Interactive protrusive structures during leukocyte adhesion and transendothelial migration.

Abstract

Leukocyte transendothelial migration during homing and inflammation requires drastic cell morphological changes, involving cytoskeletal-directed clustering of adhesion receptors in specialized protrusive membrane structures in leukocytes and endothelial cells. Extravasation is an active process not only for leukocytes but also for endothelial cells, which promote the rapid and efficient entry of leukocytes to the target tissues, without disturbing the integrity of the endothelial barrier. Herein, we have revised the specialized protrusive structures (microvilli, endothelial docking structures, leukocyte lamellipodia and uropod) involved in the different stages of leukocyte extravasation. The adhesion receptor redistribution, cytoskeletal remodelling and intracellular signaling events that participate in this phenomenon are also discussed.

Full text

Page 1

[Frontiers in Bioscience 9, 1849-1863, May 1, 2004]
1849
INTERACTIVE PROTRUSIVE STRUCTURES DURING LEUKOCYTE ADHESION AND TRANSENDOTHELIAL
MIGRATION
Olga Barreiro, Miguel Vicente-Manzanares, Ana Urzainqui, María Yáñez-Mó, and Francisco Sánchez-Madrid
Servicio de Inmunología, Hospital de la Princesa, Universidad Autónoma de Madrid, 28006 Madrid, Spain
TABLE OF CONTENTS
1. Abstract
2. Introduction
3. Initial interactions between circulating leukocytes and the endothelium: tethering and rolling
3.1. Selectins and their ligands as critical mediators of tethering and rolling
3.2. Membrane topography of adhesion molecules involved in rolling
3.3. Selectins and PSGL-1 as signaling receptors
4. Activation, arrest and firm adhesion of leukocytes
4.1. Chemokines trigger the arrest of leukocytes on endothelium
4.2. Integrin affinity and avidity changes as leukocyte strategies to achieve firm attachment and spreading
4.3. The docking structure: the endothelial contribution to the leukocyte firm adhesion process
4.3.1. VCAM-1 and ICAM-1 play an essential role in leukocyte capture
4.3.2. Structural components and signaling pathways involved in the generation and maintenance of the
docking structure
4.3.3. VCAM-1 and ICAM-1 outside-in signaling
5. Transendothelial migration
5.1. Leukocytes undergo drastic cytoskeletal rearrangements to extravasate across the endothelial barrier
5.2. Endothelial cell lateral junctions regulation
5.2.1. Tight junction proteins: role of JAM family in transendothelial migration
5.2.2. Adherens junction disappearance at leukocyte contacts: proteolysis or displacement?
5.2.3. Other molecules with a major role in the passage of leukocytes across endothelium
6. Concluding remarks and perspectives
7. Acknowledgements
8. References
1. ABSTRACT
Leukocyte transendothelial migration during
and inflammation requires
morphological changes, involving cytoskeletal-directed
clustering of adhesion receptors in specialized protrusive
membrane structures in leukocytes and endothelial cells.
Extravasation is an active process not only for leukocytes
but also for endothelial cells, which promote the rapid and
efficient entry of leukocytes to the target tissues, without
disturbing the integrity of the endothelial barrier. Herein,
we have revised the specialized protrusive structures
(microvilli, endothelial docking structures, leukocyte
lamellipodia and uropod) involved in the different stages of
leukocyte extravasation.
redistribution, cytoskeletal remodelling and intracellular
signaling events that participate in this phenomenon are
also discussed.
homing drastic cell
The adhesion receptor
2. INTRODUCTION
Cell adhesion receptors regulate many cellular
processes such as activation,
differentiation and death (1, 2), by both signal transduction
and the modulation of intracellular signaling cascades
triggered by different growth factors (3). Cellular
migration, growth,
interactions are critical for regulation of hematopoiesis (4,
5) and inflammatory responses (6, 7). The coordinate
function of adhesion receptors, cytoskeleton and signaling
molecules is crucial for leukocyte extravasation, a central
process in immunity. Hence, the correct integration of
“outside-in” and “inside-out” signals in leukocytes and
endothelium during each stage of extravasation is critical to
allow the completion of this phenomenon, the so-called
“multi-step paradigm” (6, 8). The present review focuses
on the molecular mechanisms and the specialized
protrusive structures that govern this dynamic process in
both leukocytes and endothelial cells.
3.
CIRCULATING
ENDOTHELIUM: TETHERING AND ROLLING
INITIAL INTERACTIONS
LEUKOCYTES
BETWEEN
AND THE
Free-flowing leukocytes contact with and adhere
to the vascular wall under shear forces to initiate an
inflammatory response or to migrate into a secondary
lymphoid organ (homing). Leukocyte tethering and rolling
on activated endothelial cells are the first steps of the
sequential process of extravasation, followed by the firm
adhesion and transendothelial migration of leukocytes (9).

Page 2

Protrusive structures in leukocyte extravasation
1850
Figure 1. The first step of the extravasation process. As shown in the diagram, free-flowing leukocytes establish transient
contacts with activated endothelial cells (tethering), being slowed down. These initial contacts allow leukocytes to roll on the
endothelial wall to become activated and finally arrest. The main molecules that participate during this process are presented in
detail in the inset: E- and P-selectin in endothelium as well as L-selectin and PSGL-1 in leukocytes are localized at specialized
protrusions (microvilli) supported by the actin cytoskeleton, which is linked to the adhesion receptors via ERM proteins, alpha-
actinin and other cytoskeletal components. All these molecules interact with their corresponding receptors through carbohydrate
residues such as Sialyl Lewisx. Thus, L-selectin binds to several endothelial counterreceptors (E-selectin among them) and
leukocyte PSGL-1 interacts with endothelial E- and P-selectin. In addition, The binding of PSGL-1 with its ligands allows the
recruitment of the tyrosine kinase Syk via ERM proteins, which triggers signaling cascades that culminate in the activation of
expression of several genes (e.g., SRE and c-fos) in leukocytes.
These initial contacts are largely mediated by selectins and
their ligands. All the issues addressed in this chapter are
schematically depicted in Figure 1.
3.1. Selectins and their ligands as critical mediators of
tethering and rolling
Selectins (P-, E- and L-selectin) are cell adhesion
molecules that predominantly
interactions of leukocytes with endothelium. They are type
I transmembrane glycoproteins that bind to sialylated
carbohydrate moieties present on ligand molecules in a
calcium-dependent manner. Selectins and their ligands
interact with variable affinity, and due to their rapid
association and dissociation rates mediate transient contacts
between leukocytes and endothelium (“tethering”) (10, 11).
Tethering results in the slowing of leukocytes in the
bloodstream and their rolling on the surface of
endothelium, which favors subsequent interactions with
endothelial cells mediated by integrins and their ligands,
mediate the initial
increasing the adhesiveness of leukocytes, that leads to
their final arrest on the vessel wall (12).
P-selectin, which is constitutively expressed by
platelets and endothelial cells in secretory granules, is
translocated to the cell surface within minutes upon cell
activation (13). Once expressed on the surface of
endothelial cells, P-selectin is rapidly internalized by
endocytosis. Therefore, this adhesion receptor has an
important role in early leukocyte recruitment during
inflammation (14). E-selectin is also expressed by
endothelial cells, but it is synthesized de novo upon cell
activation (15). After stimulation with IL-1 or TNF-alpha,
it is maximally expressed on the membrane at 4h, and then
is slowly internalized and degraded (16). L-selectin is
constitutively present in most leukocytes, but it is rapidly
shed upon cell activation, thus facilitating the progression
to adhesion and transendothelial migration (TEM) (17-19).
It was first described as a lymphocyte homing receptor, but

