(Journal of Leukocyte Biology. 2001;69:860-866.)
© 2001
by Society for Leukocyte Biology
Novel chemokine functions in lymphocyte migration through vascular endothelium under shear flow
Guy Cinamon,
Valentin Grabovsky,
Eitan Winter,
Suzanna Franitza,
Sara Feigelson,
Revital Shamri,
Oren Dwir and
Ronen Alon
Department of Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel
Correspondence: Ronen Alon, Department of Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel. Email: ronalon{at}wicc.weizmann.ac.il
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ABSTRACT
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The recruitment of circulating leukocytes at vascular sites in target
tissue has been linked to activation of Gi-protein signaling in
leukocytes by endothelial chemokines. The mechanisms by which apical
and subendothelial chemokines regulate leukocyte adhesion to and
migration across endothelial barriers have been elusive. We recently
found that endothelial chemokines not only stimulate integrin-mediated
arrest on vascular endothelial ligands but also trigger earlier very
late antigen (VLA)-4 integrin-mediated capture (tethering) of
lymphocytes to vascular cell adhesion molecule 1 (VCAM-1)-bearing
surfaces by extremely rapid modulation of integrin clustering at
adhesive contact zones. This rapid modulation of integrin avidity
requires chemokine immobilization in juxtaposition with the VLA-4
ligand VCAM-1. We also observed that endothelial-bound chemokines
promote massive lymphocyte transendothelial migration (TEM). It is
interesting that chemokine-promoted lymphocyte TEM requires continuous
exposure of lymphocytes but not of the endothelial barrier to fluid
shear. It is noteworthy that lymphocyte stimulation by soluble
chemokines did not promote lymphocyte TEM. Our results suggest new
roles for apical endothelial chemokines both in triggering lymphocyte
capture to the endothelial surface and in driving post-arrest events
that promote lymphocyte transmigration across endothelial barriers
under shear flow.
Key Words: inflammation • trafficking • integrins • G proteins
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INTRODUCTION
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Leukocyte recruitment at inflamed or lymphoid tissues is mediated
by sequential adhesive interactions between multiple vascular receptors
and their endothelial counter-ligands [1
2
3
]. This
adhesive cascade allows the circulating immune cell to arrest on the
vessel wall at the site of emigration to the target tissue. Specific
stimulatory signals are presented to the recruited leukocyte and
up-regulate in situ its integrin avidity to the ligand, enabling the
cell to generate shear-resistant adhesion to the vessel wall.
Chemoattractive cytokines, primarily chemokines, and their
Gi-protein-coupled receptors (GPCRs) on leukocytes, are
critically involved in triggering leukocyte-integrin avidity at
endothelial adhesive zones to vascular-endothelial-integrin ligands.
This has been demonstrated over the past decade in numerous in vivo and
in vitro studies [4
5
6
7
8
9
10
11
]. These studies have
conclusively shown that integrin-mediated arrest of hematopoietic cells
on their target endothelium is effectively suppressed by blocking the
Gi-protein pathway of the circulating cells. Subsequent to arrest on
endothelium, leukocytes undergo transendothelial migration, a
nonproteolytic process in which adherent blood-borne cells cross the
endothelial lining of the vessel wall, usually through intercellular
borders at or near their original site of recruitment on the vascular
endothelium [12
13
14
]. Endothelium-associated chemokines
might participate in this process, by continuously signaling to the
arrested leukocytes to trigger promigratory actin-remodeling events
[15
]. The cytoskeletal reorganization should allow the
arrested leukocyte to move from the initial recruitment site, undergo
morphological changes, initiate its TEM across the endothelial layer,
and complete diapedesis—all while maintaining resistance to
shear-induced detachment from the vessel wall.
