cells,
consistent with a minor motility-promoting activity of chemokine-
activated LFA-1 (Fig. 4b).
In contrast, when T lymphocytes were settled on identical sub-
strates under continuous application of shear stress, CCL21 triggered
LFA-1-mediated adhesive interactions with ICAM-1 (Fig. 4b). Like-
wise, T lymphocytes that had already spread on high-density ICAM-1
and CCL21 for 3–4 min in shear-free conditions and were then
subjected to shear forces rapidly developed strong LFA-1 adhesions
(Fig. 4c). Thus, LFA-1, although mainly ‘silent’ in shear-free condi-
tions in the presence of ICAM-1 and CCL21 immobilized together,
was readily activated by the application of shear force.
FRCs in the T cell zone express also the VLA-4 ligand VCAM-1
(ref. 13). However, the motility of T cells encountering immobilized
CCL21 presented in juxtaposition to high-density VCAM-1 was not
affected by the presence of VCAM-1, and VLA-4 blockade had no
effect on the motility of cells over surfaces coated with VCAM-1 and
CCL21 (Fig. 5a and data not shown). Nevertheless, similar to LFA-1,
VLA-4 was readily activated in T lymphocytes in the same conditions
in the presence of shear stress (Fig. 5b). As ligand-occupied VLA-4 can
augment LFA-1-mediated T cell motility and adhesiveness22, we next
assessed whether CCL21-stimulated T lymphocytes stopped on a
surface bearing equal densities of VCAM-1 and ICAM-1. In the
absence of shear stress, the presence of both integrin ligands failed
to arrest or even decelerate T lymphocytes moving over surface-bound
CCL21 (Fig. 5c). However, as seen on surfaces coated with individual
integrin ligands, in the presence of shear stress, CCL21 triggered
adhesion of both VLA-4 and LFA-1 to the surface coated with
VCAM-1 and ICAM-1 (Fig. 5d). In shear-free conditions, blockade
of VLA-4 marginally reduced the velocity of T cells migrating over
surface-bound CCL21 and high-density VCAM-1 but did not alter the
motility of T cells exposed to immobilized CCL21, VCAM-1 and
ICAM-1 (Fig. 5a,c). Thus, in the presence of motility-promoting
CCL21 signals, both VLA-4 and LFA-1 are similarly ‘silent’ on
lymphocytes migrating in shear-free conditions.
This mechanical shear stress regulation of integrins was not unique
to T lymphocytes. We confirmed a similar effect in neutrophils
arrested on the integrin Mac-1 ligand fibrinogen immobilized together
with the potent chemokine interleukin 8 (IL-8). In the absence of
shear stress, neutrophil spreading and locomotion were entirely
dependent on IL-8 but not on b2integrins, whereas under shear
stress, neutrophils developed robust b2-mediated adhesion to fibrino-
gen in response to the same IL-8 (Supplementary Fig. 7 online and
data not shown).
Microtubules are dispensable for CCL21-promoted motility
Microtubules are key cytoskeleton components that regulate both
polarity and directional movement. In leukocytes, microtubules
80
Shear-free
ICAM-1 LamininICAM-1 CCL21 ICAM-1
Motile
Arrested
9.2 ± 0.8
7.0 ± 0.11
**
Total cells (%) 70
60
50
40
30
20
10
0
30
25
20
15
10
5
0
Shear stress
Transient
Firm
Tether frequency (%)
30
25
20
15
10
5
0
Transient
Firm
Tether frequency (%)
30
25
20
15
10
5
0
Transient
Firm
Tether frequency (%)
80
Motile
Arrested
8.9 ± 0.67.9 ± 1.1
Total cells (%) 70
60
50
40
30
20
10
0
80
Motile
Arrested
8.9 ± 1.4
8.7 ± 1.1
Total cells (%) 70
60
50
40
30
20
10
0
CCL21–
–
+
–
+
+
β2 blockade
–
–
+
–
+
+
Initially spread
Resisting detachment
60
40
20
Initially settled cells (%)
0
CCL21
ICAM-1
Anti-β2
+
–
–
+
+
–
+
+
+
a
b
c
Figure 4 LFA-1 mediates stable adhesions in lymphocytes moving on
immobilized CCL21 and ICAM-1 under shear stress but not in shear-
free conditions. (a) Immunohistochemistry of lymph node sections
(antibodies, above images). Scale bars, 30 mm. Data are representative of
three experiments. (b) Left, fraction of T cells moving (Motile) or polarized
and arrested (Arrested) on CCL21 alone (600 sites/mm2) or with ICAM-1
(top, 320 sites/mm2; middle, 160 sites/mm2; bottom, 0 sites/mm2), among
T cells pretreated with control antibody (–) or the b2-blocking antibody
TS1.18 (+). Numbers above bars indicate the velocity of all motile T cells
(mean + s.e.m.). **, P o 0.01. Right, fraction of T cells establishing either
transient or firm adhesions when interacting under shear stress (0.5 dyn/
cm2) with substrates and blocking conditions identical to those at left. Error
bars: left, mean + s.d. of four fields; right, mean + range of two fields. Data
are one representative of three experiments. (c) T lymphocytes allowed
to spread for 5 min on CCL21 (600 sites/mm2) with or without ICAM-1
(320 sites/mm2) and then exposed for 60 s to a shear stress of 0.2 dyn/cm2.