Page 3

Protrusive structures in leukocyte extravasation
1851
it also participates in leukocyte recruitment at later stages
of the inflammatory response (20).
The best characterized selectin ligand is PSGL-1
(P-selectin glycoprotein ligand-1),
sialomucin expressed by almost all leukocytes (21) and
platelets (22). PSGL-1 is an important functional ligand in
vivo for all three selectins (23-26). In addition, CD24 and
ESL-1 (E-selectin ligand-1) seem to be ligands in myeloid
cells for P-selectin and E-selectin, respectively (27-29).
Finally, it has been described that E-selectin, GlyCAM-1,
MAdCAM-1, CD34 and Sgp200 specifically interact with
L-selectin (reviewed in 30).
a homodimeric
Although selectins and their ligands are the
primary mediators of leukocyte rolling, alternative cell
adhesion pathways are involved in this phenomenon. It has
been described that alpha4beta1 (VLA-4) and alpha4beta7
integrins can also mediate leukocyte tethering, rolling and
arrest through their interaction with VCAM-1 and
MAdCAM-1 in the absence of selectins (31). On the other
hand, the interaction of LFA-1(alphaLbeta2)/ICAM-1
cooperates with L-selectin in leukocyte rolling by
stabilizing the tethering phase and decreasing the rolling
velocity (32, 33). In addition, the chemokines CX3CL1
(fractalkine) and CXCL16 also mediate both rolling and
firm adhesion by interacting with CX3CR1 and CXCR6,
respectively (34-37).
3.2. Membrane topography of adhesion molecules
involved in rolling
Adhesion receptor distribution on cell membrane
has a key role in leukocyte interactions and is an important
regulatory mechanism for leukocyte trafficking (38).
Selectins are clustered at the tips of microvilli, and this
localization is critical for tethering and rolling.
L-selectin is anchored to the actin cytoskeleton
through the constitutive association of its cytoplasmic tail
to alpha-actinin, and its cell activation-dependent binding
to moesin (39, 40). Although the association with alpha-
actinin is not essential for its targeting to microvilli (39), its
cytoplasmic anchorage to the actin cytoskeleton is
necessary to control L-selectin function (41, 42). PSGL-1 is
also localized at the tips of microvilli, and this subcellular
distribution has been found to be important for the
initiation of tethering and rolling of leukocytes (43, 44).
Furthermore, the capability of alpha4, but not beta2
integrins, to initiate leukocyte adhesion under flow is also
explained by its selective topographic localization at
microvilli (30). Finally, less is known about the
involvement of cytoskeleton in the efficient presentation in
microvilli of E- and P-selectin by endothelial cells.
However, it has been described that leukocyte adhesion
induces E-selectin linkage to the actin cytoskeleton through
alpha-actinin, paxillin, vinculin, and FAK, but not talin
(45).
3.3. Selectins and PSGL-1 as signaling receptors
It has been demonstrated that L-selectin activates
multiple signaling pathways involved in the reorganization
of the actin cytoskeleton, such as the MAPK cascade (46),
the tyrosine kinase p56lck and Ras (47) or the Rho GTPase
Rac2 (48). In this regard, it has been described that
neutrophils from Rac2-/- mice show deficient actin
polymerization and L-selectin-mediated rolling (49). On
the other hand, PSGL-1 activates the MAPK pathway (50),
and acts as a negative regulator of human hematopoietic
progenitor cells (5). In addition, it has been demonstrated
that PSGL-1 induces a rapid synthesis of uPAR and
different cytokines such as TNF-alpha, IL-8 and MCP-1 in
neutrophils, monocytes and T cells (51-54). Moreover, it
has been shown that PSGL-1 induces activation of beta-2
integrins and binding to ICAM-1 in neutrophils (55, 56).
Our group has also described the interaction of PSGL-1
with ERM proteins, which link membrane molecules with
the actin cytoskeleton (57, 58). This interaction is of critical
importance for the leukocyte activation that occurs before
extravasation, because it allows the recruitment of the
tyrosine kinase Syk by association to ERM proteins
through their phosphorylated ITAM-like motifs. Therefore,
after PSGL-1 ligation to P-selectin or E-selectin, Syk
conveys rolling-emanating signals to the activation of gene
expression programs (59).This phenomenon suggests that
the intracellular signals induced through PSGL-1 have a
priming effect on leukocyte activation, up-regulating the
expression of different molecules further involved in
extravasation and effector functions (60). Since it has been
demonstrated that the cytoplasmic tail of L-selectin also
interacts with moesin (40), it is very likely that selectins
use a similar strategy to trigger intracellular signaling
cascades. In this regard, it has been shown that E-selectin is
dephosphorylated upon endothelial cell interaction with
leukocytes, supporting its role as a signal transduction
molecule (61). In addition, P-selectin also functions as a
signaling receptor, mediating stimulation through its
interaction with ligands expressed by leukocytes (62).
4. ACTIVATION, ARREST AND FIRM ADHESION
OF LEUKOCYTES
During their rolling, leukocytes are stimulated by
chemokines and integrin ligands expressed on the surface
of endothelial cells. These outside-in signals induce an
important increase in the affinity and/or avidity of
leukocyte integrins (inside-out signals) that allows the
shear-resistant arrest of these cells and their firm adhesion
to activated endothelium. The adhesion mediated by
integrins and their ligands, and their subsequent signaling
processes involve a profound remodelling of cytoskeleton
in both endothelial cells and leukocytes. In Figure 2, the
major adhesive, structural and signaling molecules that
participate in the leukocyte firm adhesion to endothelium
are summarized.
4.1. Chemokines trigger the arrest of leukocytes on
endothelium
The main in situ modulators of integrin function
are chemokines. These chemotactic cytokines act through
G-protein-coupled receptors (GPCR), and induce an array
of activatory signals within fractions of seconds, leading to
an enhancement of adhesion and shape changes in
leukocytes (63, 64). Since it is unlikely that soluble
chemokine gradients present in the blood flow regulate

Page 4

Protrusive structures in leukocyte extravasation
1852
Figure 2. Activation, arrest and firm adhesion of leukocytes on endothelium: The slowing-down of leukocytes facilitates their
interaction with chemokines exposed on endothelium, triggering the activation of leukocyte integrins by increasing their affinity
and avidity to allow the final arrest of adherent leukocytes. This activated state involves a drastic morphological change from the
round shape of circulating leukocytes to the polarized shape typical of migrating cells. The acquisition of polarity implies the
segregation of adhesion molecules (ICAM-1, ICAM-3, CD43, CD44, PSGL-1, etc) to the rear pole of the cell (uropod), which is
lumen-orientated for the recruitment of bystander leukocytes; whereas the integrins localized to the contact area with
endothelium to allow the spreading of the cell body onto the vascular wall. On the other hand, the endothelium also plays an
active role in firm adhesion by creating docking structures around the attached leukocytes. These endothelial docking structures
are formed as a result of VCAM-1 and ICAM-1 engagement by their integrin counterreceptors (VLA-4 and LFA-1, respectively),
and are supported by a cortical actin scaffold in which ERM proteins, alpha-actinin, vinculin,VASP and other actin-related
proteins participate. In addition, members of the tetraspanin family also cooperate with the endothelial adhesion receptors in the
docking structure formation. The principal regulatory molecules involved in the formation of the above-mentioned structure and
in leukocyte integrin activation (as discussed in the text) are also shown in the inset.
leukocyte trafficking to specific target tissues, it is thought
that chemokines mainly function immobilized at the
emigration site (65). At sites of inflammation or in
secondary lymphoid tissues, chemokines are present in the
subendothelial tissues, as well as in the luminal surface of
endothelium. These chemokines are synthesized or
transported (transcytosis) by endothelial cells and bound to
glycosaminoglycans (GAGs) and, possibly, to the Duffy
antigen/receptor for chemokines (DARC) (66). Chemokine-
binding sites are concentrated on endothelial microvilli, as
occurs with chemokine receptors in leukocytes (67). The
presence of particular subsets of chemokines on
endothelium contributes to the selective recruitment of
leukocytes, a critical phenomenon for the inflammatory
response and lymphocyte homing (reviewed in 68).
Chemokines may differentially modulate distinct integrins
in the same microenvironment, leading to transient or
sustained adhesion of leukocytes (69). Furthermore,
CXCL12 (SDF-1alpha), CCL19 (MIP-3beta), CCL21
(SLC) and CCL20 (MIP-3alpha) have all been shown to
induce LFA-1-mediated cell arrest in different lymphoid
subpopulations (63, 65, 70). In addition, SDF-1alpha up-
regulates VLA-4 affinity for VCAM-1, promoting
monocyte arrest (71). The mechanism involved in such
regulation is not clear, but may involve small Ras GTPases
such as Rap1 (72-74), Rho GTPases (75) and other
signaling molecules.
4.2. Integrin affinity and avidity changes as leukocyte
strategies to achieve firm attachment and spreading
Integrins comprise a family of alpha-beta
heterodimeric transmembrane molecules whose activation
can be tuned to bind to different Ig-like or extracellular