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IMMOBILIZED ENDOTHELIAL CHEMOKINES INDUCE INTEGRIN CLUSTERING AND
AVIDITY DURING SUBSECOND CONTACTS THROUGH Gi-PROTEIN ACTIVATION
ON TETHERED LEUKOCYTES
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Several potent lymphocyte chemokines, functionally displayed on
cytokine-activated human umbilical-vein endothelial cells (HUVECs), can
instantaneously augment VLA-4 integrin-dependent adhesion of
lymphocytes and CD34+ hematopoietic progenitor cells
captured from the blood flow, resulting in firm arrest of these cells
[11
16 ]. This ability of lymphocytes and hematopoietic
progenitor cells to develop shear-resistant adhesion to vascular cell
adhesion molecule 1 (VCAM-1)-bearing surfaces has been recently linked
by us to rapid potentiation of VLA-4 avidity at transient
leukocyte-substrate contact zones containing immobilized chemokines
[11
]. Chemokine potentiation of VLA-4 avidity is
facilitated by VLA-4 clustering triggered by a Gi-protein-signaling
event that occurs within as little as <0.1 s, the fastest
inside-out integrin-signaling event reported to date. It is interesting
that saturating levels of soluble chemokines failed to trigger VLA-4
avidity to VCAM-1 under shear flow, suggesting that localized rather
than global signals must be transmitted from occupied GPCRs to trigger
VLA-4 clustering at adhesive zones (Fig. 1
). Thus, chemokines need to be presented in juxtaposition to the
endothelial ligand within the adhesive contact zone to rapidly
transduce VLA-4 clustering within the zone [11
]. It is
therefore likely that endothelium-displayed chemokines, unlike soluble
chemokines, trigger VLA-4 adhesion at dynamic endothelial sites by
rapidly increasing the effective mobility and clustering properties of
the integrin within local VCAM-1-containing contact sites (Fig. 1)
.
VLA-4 clustering induced within subseconds of contact with a ligand
results in enhanced lymphocyte tethering to and rolling on
VCAM-1-bearing surfaces under physiological shear flow
[11
]. This is the first demonstration that endothelial
chemokines may function at an earlier stage than was previously
realized in augmenting reversible leukocyte interactions with vascular
endothelium before cell arrest on the vessel wall.
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Figure 1. Model for subsecond stimulation of VLA-4 clustering at adhesive
contacts by chemokine binding to integrin-associated GPCRs. Fast
changes in integrin clustering at short-lived adhesive contact zones
must occur to convert nonfunctional binding events into functional
tethers. An initial VLA-4–VCAM-1 bond that is too weak to support a
functional tether (1) is followed by a local chemokine signal
transmitted through the chemokine GPCR (2). The G-protein signal
triggered by the GPCR is transmitted directly to the cytoplasmic tail
of a neighboring VLA-4 molecule or to an effector target near this
second VLA-4 (3). As a result, the mobility of the neighboring VLA-4 is
increased such that it can cluster at the site of initial bond
formation (4). A preformed GPCR–VLA-4 complex is probably stabilized
within a raft domain on the tip of a leukocyte
microvillus._art>
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This fast coupling may depend on segregation of the GPCR and its target
integrin within preformed supramolecular structures, possibly localized
on leukocyte-surface microvilli (Fig. 1)
—preferential sites
of leukocyte-endothelial contacts under shear flow [17
,
18
]. VLA-4 as well as other lymphocyte integrins, like
lymphocyte-function-associated antigen 1 (LFA-1) and 
4ß7, may be
segregated at these or other surface locations, within specific lipid
raft microdomains. Such domains are enriched with
heterotrimeric G proteins, integrin-associated tetraspannins, and Src
kinases, all putative modulators of integrin adhesiveness at dynamic
contacts [19
20
21
22
23
].
Because VLA-4 and LFA-1 are topographically segregated on the leukocyte
surface, it is likely that each integrin is preferentially associated
with a different subset of GPCRs capable of inducing its clustering.
Consistent with this notion, chemokines can trigger reversible
subsecond VLA-4 tethers to VCAM-1, which support lymphocyte capture and
subsequent rolling interactions on VCAM-1-bearing surfaces, whereas
chemokine-stimulated LFA-1 adhesion to intercellular-adhesion-molecule
1 (ICAM-1) results in irreversible adhesion and firm arrest
[7
]. Chemokine-triggered arrest is linked to
augmentation of both LFA-1 clustering and binding affinity to ICAM-1
[10
]. An interesting possibility is that the ability of
VLA-4 to arrest lymphocytes on VCAM-1 might also be caused by
high-affinity states preexistent on subsets of circulating lymphocytes
[24
]. However, VLA-4 affinity is internally controlled
by the lymphocyte activation state [25
] and is not
modulated by chemokine [11
]. Thus, to promote reversible
leukocyte rolling, chemokine stimulation of VLA-4 should promote
integrin adhesiveness without inducing high affinity to ligands.