Anti-b2, pretreatment of T cells with the b2-blocking antibody TS1.18. Data
represent the mean of three fields of view and are one representative of
three experiments.
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regulate directional motility23,24and integrin adhesiveness25. Micro-
tubule disassembly releases b2integrins from cytoskeletal constraints,
thereby increasing their avidity for ICAM-1. Given the key involve-
ment of the actin cytoskeleton in CCR7-triggered LFA-1 adhesiveness
and in lymphocyte motility over CCL21 (refs. 15,26; data not shown),
we sought to delineate the function of microtubules in CCL21-
stimulated T cell motility. Microtubule depolymerization induced by
nocodazole (Fig. 6a) did not alter CCL21-triggered lymphocyte
spreading or locomotion in shear-free conditions (Fig. 6b). However,
in conditions of shear stress, disruption of microtubule polymerization
significantly decreased the CCL21-triggered LFA-1-induced arrest of
T cells on ICAM-1 (Fig. 6c). Notably, T cell motility involved myosin
II–mediated contractility (Supplementary Fig. 8 online). These find-
ings collectively indicate that CCL21-triggered T cell motility does not
require an intact microtubule cytoskeleton; however, although integrin
independent, it still involves contractile motion.
CCL21-induced T cell locomotion over lymph node cells
Next we sought to confirm that CCL21 presented by DCs or stromal
cells expressing ICAM-1 effectively promotes in vitro T cell motility
and prevents LFA-1-mediated T cell arrest. Immature DCs, abundant
in the lymph node T cell zone, spread rapidly and extended many
protrusions when settled on immobilized CCL21 or CCL19 (data
not shown). These DCs expressed ICAM-1 (Fig. 7a) but did not
attract T cells unless they were overlaid with CCL21 (Fig. 7a,b and
Supplementary Video 9 online). Most T cells that moved toward and
over autologous DCs in response to immobilized CCL21 did so
independently of LFA-1 blocking (Fig. 7b). In addition, the small
fraction of T cells that arrested during locomotion over the DC surface
did not require functional LFA-1 (Fig. 7c). Similar to DCs, ICAM-1-
expressing human bone marrow stromal cells (Fig. 7d) supported
robust lymphocyte motility only when overlaid with CCL21 (Fig. 7d,e
and Supplementary Video 10 online). This motility and the residual
lymphocyte arrest occurred independently of LFA-1 (Fig. 7e,f). Thus,
lymphocyte motility over and arrest on ICAM-1-expressing autolo-
gous DCs and stroma triggered by CCL21 did not require LFA-1.