Page 5

Protrusive structures in leukocyte extravasation
1853
matrix ligands. Integrin-mediated cell adhesion is tightly
regulated by conformational changes (affinity) and
clustering (avidity), being independent of surface
expression levels (76). Circulating leukocytes avoid non-
specific contacts with vascular walls by maintaining their
integrins in non-adhesive states. The in situ activation of
integrins during leukocyte rolling can be driven by multiple
factors. Among affinity regulatory signals, divalent cations
such as Mn2+, Mg2+ or Ca2+ (77-79) rank as important
elements at least in vitro. On the other hand, the binding of
integrins to endothelial ligands can be enhanced
independently of integrin affinity by increasing receptor
density (avidity) at the contact area. Integrin avidity can be
defined as a rapid interplay between preformed, ligand-
induced, and chemokine-triggered avidity states (reviewed
in 42). Other potential modulators of VLA-4 and LFA-1
avidity at dynamic contacts, requiring concomitant
chemokine triggering, seem to be CD47 and L-selectin (80,
81). There is still another level of regulation known as
integrin cross-talk. Thus, the interaction of high affinity
VLA-4 with VCAM-1 may trigger LFA-1 clustering,
enhancing its avidity to ICAM-1, and promoting leukocyte
firm adhesion to endothelium. On the contrary, LFA-
1/ICAM-1 engagement decreases the binding of VLA-4 to
VCAM-1, allowing leukocyte migration towards the
transmigration sites (82, 83).
The clustering of integrins is dependent on their
release from the actin cytoskeleton. In this regard, there are
relevant differences between VLA-4 and LFA-1. LFA-1
may preform microclusters stabilized by H-ras and
cytohesin-1, which are activated via PI3-K or PKC (84-86).
However, the macroclustering of LFA-1 prior to ligand
engagement is prevented by cytoskeleton anchorage, with
the involvement of non-phosphorylated PKC substrates and
talin. Consequently, it is necessary the activation of PKC
and calpain to release LFA-1 from the actin cytoskeleton
(87). Conversely, VLA-4 clustering cannot be induced
prior to ligand binding. Furthermore, although PKC signals
participate in the release of VLA-4 from cytoskeleton,
calpain or PI3-K are not implicated. In addition, it has been
described that VLA-4 constitutively interacts with paxillin,
while LFA-1 is able to interact with alpha-actinin
(reviewed in 42).
Once integrin avidity has been up-regulated and
an effective engagement with ligand occurs, the integrin
anchorage to actin cytoskeleton is restored and outside-in
signaling leads to actin remodelling and cell spreading.
Then, leukocytes undergo a profound change in their
morphology, acquiring a polarized, motility-related shape
(reviewed in 88). This cell shape change favors leukocyte
extravasation, and is also involved in the recruitment of
bystander leukocytes through the trailing edge (uropod)
(89).
4.3. The docking structure: the endothelial contribution
to the leukocyte firm adhesion process
Endothelium had been considered as a mere
physiological barrier, a passive partner for leukocytes
during TEM. However, it is now evident that endothelium
is a key active element in different physiological processes,
including leukocyte extravasation. Our group has
contributed to gain insight into the molecular mechanisms
underlying the active role of endothelial cells in the TEM
of leukocytes. We have found that activated endothelial
cells generate “docking” structures that efficiently attach
leukocytes, partially engulfing them. VCAM-1 and ICAM-
1, together with cytoskeletal and signaling molecules, are
essential constituents of these cup-like structures based on
microspikes that emerge from the endothelial apical
surface, and which are dynamically involved in capturing
leukocytes prior to TEM (90).
4.3.1. VCAM-1 and ICAM-1 play an essential role in
leukocyte capture
VCAM-1 and ICAM-1, members of the Ig
superfamily, are the two major endothelial adhesion
molecules involved in the binding to leukocyte integrins
VLA-4 and LFA-1, respectively (91, 92). ICAM-1 but not
VCAM-1 is expressed at low levels in resting endothelium,
and both molecules are induced upon cell activation by pro-
inflammatory cytokines such as IL-1 and TNF-alpha(93,
94). We have recently found that these integrin ligands are
laterally associated with different tetraspanins (CD9, CD81
and CD151), forming protein microdomains in the apical
surface of endothelium (Barreiro and Yáñez-Mó,
unpublished data). Furthermore, it has been described that
VCAM-1 and ICAM-1 are anchored to the actin
cytoskeleton through members of the ERM family, mainly
ezrin and moesin (90, 95, 96). All these molecules, which
are clustered at endothelial microvilli and microspikes,
contact and surround the adherent leukocyte, as key
elements of the docking structure. The cytoskeletal linkage
of VCAM-1 and ICAM-1 is critical for the generation of
this structure upon leukocyte adhesion, but it is not
necessary for the proper presentation of VCAM-1 and
ICAM-1 at the apical surface, since this localization seems
to be independent of ligand engagement and actin
anchorage (Barreiro and Yáñez-Mó, unpublished data).
Dynamic experiments have demonstrated the
involvement of ICAM-1 (through its interaction with LFA-
1) not only in the firm adhesion of leukocytes but in their
transendothelial migration and subsequent movement
underneath the endothelial monolayer. Moesin has a similar
behaviour, suggesting that ICAM-1 is anchored to the actin
cytoskeleton via ERM proteins during the whole process.
On the contrary, VCAM-1 is excluded of the late steps of
leukocyte extravasation and only participates in the
formation of the endothelial docking structure that firmly
attaches the lymphocyte to the endothelium (90).
4.3.2. Structural components and signaling pathways
involved in the generation and maintenance of the
docking structure
The sequential steps involved in the generation of
the endothelial docking structure could be as follows. The
initial interaction of VCAM-1 and ICAM-1 with their
ligands (VLA-4 and LFA-1 integrins, respectively) triggers
their clustering at the leukocyte-endothelium contact area,
together with phosphorylated activated ERM proteins.
Then, these adaptor proteins in concert with alpha-actinin
and vinculin participate in the rearrangement of the actin
cytoskeleton to generate the docking structure. The

Page 6

Protrusive structures in leukocyte extravasation
1854
participation of other focal adhesion proteins such as talin
or paxillin remains to be elucidated. On the other hand, it
has been also described that VASP, which cooperates with
the WASP-Arp2/3 complex in actin polymerization at
nascent protrusions (97), is concentrated at the docking
structure (90). These data suggest that this endothelial
structure is supported by actin polymerization. However,
microtubules do not appear to be involved in this process.
Interestingly, the endothelial docking structure seems to be
reminiscent of nascent complement receptor-mediated
phagosomes, in that the subcellular distribution of all these
structural proteins is similar in both structures (98).
Regarding the signaling pathways involved in the
generation and maintenance of the docking structure, it has
been described the preferential accumulation of PI(4,5)P2 at
the tips of the microspikes of this structure, where could
participate in the activation of the ERM proteins.
Furthermore, the essential role of the Rho/p160 ROCK
signaling pathway in the formation of this protrusive
structure, has been also documented (90). These results
concur with the regulation of the VCAM-1, ICAM-1, and
E-selectin clustering by the GTPase Rho during monocyte
adhesion (99). Finally, further analyses are necessary to
understand the mechanisms underlying the disruption of the
endothelial docking structure to allow leukocyte diapedesis.
4.3.3. VCAM-1 and ICAM-1 outside-in signaling
VCAM-1 and ICAM-1 are capable of transducing
signals after ligand binding. VCAM-1 is involved in the
opening of the “endothelial passage” through which
leukocytes can extravasate. In this regard, VCAM-1
ligation induces NADPH oxidase activation and the
production of reactive oxygen species (ROS) in a Rac-
mediated manner, with subsequent activation of matrix
metalloproteinases and loss of VE-cadherin-mediated
adhesion. This signaling pathway can be blocked by
TGFbeta1 and IFNgamma (100-103). On the other hand,
cross-linking of both VCAM-1 and ICAM-1 induces a
rapid increase in intracellular Ca2+ concentration (62, 104).
ICAM-1-mediated calcium signaling has been mostly
studied in brain endothelial cells. In this cellular model, it
has been found that ICAM-1-mediated calcium increase
triggers activation of Src and subsequent phosphorylation
of cortactin (104). ICAM-1 is also able to activate RhoA
inducing stress fiber formation (105) and phosphorylation
of FAK, paxillin and p130Cas, which in turn trigger
different signaling pathways involving JNK or p38 (106-
108). Moreover, ICAM-1 cross-linking stimulate c-fos and
rhoA transcription (105). As reported for PSGL-1 (59),
ICAM-1 might enhance c-fos expression through the
recruitment of Syk to the ICAM-1/ERM complex, but such
possibility deserves further investigation. Finally, the
ICAM-1 cross-linking can induce its own expression as
well as that of VCAM-1, as a regulatory mechanism to
facilitate leukocyte TEM (109).
5. TRANSENDOTHELIAL MIGRATION
The signals involved in the firm adhesion of
leukocytes to endothelium must be reverted, weakening the
original contact sufficiently to allow the migration and
extravasation of leukocytes. During TEM, endothelial
junctions must be loosen to a limited extent, thereby
avoiding cell monolayer damage or important changes in
permeability. Thus, the leukocyte and endothelium
membranes are kept in close contact and show prominent
associated cytoskeletal structures. Subsequently, the
endothelial membranes reseal their connections over the
trailing end of the leukocyte.
5.1.
rearrangements to extravasate across the endothelial
barrier
In leukocytes, integrin-dependent adhesion is
required for changes in cytoskeleton plasticity and cell
motility (110). In addition, it has been recently described
that immobilized chemokines play a pivotal role in this
process, since SDF-1alpha presented on the apical surface
of endothelial cells can trigger lymphoid TEM under shear
stress conditions in the absence of a chemoattractant
gradient across the endothelium, whereas soluble
chemotactic gradients do not. This process has been
designated as “chemorheotaxis”
monocytes (112) and neutrophils (113) do not require
endothelial apical chemokines to undergo TEM, hence
postulating this phenomenon as lymphocyte-specific.
Leukocytes undergo drastic cytoskeletal
(111). However,
The regulation of the deformation of the
leukocyte cytoskeleton during TEM has not been well
studied. The possible activation of regulators of the actin
cytoskeleton such as small Rho GTPases by integrins or
chemokines remains to be elucidated. However, an
attractive hypothesis would comprise the activation of
Cdc42 by integrins or chemokines, which would cause the
extension of a thin exploratory pseudopodium between
endothelial cells that, by sequential Rac1 activation, would
evolve into a lamella squeezed within an endothelial
monolayer gap. This leading lamella and the leukocyte
membrane in contact with endothelium are enriched in
LFA-1 (114, 115). Finally, the stretching of cell body and
tail retraction would result from delayed, tail-oriented
RhoA-ROCK activation and actomyosin-based contraction
(116).
5.2. Endothelial cell lateral junctions regulation
The components of the endothelial lateral
junctions can be divided in tight, adherens and gap
junctions, each containing distinct molecular constituents,
although they do not exhibit a well-organized basolateral
organization as in epithelium. These molecular complexes
are dynamically organized, associate with the actin
cytoskeleton and, except for gap junctions, actively
participate in leukocyte TEM. Recent reports strongly
suggest that leukocytes and endothelium communicate each
other during TEM. The most characterized intracellular
signals generated by TEM in endothelium are the
mobilization of intracellular Ca2+ and the reorganization of
actin, myosin and associated molecules (117, 118).
Figure 3 establishes a comparison between the
interendothelial junctions in the vascular wall and the
heterotypic leukocyte-endothelium interactions during
transendothelial migration. The morphological changes that
leukocytes undergo to pass across the endothelial barrier, as