Chemokine stimulation of VLA-4-mediated rolling is consistent with the
previously reported functional hierarchy of these integrins, indicating
that VLA-4 adhesion precedes LFA-1 adhesion strengthening in various
multistep, adhesive cascades of leukocytes on endothelial surfaces
[26
]. VLA-4 and LFA-1 are also differentially modulated
by soluble chemokine signals. Whereas LFA-1 affinity and clustering can
be triggered by soluble chemokines with similar efficiency to
immobilized chemokines [10
], VLA-4 fails to respond to
such chemokines under physiological shear flow [11
].
In another interesting finding, we demonstrated that subsecond
signaling by chemokines does not involve Ca2+ mobilization
from intracellular stores, phosphatidylinositol 3 (PI3)-kinase
activity, or PTK activation [11
]. Consistent
with our results, chemokine-stimulation of LFA-1 affinity was also
PI3-kinase independent and did not implicate a rise in intracellular
free Ca2+, although the kinase was instrumental in LFA-1
mobility within the membrane and its polar patching on lymphocytes
arrested on ICAM-1-bearing surfaces [10
]. These
relatively slow processes (>10 s) appeared therefore to contribute to
LFA-1-mediated adhesion strengthening at a post-tethering level
[10
]. Thus, none of the classical effector systems
triggered by chemokine signaling through GPCRs were necessary to
promote rapid endothelial adhesion of lymphocytes mediated by either
VLA-4 or LFA-1. Although Gi-protein activation of VLA-4 and LFA-1
adhesiveness has been linked to rapid stimulation of the guanosine
triphosphatase RhoA [27
], the role of this or other
Rho-like guanosine triphosphatases in subsecond chemokine-induced VLA-4
clustering or in rapidly triggered LFA-1 affinity has not been
demonstrated. It is possible that these rapid modulations of integrin
activity are controlled by GPCR signaling to a subset of Rho,
constitutively associated with preformed membranal-integrin–GPCR
complexes. We raise this hypothesis because Rho is a cytoplasmic
protein [27
], and therefore its recruitment to the
membrane would be too slow to account for its putative role in
triggering integrin avidity at subsecond adhesive zones.
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ANALYSIS OF LEUKOCYTE TEM UNDER SHEAR FLOW BY AN ALTERNATIVE
EXPERIMENTAL SYSTEM TO TRANSWELL CHEMOTAXIS CHAMBERS
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In addition to stimulating integrin avidity to endothelial
ligands, chemokines control leukocyte motility and microenvironmental
localization within extravascular target tissues [28-31]. Chemotactic
migration triggered by chemokines may predominate in extravascular
tissues. Indeed, gradients of soluble chemokines introduced at basal
compartments of endothelial cells (ECs) can direct leukocyte migration
across endothelial barriers as well as extracellular matrices in vitro
[32
33
34
]. However, at present there is no in vivo
evidence that putative chemotactic gradients across vascular
endothelium play any physiological role in directing leukocyte
transendothelial migration under vascular shear flow [32
,
35
]. Transendothelial chemotaxis has been traditionally
studied in transwell Boyden chamber assays in the absence of shear
flow. The lack of a physiological mechanical context and the prolonged
time scales of transwell assays provide a poor model for physiological
leukocyte transendothelial migration (TEM). Also unclear is whether
leukocytes adherent to vascular endothelium under shear flow can sense
and respond to sublumenal tissue chemokines within the short time frame
of their contact with the endothelium, translating these putative
signals to efficient diapedesis towards the chemokine source. The
notion that apical chemokines are critically involved in regulating
leukocyte adhesion to vascular endothelial ligands allowed us to raise
the intriguing possibility that apical chemokines could in fact signal
promigratory stimuli to adherent leukocytes even in the absence of
chemokine gradients, after leukocyte arrest on the endothelial-cell
surface.
Leukocytes generally move from their original site of arrest on the
endothelial surface to their site of diapedesis [36
].