Lymphocyte motility in resting lymph node T cell zones
To assess the contribution of LFA-1 and VLA-4 to in vivo T cell
motility in the resting lymph node, we next compared the interstitial
motility of wild-type T cells and CD18-deficient T cells (lacking
LFA-1) adoptively transferred into wild-type or ICAM-1-deficient
recipient mice. As LFA-1 is the only CD18-containing integrin on
resting T cells, this system allowed us to assess the contribution of
LFA-1 to T cell motility patterns in the T cell zone. We assessed the
motility of transferred cells in the T cell zones of explanted recipient
lymph nodes by two-photon microscopy at 24 h after transfer
(Supplementary Video 11 online). Only a minor fraction of trans-
ferred lymphocytes (1–3%) transiently arrested during migration, and
they did so independently of CD18 (data not shown). The motile
*
abcd
Total events (%)
0
Shear-free
VCAM-1
CCL21
VLA-4 blockade
–
–
+
–
+
+
30
20
10
40
50
Motile
Arrested
80
10 ± 1.17.6 ± 0.4
60
70
Transient
Rolling
Firm
Tether frequency (%)
0
Shear stress
VCAM-1
CCL21
VLA-4 blockade
–
–
+
–
+
+
30
10
20
40
50
Total events (%)
0
Shear-free
VCAM-1 + ICAM-1
CCL21
β2 + VLA-4 blockade
β2 + VLA-4 blockade
–
–
+
–
+
+
30
20
10
40
50
Motile
Arrested
9.7 ± 2.0 9.5 ± 0.4
60
70
90
80
Transient
Firm
Tether frequency (%)
0
Shear stress
VCAM-1 + ICAM-1
CCL21–
–
+
–
+
+
30
10
20
40
50
Figure 5 VLA-4 is not adhesive in T cells migrating on CCL21 and juxtaposed integrin ligands in shear-free conditions. (a) Fraction of PBLs either moving
or polarized and arrested on CCL21 (600 sites/mm2) plus high-density VCAM-1 (320 sites/mm2) in the presence or absence of the VLA-4 inhibitor Bio1211
(1 mg/ml; VLA-4 blockade). *, P o 0.04. (b) Fraction of T cells establishing transient, rolling or firm adhesions when interacting under shear stress with
substrates and treatments identical to those in a. (c) Fraction of PBLs either moving or polarized and arrested on CCL21 (600 sites/mm2) immobilized
together with both VCAM-1 and ICAM-1 (160 sites/mm2each), among PBLs pretreated with control antibody (–) or a blocking ‘cocktail’ (Bio1211 (VLA-4
blockade) mixed with TS1.18 (b2blockade); +) and settled without washing. (d) Fraction of T cells establishing transient or firm adhesions when interacting
under shear stress with substrates and treatments identical to those in c. Numbers above bars (a,c) indicate the velocity of moving T cells (mean ± s.e.m.).
Data are the mean + s.d. of four fields (a,c) or the mean + range of two fields (b,d) and are from one representative of three experiments.
*
**
Motile lymphocytes
(% of total cells)
20
0
Nocodazole
Shear-free
7.8 ± 0.6
11.2 ± 0.9
11.0 ± 1.3
9.7 ± 3.4
ICAM-1
Nocodazole
β2 blockade
–
–
–
+
–
–
+
+
–
+
–
+
Control
a
bc
40
60
80
Tether frequency (%)
0
Shear stress
Transient
Firm
ICAM-1
Nocodazole
β2 blockade
–
–
–
+
–
–
+
+
–
+
–
+
10
20
30
Figure 6 Microtubules contribute to firm CCL21-induced LFA-1
adhesiveness under shear stress but are not required for lymphocyte
motility on CCL21 and ICAM-1. (a) Visualization of microtubules in
human PBLs pretreated for 40 min with the microtubule-depolymerizing
agent nocodazole or control reagent, then fixed and stained with anti-
tubulin. Original magnification, ?40. Images are of two representative of
60 cells of each group analyzed in two experiments. (b) Effect of nocodazole
pretreatment on the fraction and velocity of PBLs moving on ICAM-1
(320 sites/mm2) and CCL21 (600 sites/mm2). b2blockade, antibody TS1.18
(blocking LFA-1). Numbers above bars indicate the velocity of moving
T cells (mean ± s.e.m.). P ¼ 0.075, nocodazole versus vehicle control.
(c) Fraction of T cells establishing transient or firm adhesions under
shear stress upon interaction with substrates identical to those in b.
**, P o 0.003 (total tethers); *, P o 0.004 (firm tethers). Data are the
mean + s.d. of four fields (b) or the mean + range of two fields (c) and are
from one representative of three experiments (b,c).
1080VOLUME 8NUMBER 10OCTOBER 2007NATURE IMMUNOLOGY
A RT I C L E S
© 2007 Nature Publishing Group http://www.nature.com/natureimmunology