Page 7

Protrusive structures in leukocyte extravasation
1855
Figure 3. Comparison between the interendothelial junctions in the vascular wall and the heterotypic leukocyte-endothelium
interactions during transendothelial migration. Only the molecules involved in both processes are shown, including members of
the tight junctions (ESAM and the JAM family), the VE-cadherin complex and other molecules such as PECAM-1 and CD99. Most of
them interact homophilically, being express by the endothelium as well as by leukocytes. On the other hand, the processes of
lamellipodia formation at the leukocyte leading edge and tail retraction, the phenomenon of chemorheotaxis and the existence of a
subendothelial soluble chemoattractant gradient to guide the extravasated leukocytes to the target tissue are also illustrated.
well as the effect of chemokines and chemoattractant
gradients in this process, have been also specified.
5.2.1. Tight junction proteins: role of JAM family in
transendothelial migration
Among the components of tight junctions
(occludin, claudins, ZO-1, -2, -3, etc), two novel groups of
adhesion molecules belonging to the Ig superfamily have
been recently identified: ESAM (Endothelial cell-Selective
Adhesion Molecule) and JAM (Junctional Adhesion
Molecule) proteins. In contrast with other members of tight
junctions, they play a role in paracellular permeability as
well as in lymphocyte homing and TEM. ESAM is
involved in the homotypic interaction of endothelial cells,
but no heterophilic ligands have been described yet (119,
120).To date, three members of the JAM family have been
identified: JAM-1, huJAM-2 (which corresponds to
mJAM-3) and huJAM-3 (which corresponds to mJAM-2)
(reviewed in 121). JAM-1 is expressed in epithelium,
endothelium, erythrocytes, PMNs, monocytes, lymphocytes
and platelets; huJAM2 is preferentially expressed in high
endothelial venules (HEV); and JAM3 is detected in
endothelium and activated T lymphocyte subsets (122-
27). All members of the family contain a PDZ-binding
motif in their cytoplasmic tail, which is involved in their
association with components of the tight junctions such as
ZO-1 and AF-6, suggesting a role for these molecules in
recruiting and stabilizing JAMs to site of junction
formation (128, 129). The JAM family proteins can
interact homophilically at endothelial tight junctions to
regulate paracellular permeability or heterophilically with
counterreceptors from leukocytes to support TEM. In this
regard, it has been described that JAM-1 is capable to
interact with itself or with LFA-1 (130). huJAM-2 and
huJAM-3 are binding partners, and this interaction could
have relevance for lymphocyte homing, due to the
restricted expression of huJAM-2 in high endothelial
venules (124). Furthermore, huJAM-3 is also capable to
interact with itself (131) and with alphaMbeta2 and
alphaXbeta2 integrins (132). In turn, huJAM-2 interacts
with alpha4beta1 integrin (133). The fact that all the
members of the JAM family interact with leukocyte
integrins and that are relocalized towards the apical
surface upon endothelium activation argues for their
fundamental role in the regulation of leukocyte
adhesion.

Page 8

Protrusive structures in leukocyte extravasation
1856
5.2.2. Adherens junction disappearance at leukocyte
contacts: proteolysis or displacement?
Adherens junctions are primarily involved in the
regulation of endothelial cell monolayer permeability. The
main component of these molecular complexes is VE-
cadherin, which mediates Ca2+-dependent homophilic
adhesion with its extracellular domain and actin
cytoskeleton linkage through the interaction of its
cytoplasmic tail with alpha-, beta-, gamma-catenin, and
p120/100 (134). During leukocyte TEM, VE-cadherin
complexes are locally disrupted, generating a localized gap
necessary for leukocyte passage, which reseals after TEM.
According to these observations, VE-cadherin acts as a
“gatekeeper” for leukocyte transmigration. Whether or not
this disruption of adherens junctions involves protein
degradation has been extensively discussed (135-137).
However, the rapid recovery of adherens junctions after the
passage of leukocytes seems to point to the existence of a
zipper mechanism, which implies the local and transient
displacement of molecular complexes from the leukocyte-
endothelium area (“trapdoor model”) (138, 139). However,
the possibility of a certain degree of complex proteolysis
cannot be excluded. Moreover,
mechanisms such as transcytosis or preferential passage
through tricellular corners, where tight junctions and
adherens junctions are less organized, cannot be ruled out
(140-142).
other alternative
5.2.3. Other molecules with a major role in the passage
of leukocytes across endothelium
Apart from the above-mentioned molecules, there
are other endothelial proteins critical for leukocyte TEM
that do not belong to tight junctions or adherens junctions
complexes. PECAM-1 is another member of the
immunoglobulin superfamily that is expressed in
endothelium as well as in leukocytes, and that actively
participates in TEM (143). It can associate homophilically
or with alphavbeta3 in cis- (144, 145). PECAM-1
transduces negative intracellular signals via the ITIM motif
of its cytoplasmic tail (146). In addition, PECAM-1 can
regulate adherens junctions by associating to beta-catenin
(147). Finally, a recent report describes the existence of a
molecular network just below the endothelial plasma
membrane that is connected at intervals with the junctional
surface. PECAM-1 has been found in this compartment,
constitutively recycling along the endothelium borders.
During TEM, PECAM-1 recycling molecules are targeted
to points of contact with leukocytes. This mechanism could
explain how endothelial cells change their borders rapid
and reversely, but remaining tightly apposed to leukocytes
to allow their migration (148).
CD99, a highly O-glycosylated type I
transmembrane protein, has been found to play a critical
role in monocyte TEM, acting at a later stage than
PECAM-1 (149). This protein is expressed in leukocytes,
where triggers the activation of alpha4beta1 integrin and
regulates the activity of LFA-1, and endothelium,
interacting homophilically at interendothelial contacts
(reviewed in 145). However, little is known about its
precise function on endothelial cells or its involvement in
signaling transduction .
6. CONCLUDING REMARKS AND PERSPECTIVES
Over the last decade, a huge effort has been made
for the study of the basic adhesive mechanisms underlying
vascular function. In this regard, the dissection of the
phenomena involved in leukocyte extravasation has
significantly improved our knowledge of different
pathophysiological conditions. The recent description of
new molecular complexes and subcellular structures
implicated in this process, as well as the characterization of
new intracellular signaling pathways or cytoskeletal
components, have added more complexity to the
extravasation mechanism and opened new insights to future
investigations. Furthermore, molecules that are newly being
involved in this process could constitute potential
molecular targets for therapeutic intervention. Finally,
some controversies and obscure points regarding, e.g.,
differences in the behaviour of monocytes, neutrophils or
lymphocytes during TEM
interendothelial junctions to allow the passage of
leukocytes still remain partially unsolved.
or the regulation of
7. ACKNOWLEDGEMENTS
We would like to thank Drs. R. González-Amaro,
M. Gómez, M. Rey, and D. Sancho for critical reading of
the manuscript. The authors’ laboratory was supported by
grants BMC-2002 00563 from the Ministerio de Ciencia y
Tecnología, Ayuda a la Investigación Básica Juan March
2002, FIPSE 36289/02,
Cardiovascular to Dr. F. Sánchez-Madrid, and fellowships
from Fundación Mapfre Medicina to O. Barreiro, and from
Comunidad Autónoma de Madrid to M. Yáñez-Mó.
and FIS CO3/01-Red
8. REFERENCES
1. Frenette, P. S. & D. D. Wagner: Adhesion molecules--
Part I. N Engl J Med 334, 1526-9 (1996)
2. Frenette, P. S. & D. D. Wagner: Adhesion molecules---
Part II: Blood vessels and blood cells. N Engl J Med 335,
43-5 (1996)
3. Aplin, A. E., A. Howe, S. K. Alahari & R. L. Juliano:
Signal transduction and signal modulation by cell adhesion
receptors: the role of integrins, cadherins, immunoglobulin-
cell adhesion molecules, and selectins. Pharmacol Rev 50,
197-263 (1998)
4. Verfaillie, C. M.: Adhesion receptors as regulators of
the hematopoietic process. Blood 92, 2609-12 (1998)
5. Levesque, J. P., A. C. Zannettino, M. Pudney, S. Niutta,
D. N. Haylock, K. R. Snapp, G. S. Kansas, M. C. Berndt &
P. J. Simmons: PSGL-1-mediated adhesion of human
hematopoietic progenitors to P-selectin results in
suppression of hematopoiesis. Immunity 11, 369-78 (1999)
6. Butcher, E. C.: Leukocyte-endothelial cell recognition:
three (or more) steps to specificity and diversity. Cell 67,
1033-6 (1991)