Even if previously arrested at sites of diapedesis, the adherent
leukocyte must reorient its cell body and leading edge towards and
within the endothelial-migration zone. Because shear flow exerts
continuous disruptive forces on arrested leukocytes, these adhesive
processes must be carefully balanced to maintain migrating leukocyte
attachment to the vessel wall. Yet, at the same time, vascular
adherence must not exceed a level that would restrain leukocyte
spreading and locomotion to the site of diapedesis. Multiple
proadhesive and motility signals must therefore be transmitted in a
highly coordinated manner to the migrating leukocyte along its vascular
migratory path. The molecular basis of leukocyte spreading, locomotion,
and positioning at these endothelial sites of diapedesis under
continuous disruptive shear flow has been obscure. As a multistep
process, leukocyte TEM has been difficult to dissect in vivo or in
vitro under physiological shear-flow settings. The existence of
vascular endothelial models that lack endogenous chemokines for T
lymphocytes and can be reconstituted with specific chemokines
[16
] enabled us to use these models to dissect the
molecular basis of lymphocyte migration across vascular endothelium
under physiological shear flow. We used cytokine-activated endothelial
monolayers reconstituted with chemokines to dissect the steps by which
apical chemokine presentation promotes lymphocyte migration on and
across inflamed endothelium. The experimental set up illustrated in
Figure 2A
is based on individual cell tracking of freshly isolated
peripheral blood lymphocytes (PBLs) accumulated on a monolayer
of cytokine-activated primary ECs [tumor necrosis factor
(TNF)-activated HUVECs] on which a chemokine of interest has been
overlaid. Both morphological changes and migratory properties of the
leukocytes adhering to the endothelial monolayers are monitored at a
single-cell level by phase-contrast videomicroscopy
[16
]. Analysis of leukocyte motion and shape is
conducted over a time scale of minutes, corresponding to in vivo
diapedesis processes of leukocytes extravasating at postcapillary
venules of lymphoid organs or inflamed peripheral tissues
[37
].
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Figure 2. Analysis of leukocyte TEM under physiological shear flow in a
parallel-plate flow chamber assay. The parallel-plate flow chamber
assay allowed us to resolve the separate steps in the migratory cascade
of leukocytes accumulating on an endothelial monolayer under shear flow
in the absence or presence of exogenous chemokine overlaid on the
apical surface of the monolayer. The indicated steps (i through vi)
were monitored by high-power videomicroscopy of all cells from their
initial accumulation in the field of view at low physiological flow (i)
until final diapedesis (vi): (i) rolling and arrest; (ii) detachment
from original site; (iii) spreading; (iv) firm stationary adhesion; (v)
locomotion over the ECs; (vi) transmigration through the ECs. The time
course of various parts of the assay, i.e., accumulation phase
and migration phase, are indicated. Below (B) are frames taken from
time-lapse digitized video recordings of lymphocytes adhered on
TNF--activated HUVEC monolayers overlaid with SDF-1 (100 ng/mL) and
subjected to continuous shear stress of 5 dyn/cm2 for the
indicated period. Lymphocytes are numbered in a counterclockwise
manner, and numbers designate their central positions at
t = 0'. The paths traveled by each lymphocyte from
original point of arrest (t = 0') to final position
underneath or over the EC monolayer (t = 6') are
depicted with dashed lines and arrowheads. At t = 6',
lymphocytes 4–6 had completed TEM, whereas lymphocytes 1–3 moved
variable distances over the endothelial surface.
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APICAL CHEMOKINES PROMOTE LYMPHOCYTE LOCOMOTION TO SITES OF
TRANSMIGRATION AND TRIGGER ROBUST TRANSENDOTHELIAL MIGRATION UNDER
SHEAR FLOW
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Stromal cell-derived factor (SDF)-1 overlaid on
cytokine-activated HUVECs not only triggers firm integrin-dependent
adhesion of PBLs but, within 1 min after recruitment, stimulates
massive spreading of almost all arrested lymphocytes (Fig. 2)
. The
spread lymphocytes then begin to slowly migrate on the endothelial
surface (Fig. 2B)
. Neither cell spreading nor locomotion are directed
with the direction of flow (Fig. 2B
, lymphocytes 4–6), and almost all
recruited lymphocytes move some distance from their original sites of
arrest before initiating TEM (Fig. 2B)
. Within 2–3 min of locomotion
on the endothelial surface, lymphocytes are observed to migrate
underneath the endothelial monolayer without returning to the apical
endothelial surface (Fig. 2
and Fig. 3
). Under optimal conditions of chemokine stimulation, about 60% of
originally accumulated PBLs can transmigrate through the ECs during the
first 20-min period (Fig. 3)
. Similar observations are obtained with
PBLs transmigrating through HUVECs overlaid with another potent T-cell
chemokine, EBI-1 molecular ligand chemokine
[38
]. The average time required for a
lymphocyte positioned at its site of emigration to complete the
transendothelial passage is 1.5 ± 0.5 min. Although
transmigrating lymphocytes migrate variable distances on ECs before
initiating TEM, there is no correlation between the distance traveled
by moving lymphocytes and their ability to undergo TEM. Chemokine
signaling through GPCRs on the adherent lymphocytes is essential for
both initial PBL arrest on the substrate and subsequent locomotion and
transmigration, since pertussis toxin pretreatment of lymphocytes
completely eliminates chemokine-triggered adhesion, locomotion, and
transmigration across the endothelial barrier (Fig. 3
, third
treatment). It is noteworthy that lymphocyte exposure to soluble
chemokines at doses that activate GPCR signaling is insufficient to
promote TEM [38
]. Furthermore, triggering of TEM by
apical SDF-1 requires a threshold density higher than that required for
triggering VLA-4-dependent arrest of lymphocytes on the endothelial
surface [38
]. Thus, the ability of endothelial
chemokines to promote lymphocyte TEM appears to depend on proper
solid-state presentation to the migrating lymphocyte at the apical
endothelial surface. This restricted requirement of chemokine
presentation for both induction of integrin clustering and lymphocyte
TEM suggests that locally presented chemokine signals may increase the
effective mobility of integrins, as well as other vascular receptors,
on migrating lymphocytes, which can facilitate their movement over and
through endothelial sites.
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Figure 3. Apical endothelial chemokines promote lymphocyte TEM under
physiological flow conditions. PBLs accumulated for 40 s at 0.75
dyn/cm2 on TNF--stimulated (24 h, 2 ng/mL) HUVECs alone
or previously overlaid with SDF-1 (100 ng, 5 min) were subjected to
physiological shear stress (5 dyn/cm2) for 20 min. Cells
which remained bound to the ECs during the entire assay (cells per
field) were divided into three categories: arrested, "locomoting"
(without transmigration), and transmigrating (TEM; see Fig. 2
). For
inhibition of Gi-protein signaling, lymphocytes were incubated with
pertussis toxin. The fractions of PBLs of the total number of
accumulated lymphocytes that remained bound to the ECs during the
entire assay are indicated on top of each set of bars. The percentages
of TEM lymphocytes of initially accumulated lymphocytes are expressed
on top of the filled bars. Results shown are the means ±
SE of multiple independent experiments, i.e., using
different donors and EC preparations. * and ** stand for
P < 0.02 and 0.0001, respectively, compared with PBL
TEM across HUVECs without adsorbed SDF-1.
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These results are the first demonstration that prototypic lymphocyte
chemokines displayed to adherent lymphocytes on the apical endothelial
surface can trigger robust transendothelial migration of lymphocytes to
extravascular spaces. We are intrigued that this mode of migration
occurs despite the minute levels of spontaneous T-cell-stimulatory
signals within this endothelial model (Fig. 3
, first treatment). These
results thus indicate an essential role for apical chemokines in
driving lymphocyte transmigration across endothelial barriers. Indeed,
in this experimental setting, lymphocytes cross the ECs away from a
lumenal endothelial surface, which displays high-density chemokines,
towards a subendothelial compartment devoid of the chemokine, i.e., in
a direction opposite to the chemokine gradient. Taxis away from
high-concentration chemokine depot (chemofugetaxis) has been
described in shear-free systems [39
]. However, in our
model of lymphocyte TEM, T cells failed to migrate from the apical
(chemokine-rich) endothelial compartment to the sublumenal
(chemokine-poor) compartment in the absence of shear flow (Fig. 4
). Furthermore, leukocyte migration driven by chemofugetaxis is
sensitive to inhibition of PI3-K signaling, whereas lymphocyte TEM
under shear flow is not [38
]. It thus appears that the
chemokine-triggered lymphocyte TEM observed by us is not a repellent
chemokine-dependent migratory mechanism such as fugetaxis.