Page 9

Protrusive structures in leukocyte extravasation
1857
7. Butcher, E. C. & L. J. Picker: Lymphocyte homing and
homeostasis. Science 272, 60-6 (1996)
8. Springer, T. A.: Traffic signals for lymphocyte
recirculation and leukocyte emigration: the multistep
paradigm. Cell 76, 301-14 (1994)
9. Springer, T. A.: Traffic signals on endothelium for
lymphocyte recirculation and leukocyte emigration. Annu
Rev Physiol 57, 827-72 (1995)
10. Mehta, P., R. D. Cummings & R. P. McEver: Affinity
and kinetic analysis of P-selectin binding to P-selectin
glycoprotein ligand-1. J Biol Chem 273, 32506-13 (1998)
11. Nicholson, M. W., A. N. Barclay, M. S. Singer, S. D.
Rosen & P. A. van der Merwe: Affinity and kinetic analysis
of L-selectin (CD62L) binding to glycosylation-dependent
cell-adhesion molecule-1. J Biol Chem 273, 763-70 (1998)
12. Vestweber, D. & J. E. Blanks: Mechanisms that
regulate the function of the selectins and their ligands.
Physiol Rev 79, 181-213 (1999)
13. McEver, R. P., J. H. Beckstead, K. L. Moore, L.
Marshall-Carlson & D. F. Bainton: GMP-140, a platelet
alpha-granule membrane protein, is also synthesized by
vascular endothelial cells and is localized in Weibel-Palade
bodies. J Clin Invest 84, 92-9 (1989)
14. Mayadas, T. N., R. C. Johnson, H. Rayburn, R. O.
Hynes & D. D. Wagner: Leukocyte rolling and
extravasation are severely compromised in P selectin-
deficient mice. Cell 74, 541-54 (1993)
15. Bevilacqua, M. P., J. S. Pober, D. L. Mendrick, R. S.
Cotran & M. A. J. Gimbrone: Identification of an inducible
endothelial-leukocyte adhesion molecule. Proc Natl Acad
Sci USA 84, 9238-42 (1987)
16. Subramaniam, M., J. A. Koedam & D. D. Wagner:
Divergent fates of P- and E-selectins after their expression
on the plasma membrane. Mol Biol Cell 4, 791-801 (1993)
17. Kishimoto, T. K., M. A. Jutila, E. L. Berg & E. C.
Butcher: Neutrophil Mac-1 and MEL-14 adhesion proteins
inversely regulated by chemotactic factors. Science 245,
1238-41 (1989)
18. Jung, T. M. & M. O. Dailey: Rapid modulation of
homing receptors (gp90MEL-14) induced by activators of
protein kinase C. Receptor shedding due to accelerated
proteolytic cleavage at the cell surface. J Immunol 144,
3130-6 (1990)
19. Walcheck, B., J. Kahn, J. M. Fisher, B. B. Wang, R. S.
Fisk, D. G. Payan, C. Feehan, R. Betageri, K. Darlak, A. F.
Spatola & T. K. Kishimoto: Neutrophil rolling altered by
inhibition of L-selectin shedding in vitro. Nature 380, 720-
3 (1996)
20. Ley, K., D. C. Bullard, M. L. Arbones, R. Bosse, D.
Vestweber, T. F. Tedder & A. L. Beaudet: Sequential
contribution of L- and P-selectin to leukocyte rolling in
vivo. J Exp Med 181, 669-75 (1995)
21. McEver, R. P. & R. D. Cummings: Role of PSGL-1
binding to selectins in leukocyte recruitment. J Clin Invest
100, S97-103 (1997)
22. Frenette, P. S., C. V. Denis, L. Weiss, K. Jurk, S.
Subbarao, B. Kehrel, J. H. Hartwig, D. Vestweber & D. D.
Wagner: P-selectin glycoprotein ligand 1 (PSGL-1) is
expressed on platelets and can mediate platelet-endothelial
interactions in vivo. J Exp Med 191, 1413-22 (2000)
23. Xia, L., M. Sperandio, T. Yago, J. M. McDaniel, R. D.
Cummings, S. Pearson-White, K. Ley & R. P. McEver: P-
selectin glycoprotein ligand-1-deficient mice have impaired
leukocyte tethering to E-selectin under flow. J Clin Invest
109, 939-50 (2002)
24. Hirata, T., G. Merrill-Skoloff, M. Aab, J. Yang, B. C.
Furie & B. Furie: P-selectin glycoprotein ligand 1 (PSGL-
1) is a physiological ligand for E-selectin in mediating T
helper 1 lymphocyte migration. J Exp Med 192, 1669-76
(2000)
25. Norman, K. E., A. G. Katopodis, G. Thoma, F.
Kolbinger, A. E. Hicks, M. J. Cotter, A. G. Pockley & P. G.
Hellewell: P-selectin glycoprotein ligand-1 supports rolling
on E- and P-selectin in vivo. Blood 96, 3585-91 (2000)
26. Sperandio, M., M. L. Smith, S. B. Forlow, T. S. Olson,
L. Xia, R. P. McEver & K. Ley: P-selectin glycoprotein
ligand-1 mediates L-selectin-dependent leukocyte rolling in
venules. J Exp Med 197, 1355-63 (2003)
27. Levinovitz, A., J. Muhlhoff, S. Isenmann & D.
Vestweber: Identification of a glycoprotein ligand for E-
selectin on mouse myeloid cells. J Cell Biol 121, 449-59
(1993)
28. Steegmaier, M., A. Levinovitz, S. Isenmann, E.
Borges, M. Lenter, H. Kocher, B. Kleuser & D. Vestweber:
The E-selectin-ligand ESL-1 is a variant of a receptor for
fibroblast growth factor. Nature 373, 615-20 (1995)
29. Aigner, S., C. L. Ramos, A. Hafezi-Moghadam, M. B.
Lawrence, J. Friederichs, P. Altevogt & K. Ley: CD24
mediates rolling of breast carcinoma cells on P-selectin.
FASEB J 12, 1241-51 (1998)
30. Patel, K. D., S. L. Cuvelier & S. Wiehler: Selectins:
critical mediators of leukocyte recruitment. Semin Immunol
14, 73-81 (2002)
31. Berlin, C., R. F. Bargatze, J. J. Campbell, U. H.
vonAndrian, M. C. Szabo, S. R. Hasslen, R. D. Nelson, E.
L. Berg, S. L. Erlandsen & E. C. Butcher: alpha4 integrins
mediate lymphocyte attachment and rolling under
physiologic flow. Cell 80, 413-22 (1995)
32. Henderson, R. B., L. H. K. Lim, P. A. Tessier, F. N. E.
Gavins, M. Mathies, M. Perreti & N. Hogg: The use of

Page 10

Protrusive structures in leukocyte extravasation
1858
Lymphocyte
deficient mice to determine the role of LFA-1, Mac-1 and
alpha4 integrin in the inflammatory response of
neutrophils. J Exp Med 194, 219-26 (2001)
Function-associated Antigen (LFA-1)-
33. Kadono, T., G. M. Venturi, D. A. Steeber & T. F.
Tedder: Leukocyte rolling velocities and migration are
optimized by cooperative L-selectin and intercellular
adhesion molecule-1 functions. J Immunol 169, 4542-50
(2002)
34. Bazan, J. F., K. B. Bacon, G. Hardiman, W. Wang, K.
Soo, D. Rossi, D. R. Greaves, A. Zlotnik & T. J. Schall: A
new class of membrane-bound chemokine with a CX3C
motif. Nature 385, 640-4 (1997)
35. Matloubian, M., A. David, S. Engel, J. E. Ryan & J. G.
Cyster: A transmembrane CXC chemokine is a ligand for
HIV-coreceptor Bonzo. Nat Immunol 1, 298-304 (2000)
36. Fong, A. M., S. M. Alam, T. Imai, B. Haribabu & D.
D. Patel: CX3CR1 tyrosine sulfation enhances fractalkine-
induced cell adhesion. J Biol Chem 277, 19418-23 (2002)
37. Haskell, C. A., M. D. Cleary & I. F. Charo: Molecular
uncoupling of fractalkine-mediated cell adhesion and signal
transduction. Rapid flow arrest of CX3CR1-expressing
cells is independent of G-protein activation. J Biol Chem
274, 10053-8 (1999)
38. von Andrian, U. H., S. R. Hasslen, R. D. Nelson, S. L.
Erlandsen & E. C. Butcher: A central role for microvillous
receptor presentation in leukocyte adhesion under flow.
Cell 82, 989-99 (1995)
39. Pavalko, F. M., D. M. Walker, L. Graham, M. Goheen,
C. M. Doerschuk & G. S. Kansas: The cytoplasmic domain
of L-selectin interacts with cytoskeletal proteins via alpha-
actinin: receptor positioning in microvilli does not require
interaction with alpha-actinin. J Cell Biol 129, 1155-64
(1995)
40. Ivetic, A., J. Deka, A. J. Ridley & A. Ager: The
cytoplasmic tail of L-selectin interacts with members of the
Ezrin-Radixin-Moesin (ERM) family of proteins: cell
activation-dependent binding of Moesin but not Ezrin. J
Biol Chem 277, 2321-9 (2002)
41. Dwir, O., G. S. Kansas & R. Alon: Cytoplasmic
anchorage of L-selectin controls leukocyte capture and
rolling by increasing the mechanical stability of the selectin
tether. J Cell Biol 155, 145-56 (2001)
42. Alon, R. & S. Feigelson: From rolling to arrest on
blood vessels: leukocyte tap dancing on endothelial integrin
ligands and chemokines at sub-second contacts. Sem
Immunol 14, 93-104 (2002)
43. Stein, J. V., G. Cheng, B. M. Stockton, B. P. Fors, E.
C. Butcher & U. H. von Andrian: L-selectin-mediated
leukocyte adhesion in vivo: microvillous distribution
determines tethering efficiency, but not rolling velocity. J
Exp Med 189, 37-50 (1999)
44. Shao, J. Y., H. P. Ting-Beall & R. M. Hochmuth:
Static and dynamic lengths of neutrophil microvilli. Proc
Natl Acad Sci USA 95, 6797-802 (1998)
45. Yoshida, M., W. F. Westlin, N. Wang, D. E. Ingber, A.
Rosenzweig, N. Resnick & M. A. J. Gimbrone: Leukocyte
adhesion to vascular endothelium induces E-selectin
linkage to the actin cytoskeleton. J Cell Biol 133, 445-55
(1996)
46. Waddell, T. K., L. Fialkow, C. K. Chan, T. K.
Kishimoto & G. P. Downey: Signaling functions of L-
selectin: enhancement of tyrosine phosphorylation and
activation of MAP kinase. J Biol Chem 270, 15403-11
(1995)
47. Brenner, B., E. Gulbins, K. Schlottmann, U.
Koppenhoefer, G. L. Busch, B. Walzog, M. Steinhausen,
K. M. Coggeshall, O. Linderkamp & F. Lang: L-selectin
activates the Ras pathway via the tyrosine kinase p56lck.
Proc Natl Acad Sci USA 93, 15376-81 (1996)
48. Brenner, B., E. Gulbins, G. L. Busch, U.
Koppenhoefer, F. Lang & O. Linderkamp: L-selectin
regulates actin polymerisation via activation of the small G-
protein Rac2. Biochem Biophys Res Commun 231, 802-7
(1997)
49. Roberts, A. W., C. Kim, L. Zhen, J. B. Lowe, R.
Kapur, B. Petryniak, A. Spaetti, J. D. Pollock, J. B. Borneo,
G. B. Bradford, S. J. Atkinson, M. C. Dinauer & D. A.
Williams: Deficiency of the hematopoietic cell-specific
Rho family GTPase Rac2 is characterized by abnormalities
in neutrophil function and host defense. Immunity 10, 183-
96 (1999)
50. Hidari, K. I., A. S. Weyrich, G. A. Zimmerman & R. P.
McEver: Engagement of P-selectin glycoprotein ligand-1
enhances tyrosine phosphorylation and activates mitogen-
activated protein kinases in human neutrophils. J Biol
Chem 272, 28750-6 (1997)
51. Celi, A., G. Pellegrini, R. Lorenzet, A. De Blasi, N.
Ready, B. C. Furie & B. Furie: P-selectin induces the
expression of tissue factor on monocytes. Proc Natl Acad
Sci USA 91, 8767-71 (1994)
52. Damle, N. K., K. Klussman, M. T. Dietsch, N.
Mohagheghpour & A. Aruffo: GMP-140 (P-selectin/CD62)
binds to chronically stimulated but not resting CD4+ T
lymphocytes and regulates
proinflammatory cytokines. Eur J Immunol 22, 1789-93
(1992)
their production of
53. Weyrich, A. S., T. M. McIntyre, R. P. McEver, S. M.
Prescott & G. A. Zimmerman: Monocyte tethering by P-
selectin regulates monocyte chemotatic protein-1 and tumor
necrosis factor-alpha secretion. Signal integration and NF-
kappa B translocation. J Clin Invest 95, 2297-303 (1995)
54. Weyrich, A. S., M. R. Elstad, R. P. McEver, T. M.
McIntyre, K. L. Moore, J. H. Morrissey, S. M. Prescott &

Page 11

Protrusive structures in leukocyte extravasation
1859
G. A. Zimmerman: Activated platelets signal chemokine
synthesis by human monocytes. J Clin Invest 97, 1525-34
(1996)
55. Ruchaud-Sparagano, M. H., T. R. Walker, A. G. Rossi,
C. Haslett & I. Dransfield: Soluble E-selectin acts in
synergy with platelet-activating factor to activate neutrophil
beta 2-integrins. Role of tyrosine kinases and Ca2+
mobilization. J Biol Chem 275, 15758-64 (2000)
56. Simon, S. I., Y. Hu, D. Vestweber & C. W. Smith:
Neutrophil tethering on E-selectin activates beta 2 integrin
binding to ICAM-1 through a mitogen-activated protein
kinase signal transduction pathway. J Immunol 164, 4348-
58 (2000)
57. Alonso-Lebrero, J. L., J. M. Serrador, C. Domínguez-
Jímenez, O. Barreiro, A. Luque, M. A. d. Pozo, K. Snapp,
G. Kansas, R. Schwartz-Albiez, H. Furthmayr, F. Lozano
& F. Sánchez-Madrid: Polarization and interaction of
adhesion molecules P-selectin Glycoprotein Ligand-1 and
Intercellular Adhesion Molecule-3 with moesin and ezrin in
myeloid cells. Blood 95, 2413-9 (2000)
58. Serrador, J. M., A. Urzainqui, J. L. Alonso-Lebrero, J.
R. Cabrero, M. C. Montoya, M. Vicente-Manzanares, M.
Yañez-Mó & F. Sánchez-Madrid: A juxta-membrane
amino acid sequence of P-selectin glycoprotein ligand-1 is
involved in moesin binding and ezrin/radixin/moesin-
directed targeting at the trailing edge of migrating
lymphocytes. Eur J Immunol 32, 1560-6 (2002)
59. Urzainqui, A., J. M. Serrador, F. Viedma, M. Yáñez-
Mó, A. Rodríguez, A. L. Corbí, J. L. Alonso-Lebrero, A.
Luque, M. Deckert, J. Vázquez & F. Sánchez-Madrid:
ITAM-based interaction of ERM proteins with Syk
mediates signaling by the leukocyte adhesion receptor
PSGL-1. Immunity 17, 401-12 (2002)
60. Gonzalez-Amaro, R. & F. Sanchez-Madrid: Cell
adhesion molecules: selectins and integrins. Crit Rev
Immunol 19, 389-429 (1999)
61. Yoshida, M., B. E. Szente, J. M. Kiely, A. Rosenzweig
& M. A. J. Gimbrone: Phosphorylation of the cytoplasmic
domain of E-selectin is regulated during leukocyte-
endothelial adhesion. J Immunol 161, 933-41 (1998)
62. Lorenzon, P., E. Vecile, E. Nardon, E. Ferrero, J. M.
Harlan, F. Tedesco & A. Dobrina: Endothelial cell E- and
P-selectin and vascular cell adhesion molecule-1 function
as signaling receptors. J Cell Biol 142, 1381-91 (1998)
63. Campbell, J. J., J. Hedrick, A. Zlotnik, M. A. Siani, D.
A. Thompson & E. C. Butcher: Chemokines and the arrest
of lymphocytes rolling under flow conditions. Science 279,
381-4 (1998)
64. Grabovsky, V., S. Feigelson, C. Chen, D. A. Bleijs, A.
Peled, G. Cinamon, F. Baleux, F. Arenzana-Seisdedos, T.
Lapidot, Y. van Kooyk, R. R. Lobb & R. Alon: Subsecond
induction of alpha4integrin clustering by immobilized
chemokines stimulates leukocyte tethering and rolling on
endothelial vascular cell adhesion molecule 1 under flow
conditions. J Exp Med 192, 495-506 (2000)
65. Stein, J. V., A. Rot, Y. Luo, M. Narasimhaswamy, H.
Nakano, M. D. Gunn, A. Matsuzawa, E. J. Quackenbush,
M. E. Dorf & U. H. von Andrian: The CC chemokine
thymus-derived chemokine agent 4 (TCA-1, secondary
lymphoid tissue chemokine, 6Ckine, exodus-2) triggers
lymphocyte function-associated antigen 1-mediated arrest
of rolling T lymphocytes in peripheral lymph node high
endothelial venules. J Exp Med 191, 61-76 (2000)
66. Middleton, J., A. M. Patterson, L. Gardner, C. Schmutz
& B. A. Ashton: Leukocyte extravasation: chemokine
transport and presentation by the endothelium. Blood 100,
3853-60 (2002)
67. Singer, I. I., S. Scott, D. W. Kawka, J. Chin, B. L.
Daugherty, J. A. DeMartino, J. DiSalvo, S. L. Gould, J. E.
Lineberger, L. Malkowitz, M. D. Miller, L. Mitnaul, S. J.
Siciliano, M. J. Staruch, H. R. Williams, H. J. Zweerink &
M. S. Springer: CCR5, CXCR4, and CD4 are clustered and
closely apposed on microvilli of human macrophages and T
cells. J Virol 75, 3779-90 (2001)
68. Johnston, B. & E. C. Butcher: Chemokines in rapid
leukocyte adhesion triggering and migration. Semin
Immunol 14, 83-92 (2002)
69. Weber, C., J. Kitayama & T. A. Springer: Differential
regulation of beta1 and beta2 integrin avidity by
chemoattractants in eosinophils. Proc Natl Acad Sci USA
93, 10939-44 (1996)
70. Tangemann, K., M. D. Gunn, P. Giblin & S. D. Rosen:
A high endothelial cell-derived chemokine induces rapid,
efficient and subset-selective arrest of rolling T
lymphocytes on a reconstituted endothelial substrate. J
Immunol 161, 6330-7 (1998)
71. Chan, J. R., S. J. Hyduk & M. I. Cybulsky:
Chemoattractants induce a rapid and transient upregulation
of monocyte alpha4 integrin affinity for vascular cell
adhesion molecule 1 which mediates arrest: an early step in
the process of emigration. J Exp Med 193, 1149-58 (2001)
72. de Bruyn, K. M. T., S. Rangarajan, K. A. Reequist, C.
G. Figdor & J. L. Bos: The small GTPase Rap1 is required
for Mn2+- and antibody-induced LFA-1- and VLA-4-
mediated cell adhesion. J Biol Chem 277, 29468-76 (2002)
73. McLeod, S. J., A. H. Y. Li, R. L. Lee, A. E. Burgess &
M. R. Gold: The Rap GTPases regulate B cell migration
toward the chemokine Stromal Cell-Derived Factor-1
(CXCL12): Potential role for Rap2 in promoting B cell
migration. J Immunol 169, 1365-71 (2002)
74. Shimonaka, M., K. Katagiri, T. Nakayama, N. Fujita,
T. Tsuruo, O. Yoshie & T. Kinashi: Rap1 translates
chemokine signals to integrin activation, cell polarization,
and motility across vascular endothelium under flow. J Cell
Biol 161, 417-27 (2003)