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Figure 4. Shear stress applied on adherent lymphocytes promotes TEM triggered by
apical chemokine. Lymphocytes accumulated for 40 s at 0.75
dyn/cm2 on SDF-1 overlaid TNF-activated HUVECs were
subjected for 15 min to shear stress of 5 dyn/cm2 or were
left in shear-free conditions. The fractions of transmigrating
lymphocytes of the originally accumulated lymphocyte population were
determined at the indicated time points. Results are the means ±
SE of five independent experiments. *, **, ***:
P < 0.05, 0.005, and 0.001 respectively, compared with
PBL TEM across HUVECs in the absence of shear flow.
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CONTINUOUSLY APPLIED WALL SHEAR FORCES PROMOTE CHEMOKINE-TRIGGERED
LYMPHOCYTE TEM
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Our recent results suggest that shear flow applied on lymphocytes
migrating through chemokine-bound activated ECs is not merely
permissive for their TEM but is in fact required for TEM initiation and
progression. When T cells are left on chemokine-bound ECs in the
absence of applied shear stress, minute levels of TEM are observed
(Fig. 4) . This shear dependence of lymphocyte TEM is irrespective of
the activation or differentiation properties of HUVEC preparation
[38
]. It is interesting that application of shear flow
on the chemokine-bound ECs shortly before lymphocyte accumulation
followed by stoppage of flow does not promote lymphocyte TEM.
Furthermore, lymphocytes in the process of transmigration through
SDF-1-bound HUVECs under continuous shear flow rapidly stop migrating
when shear flow is halted [38
]. Thus, to optimally
promote lymphocyte TEM, shear stress must be continuously applied on
the adherent lymphocyte at its apical endothelial contact with the
adsorbed chemokine. It thus appears that external forces provided by
applied shear stresses may trigger mechanoresponsive elements on
migratory lymphocytes, which would combine with the localized
biochemical signals transduced to the migrating cells by apical or
junctional endothelial chemokines. Subsequent chemokine signals
encountered by the extravasating lymphocytes are likely to facilitate
its migration through the extracellular matrix (ECM) and
navigation to its final target within the tissue (Fig. 5
).
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Figure 5. Leukocyte transmigration across endothelial and stromal barriers
requires distinct distribution patterns of chemokines. Under shear flow
conditions, leukocyte adhesion to the vascular endothelial barrier is
critically dependent on stimulation of leukocyte integrins by apical
endothelial chemokine(s) (open circles). These chemokines
alone or in conjunction with subendothelial chemokines (gray circles)
also promote rapid TEM under continuous-shear-flow conditions. In the
absence of shear flow, e.g., like in transstromal migration, leukocytes
may cross the stromal barrier only in response to a basal chemokine
(alone or when two distinct chemokines are sequentially displayed to
the migrating leukocyte on the apical and basal surfaces of the
barrier). Stimulation of shear-resistant leukocyte adhesion by apical
chemokines is not required in transstromal migration, in contrast to
its requirement in transendothelial migration. Although
leukocyte TEM is shown to take place through intercellular borders,
particular subsets of leukocytes may cross the endothelial lining by
traversing the venular endothelium through a transcellular route
[50
]. Relative hypothesized rates of lymphocyte
transmigration under these different experimental conditions are
summarized in the table below. EC, endothelial cells; ECM,
extracellular matrix; ST, stromal cells.
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POSTULATED MECHANISMS UNDERLYING NAVIGATION OF EXTRAVASATING
LYMPHOCYTES TO TARGET TISSUES: REGULATION BY SEQUENTIAL CHEMOKINE
SIGNALS COUPLED TO MECHANICAL SIGNALS OF SHEAR FLOW
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Our findings of the key role played by apical endothelial
chemokines in lymphocyte TEM may be extended to other leukocyte and EC
systems. Similar functions of immobilized chemoattractants in promoting
leukocyte TEM under physiological shear flow can be carried out with
platelet-activating factor and interleukin (IL)-8 (e.g.,
neutrophil TEM [40–42]) as well as by IP-10 (e.g., TEM of
IL-2-activated lymphoblasts) [43
, 44
]. Our
results lead us to predict that the threshold levels of chemokine
signals sufficient to trigger integrin-dependent arrest on endothelial
surfaces under flow are lower than those required to promote TEM of the
same cells through identical endothelial surfaces [38
].
Presentation of the chemokine on distinct regions of the EC lining
(e.g., apical, junctional) may provide such functional
specialization among serially triggered GPCRs on the migrating
leukocytes. Reminiscent of these findings, peripheral-blood monocytes
have been recently reported to use distinct chemokine interactions to
trigger integrin-mediated arrest, subsequent spreading, or
transendothelial migration across inflamed endothelium under shear flow
[15
]. Hierarchical involvement of particular
chemokine-GPCRs has been suggested to prevail in distinct steps of the
migratory cascade of lymphocyte subsets on and across endothelial
barriers. In this respect, recent studies on extravascular-migration
model systems predict that leukocytes can integrate multiple
chemotactic signals and can navigate in a stepwise manner through
chemoattractant arrays [28
, 45
]. The
presence of several apical chemokines or the combination of several
apical and basal chemokines may be similarly necessary to promote
successful emigration through both simultaneous and sequential
engagement of respective GPCRs coexpressed on individual migrating
cells (Fig. 5)
. The in vitro experimental approach developed by us,
combined with controlled monoclonal-antibody-blocking studies performed
on different endothelial systems, should enable testing of how the
order of chemokine presentation to defined subsets of migrating
leukocytes controls the extent and rate of their TEM across various
types of vascular endothelia.
Chemokines are cationic proteins, which bind to heparan sulfate and
related glycosaminoglycan moieties of endothelial or matrix
proteoglycans [46
, 47
]. The idea that
juxtacrine signaling by immobilized chemoattractants may regulate
adhesion and migration was first demonstrated for neutrophils
interacting with inflamed endothelium [40
]. Haptotaxis
was suggested as an alternative migratory mechanism to chemotaxis by
which cells move to a region of highest adhesiveness
[48
]. Our results strongly suggest that immobilized
chemokines presented on adhesive endothelial surfaces act as
haptotactic and juxtacrine regulatory elements in lymphocytes and
possibly other leukocytes migrating on and through endothelial
surfaces. An attractive possibility is that these haptotactic functions
of chemokines are not restricted to endothelial surfaces and might
contribute to leukocyte motility through the ECM barriers underlying
the vessel wall. Although haptotactic migration has not been
demonstrated in vivo, recent observations suggest that particular
chemokines can drive directional lymphocyte migration across ECMs, even
when uniformly presented in immobilized states on these matrices
[49
]. Thus, sublumenal chemokines deposited at the site
of leukocyte extravasation (Fig. 5)
may provide haptotactic and
chemotactic signals that up-regulate both leukocyte adhesion and
motility through the underlying basal lamina. Subsequently, gradients
of locally secreted chemokines may navigate the migrating leukocyte
through the ECM to its final destination in a chemotactic manner
[45
]. In conclusion, our studies suggest that
chemokines, commonly suggested to act as mere chemoattractants
[31
] might transmit both proadhesive and promigratory
signals to recruited lymphocytes in a nonchemotactic manner (i.e.,
independently of a gradient and not implicating the classical
Gi-protein-triggered effector systems) [11
,
38
]. Taken together, these new notions and the novel
mechanoregulatory role we find for wall shear forces in promoting
chemokine-triggered lymphocyte migration suggest that chemokine
regulation of leukocyte trafficking is much more versatile than was
previously realized.
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ACKNOWLEDGEMENTS
|
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R. Alon is the incumbent of the Tauro Career Development Chair in
Biomedical Research. Parts of this work were supported by the Israel
Science Foundation, the Minnerva Foundation, Germany, and the Abisch
Frenkel Foundation. We thank Dr. S. Shwarzbaum for editorial
assistance.
Received December 30, 2000;
revised April 4, 2001;
accepted April 5, 2001.
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ABSTRACT
INTRODUCTION