Page 12

Protrusive structures in leukocyte extravasation
1860
75. Laudanna, C., J. J. Campbell & E. C. Butcher: Role of
Rho in chemoattractant-activated leukocyte adhesion
through integrins. Science 271, 981-3 (1996)
76. Sánchez-Mateos, P., C. Cabañas & F. Sánchez-Madrid:
Regulation of integrin function. Semin Cancer Biol 7, 99-
109 (1996)
77. Day, E. S., L. Osborn & A. Whitty: Effect of divalent
cations on the affinity and selectivity of alpha4 integrins
towards the integrin ligands vascular cell adhesion
molecule-1 and mucosal addressin cell adhesion molecule-
1: Ca2+ activation of integrin alpha4beta1 confers a
distinct ligand specificity. Cell Commun Adhes 9, 205-19
(2002)
78. Dransfield, I., C. Cabañas, A. Craig & N. Hogg:
Divalent cation regulation of the function of the leukocyte
integrin LFA-1. J Cell Biol 116, 219-26 (1992)
79. van Kooyk, Y., P. Weder, K. Heije & C. G. Figdor:
Extracellular Ca2+ modulates
associated antigen-1 cell surface distribution on T
lymphocytes and consequently affects cell adhesion. J Cell
Biol 124, 1061-70 (1994)
leukocyte function-
80. Ticchioni, M., V. Raimondi, L. Lamy, J. Wijdenes, F.
P. Lindberg, E. J. Brown & A. Bernard: Integrin-associated
protein (CD47/IAP) contributes to T cell arrest on
inflammatory vascular endothelium under flow. FASEB J
15, 341-50 (2001)
81. Hwang, S. T., M. S. Singer, P. A. Giblin, T. A.
Yednock, K. B. Bacon, S. I. Simon & S. D. Rosen:
GlyCAM-1, a physiologic ligand for L-selectin, activates
beta 2 integrins on naive peripheral lymphocytes. J Exp
Med 184, 1343-8 (1996)
82. Chan, J. R., S. J. Hyduk & M. I. Cybulsky: Alpha 4
beta 1 integrin/VCAM-1 interaction activates alpha L beta
2 integrin-mediated adhesion to ICAM-1 in human T cells.
J Immunol 164, 746-53 (2000)
83. Porter, J. C. & N. Hogg: Integrin cross talk: activation
of lymphocyte function-associated antigen-1 on human T
cells alters alpha4beta1- and alpha5beta1-mediated
function. J Cell Biol 138, 1437-47 (1997)
84. Kolanus, W., W. Nagel, B. Schiller, L. Zeitlmann, S.
Godar, H. Stockinger & B. Seed: Alpha L beta 2
integrin/LFA-1 binding to ICAM-1 induced by cytohesin-1,
a cytoplasmic regulatory molecule. Cell 86, 233-42 (1996)
85. Kolanus, W. & B. Seed: Integrins and inside-out signal
transduction: converging signals from PKC and PIP3. Curr
Opin Cell Biol 9, 725-31 (1997)
86. Tanaka, Y., Y. Minami, S. Mine, H. Hirano, C. D. Hu,
H. Fujimoto, K. Fujii, K. Saito, J. Tsukada, Y. van Kooyk,
C. G. Figdor, T. Kataoka & S. Eto: H-Ras signals to
cytoskeletal machinery in induction of integrin-mediated
adhesion of T cells. J Immunol 163, 6209-16 (1999)
87. Stewart, M. P., A. McDowall & N. Hogg: LFA-1-
mediated adhesion is regulated by cytoskeletal restraint and
by a Ca2+-dependent protease, calpain. J Cell Biol 140,
699-707 (1998)
88. Vicente-Manzanares, M., D. Sancho, M. Yáñez-Mó &
F. Sánchez-Madrid: The leukocyte cytoskeleton in cell
migration and immune interactions. Int Rev Cytol 216, 233-
89 (2002)
89. del Pozo, M. A., C. Cabanas, M. C. Montoya, A. Ager,
P. Sanchez-Mateos & F. Sanchez-Madrid: ICAMs
redistributed by chemokines to cellular uropods as a
mechanism for recruitment of T lymphocytes. J Cell Biol
137, 493-508 (1997)
90. Barreiro, O., M. Yañez-Mó, J. M. Serrador, M. C.
Montoya, M. Vicente-Manzanares, R. Tejedor, H.
Furthmayr & F. Sánchez-Madrid: Dynamic interaction of
VCAM-1 and ICAM-1 with moesin and ezrin in a novel
endothelial docking structure for adherent leukocytes. J
Cell Biol 157, 1233-45 (2002)
91. Marlin, S. D. & T. A. Springer: Purified intercellular
adhesion molecule-1 (ICAM-1) is a ligand for lymphocyte
function-associated antigen 1 (LFA-1). Cell 51, 813-9
(1987)
92. Elices, M. J., L. Osborn, Y. Takada, C. Crouse, S.
Luhowskyj, M. E. Hemler & R. R. Lobb: VCAM-1 on
activated endothelium interacts with the leukocyte integrin
VLA-4 at a site distinct from the VLA-4/fibronectin
binding site. Cell 60, 577-84 (1990)
93. Dustin, M. L., R. Rothlein, A. K. Bhan, C. A. Dinarello
& T. A. Springer: Induction by IL-1 and interferon-gamma:
tissue distribution, biochemistry, and function of a natural
adherence molecule (ICAM-1). J Immunol 137, 245-54
(1986)
94. Carlos, T. M. & J. M. Harlan: Leukocyte-endothelial
adhesion molecules. Blood 84, 2068-101 (1994)
95. Heiska L., K. Alfthan, M. Gronholm, P. Vilja, A.
Vaheri & O. Carpen: Association of ezrin with intercellular
adhesion molecule-1 and -2 (ICAM-1 and ICAM-2).
Regulation by phosphatidylinositol 4, 5-bisphosphate. J
Biol Chem 273, 21893-900 (1998)
96. Vaheri A., O. Carpen, L. Heiska, T.S. Helander, J.
Jaaskelainen, P. Majander-Nordenswan, M. Sainio, T.
Timonen & O. Turunen: The ezrin protein family:
membrane-cytoskeleton interactions
associations. Curr Opin Cell Biol 9, 659-66 (1997)
and disease
97. Castellano, F., C. Le Clainche, D. Patin, M. Carlier &
P. Chavrier: A WASp-VASP complex regulates actin
polymerization at the plasma membrane. EMBO J 20,
5603-14 (2001)
98. Allen, L. A. & A. Aderem: Molecular definition of
distinct cytoskeletal structures involved in complement-

Page 13

Protrusive structures in leukocyte extravasation
1861
and Fc receptor-mediated phagocytosis in macrophages. J
Exp Med 184, 627-37 (1996)
99. Wojciak-Stothard, B., L. Williams & A. J. Ridley:
Monocyte adhesion and spreading on human endothelial
cells is dependent on Rho-regulated receptor clustering. J
Cell Biol 145, 1293-307 (1999)
100. van Wetering, S., J. D. van Buul, S. Quik, F. P. Mul,
E. C. Anthony, J. P. ten Klooster, J. G. Collard & P. L.
Hordijk: Reactive oxygen species mediate Rac-induced loss
of cell-cell adhesion in primary human endothelial cells. J
Cell Sci 115, 1837-46 (2002)
101. van Wetering, S., N. van Den Berk, J. D. van Buul, F.
P. Mul, I. Lommerse, R. Mous, J. P. ten Klooster, J. J.
Zwaginga & P. L. Hordijk: VCAM-1-mediated Rac
signaling controls endothelial cell-cell contacts and
leukocyte transmigration. Am J Physiol Cell Physiol 285,
C343-52 (2003)
102. Cook-Mills, J. M.: VCAM-1 signals during
lymphocyte migration: role of reactive oxygen species. Mol
Immunol 39, 499-508 (2002)
103. Hordijk, P. L.: Endothelial signaling in leukocyte
transmigration. Cell Biochem Biophys 38, 305-22 (2003)
104. Etienne-Manneville, S., J. B. Manneville, P.
Adamson, B. Wilbourn, J. Greenwood & P. O. Couraud:
ICAM-1-coupled cytoskeletal
transendothelial lymphocyte migration involve intracellular
calcium signaling in brain endothelial cell lines. J Immunol
165, 3375-83 (2000)
rearrangements and
105. Thompson, P. W., A. M. Randi & A. J. Ridley:
Intercellular adhesion molecule (ICAM)-1, but not ICAM-
2, activates RhoA and stimulates c-fos and rhoA
transcription in endothelial cells. J Immunol 169, 1007-13
(2002)
106. Greenwood, J., S. Etienne-Manneville, P. Adamson &
P. O. Couraud: Lymphocyte migration into the central
nervous system: implication of ICAM-1 signalling at the
blood-brain barrier. Vascul Pharmacol 38, 315-22 (2002)
107. Hubbard, A. K. & R. Rothlein: Intercellular adhesion
molecule-1 (ICAM-1) expression and cell signaling
cascades. Free Radic Biol Med 28, 1379-86 (2000)
108. Wang, Q. & C. M. Doerschuk: The signaling
pathways induced by neutrophil-endothelial cell adhesion.
Antioxid Redox Signal 4, 39-47 (2002)
109. Clayton, A., R. A. Evans, E. Pettit, M. Hallett, J. D.
Williams & R. Steadman: Cellular activation through the
ligation of intercellular adhesion molecule-1. J Cell Sci
111, 443-53 (1998)
110. Sanchez-Madrid, F. & M. A. del Pozo: Leukocyte
polarization in cell migration and immune interactions.
EMBO J 18, 501-11 (1999)
111. Cinamon, G., V. Shinder & R. Alon: Shear forces
promote lymphocyte migration
endothelium bearing apical chemokines. Nat Immunol 2,
515-22 (2001)
across vascular
112. Weber, K. S., P. von Hundelshausen, I. Clark-Lewis,
P. C. Weber & C. Weber: Differential immobilization and
hierarchical involvement of chemokines in monocyte arrest
and transmigration on inflamed endothelium in shear flow.
Eur J Immunol 29, 700-12 (1999)
113. Wan, M., Y. Wang, Q. Liu, R. Schramm & H.
Thorlacius: CC chemokines induce P-selectin-dependent
neutrophil rolling and recruitment in vivo: intermediary role
of mast cells. Br J Pharmacol 138, 698-706 (2003)
114. Sandig, M., E. Negrou & K. A. Rogers: Changes in
the distribution of LFA-1, catenins, and F-actin during
transendothelial migration of monocytes in culture. J Cell
Sci 110, 2807-18 (1997)
115. Sandig, M., M. L. Korvemaker, C. V. Ionescu, E.
Negrou & K. A. Rogers: Transendothelial migration of
monocytes in rat aorta: distribution of F-actin, alpha-
catenin, LFA-1, and PECAM-1. Biotech Histochem 74,
276-93 (1999)
116. Worthylake, R. A., S. Lemoine, J. M. Watson & K.
Burridge: RhoA is required for monocyte tail retraction
during transendothelial migration. J Cell Biol 154, 147-60
(2001)
117. Worthylake, R. A. & K. Burridge: Leukocyte
transendothelial migration: orchestrating the underlying
molecular machinery. Curr Opin Cell Biol 13, 569-77
(2001)
118. Johnson-Leger, C., M. Aurrand-Lions & B. A. Imhof:
The parting of the endothelium: miracle, or simply a
junctional affair? J Cell Sci 113, 921-33 (2000)
119. Hirata, K., T. Ishida, K. Penta, M. Rezaee, E. Yang, J.
Wohlgemuth & T. Quertermous: Cloning of an
immunoglobulin family adhesion molecule selectively
expressed by endothelial cells. J Biol Chem 276, 16223-31
(2001)
120. Nasdala, I., K. Wolburg-Buchholz, H. Wolburg, A.
Kuhn, K. Ebnet, G. Brachtendorf, U. Samulowitz, B.
Kuster, B. Engelhardt, D. Vestweber & S. Butz: A
transmembrane tight junction protein selectively expressed
on endothelial cells and platelets. J Biol Chem 277, 16294-
303 (2002)
121. Luscinskas, F. W., S. Ma, A. Nusrat, C. A. Parkos &
S. K. Shaw: The role of endothelial cell lateral junctions
during leukocyte trafficking. Immunol Rev 186, 57-67
(2002)
122. Martin-Padura, I., S. Lostaglio, M. Schneemann, L.
Williams, M. Romano, P. Fruscella, C. Panzeri, A.
Stoppacciaro, L. Ruco, A. Villa, D. Simmons & E. Dejana:

Page 14

Protrusive structures in leukocyte extravasation
1862
Junctional adhesion molecule, a novel member of the
immunoglobulin superfamily that distributes at intercellular
junctions and modulates monocyte transmigration. J Cell
Biol 142, 117-27 (1998)
123. Palmeri, D., A. van Zante, C. C. Huang, S.
Hemmerich & S. D. Rosen: Vascular endothelial junction-
associated molecule, a
immunoglobulin superfamily, is localized to intercellular
boundaries of endothelial cells. J Biol Chem 275, 19139-45
(2000)
novel member of the
124. Arrate, M. P., J. M. Rodríguez, T. M. Tran, T. A.
Brock & S. A. Cunningham: Cloning of human junctional
adhesion molecule 3 (JAM3) and its identification as the
JAM2 counter-receptor. J Biol Chem 276, 45826-32 (2001)
125. Aurrand-Lions, M. A., L. Duncan, L. Du Pasquier &
B. A. Imhof: Cloning of JAM-2 and JAM-3: an emerging
junctional adhesion molecular family? Curr Top Microbiol
Immunol 251, 91-8 (2000)
126. Cunningham, S. A., M. P. Arrate, J. M. Rodríguez, R.
J. Bjercke, P. Vanderslice, A. P. Morris & T. A. Brock: A
novel protein with homology to the junctional adhesion
molecule. Characterization of leukocyte interactions. J Biol
Chem 275, 34750-6 (2000)
127. Aurrand-Lions, M. A., L. Duncan, C. Ballestrem & B.
A. Imhof: JAM-2, a novel immunoglobulin superfamily
molecule, expressed by endothelial and lymphatic cells. J
Biol Chem 276, 2733-41 (2001)
128. Bazzoni, G., O. M. Martínez-Estrada, F. Orsenigo, M.
Cordenonsi, S. Citi & E. Dejana: Interaction of junctional
adhesion molecule with the tight junction components ZO-
1, cingulin, and occludin. J Biol Chem 275, 20520-6 (2000)
129. Ebnet, K., C. U. Schulz, M. K. Meyer Zu
Brickwedde, G. G. Pendl & D. Vestweber: Junctional
adhesion molecule interacts with the PDZ domain-
containing proteins AF-6 and ZO-1. J Biol Chem 275,
27979-88 (2000)
130. Ostermann, G., K. S. Weber, A. Zernecke, A.
Schroder & C. Weber: JAM-1 is a ligand of the beta(2)
integrin LFA-1 involved in transendothelial migration of
leukocytes. Nat Immunol 3, 151-8 (2002)
131. Aurrand-Lions, M., C. Johnson-Leger, C. Wong, L.
Du Pasquier & B. A. Imhof: Heterogeneity of endothelial
junctions is reflected by differential expression and specific
subcellular localization of the three JAM family members.
Blood 98, 3699-707 (2001)
132. Santoso, S., U. J. Sachs, H. Kroll, M. Linder, A. Ruf,
K. T. Preissner & T. Chavakis: The junctional adhesion
molecule 3 (JAM-3) on
counterreceptor for the leukocyte integrin Mac-1. J Exp
Med 196, 679-91 (2002)
human platelets is a
133. Cunningham, S. A., J. M. Rodríguez, M. P. Arrate, T.
M. Tran & T. A. Brock: JAM2 interacts with alpha4beta1.
Facilitation by JAM3. J Biol Chem 277, 27589-92 (2002)
134. Lampugnani, M. G., M. Corada, L. Caveda, F.
Breviario, O. Ayalon, B. Geiger & E. Dejana: The
molecular organization of endothelial cell to cell junctions:
differential association of plakoglobin, beta-catenin, and
alpha-catenin with vascular endothelial cadherin (VE-
cadherin). J Cell Biol 129, 203-17 (1995)
135. Del Maschio, A., A. Zanetti, M. Corada, Y. Rival, L.
Ruco, M. G. Lampugnani & E. Dejana: Polymorphonuclear
leukocyte adhesion triggers the disorganization of
endothelial cell-to-cell adherens junctions. J Cell Biol 135,
497-510 (1996)
136. Allport, J. R., H. Ding, T. Collins, M. E. Gerritsen &
F. W. Luscinskas: Endothelial-dependent mechanisms
regulate leukocyte transmigration: a process involving the
proteasome and disruption of the vascular endothelial-
cadherin complex at endothelial cell-to-cell junctions. J
Exp Med 186, 517-27 (1997)
137. Moll, T., E. Dejana & D. Vestweber: In vitro
degradation of endothelial catenins by a neutrophil
protease. J Cell Biol 140, 403-7 (1998)
138. Allport, J. R., W. A. Muller & F. W. Luscinskas:
Monocytes induce reversible focal changes in vascular
endothelial cadherin complex during transendothelial
migration under flow. J Cell Biol 148, 203-16 (2000)
139. Shaw, S. K., P. S. Bamba, B. N. Perkins & F. W.
Luscinskas: Real-time imaging of vascular endothelial-
cadherin during leukocyte
endothelium. J Immunol 167, 2323-30 (2001)
transmigration across
140. Feng, D., J. A. Nagy, K. Pyne, H. F. Dvorak & A. M.
Dvorak: Neutrophils emigrate from venules by a
transendothelial cell pathway in response to FMLP. J Exp
Med 187, 903-15 (1998)
141. Burns, A. R., D. C. Walker, E. S. Brown, L. T.
Thurmon, R. A. Bowden, C. R. Keese, S. I. Simon, M. L.
Entman & C. W. Smith: Neutrophil transendothelial
migration is independent of tight junctions and occurs
preferentially at tricellular corners. J Immunol 159, 2893-
903 (1997)
142. Burns, A. R., R. A. Bowden, S. D. MacDonell, D. C.
Walker, T. O. Odebunmi, E. M. Donnachie, S. I. Simon, M.
L. Entman & C. W. Smith: Analysis of tight junctions
during neutrophil transendothelial migration. J Cell Sci
113, 45-57 (2000)
143. Muller, W. A., S. A. Weigl, X. Deng & D. M.
Phillips: PECAM-1 is required for transendothelial
migration of leukocytes. J Exp Med 178, 449-60 (1993)
144. Wong, C. W., G. Wiedle, C. Ballestrem, B.
Wehrle-Haller, S. Etteldorf, M. Bruckner, B. Engelhardt, R.
H. Gisler & B. A. Imhof: PECAM-1/CD31 trans-
homophilic binding at the intercellular junctions is
independent of its cytoplasmic domain; evidence for
heterophilic interaction with integrin alphavbeta3 in Cis.
Mol Biol Cell 11, 3109-21 (2000)

Page 15

Protrusive structures in leukocyte extravasation
1863
145. Aurrand-Lions, M. A., C. Johnson-Leger & B. A.
Imhof: Role of interendothelial adhesion molecules in the
control of vascular functions. Vascul Pharmacol 39, 239-46
(2002)
146. Newman, D. K., C. Hamilton & P. J. Newman:
Inhibition of antigen-receptor signaling by Platelet
Endothelial Cell Adhesion Molecule-1 (CD31) requires
functional ITIMs, SHP-2, and p56(lck). Blood 97, 2351-7
(2001)
147. Ilan, N., L. Cheung, E. Pinter & J. A. Madri: Platelet-
endothelial cell adhesion molecule-1 (CD31), a scaffolding
molecule for selected catenin family members whose
binding is mediated by
serine/threonine phosphorylation. J Biol Chem 275, 21435-
43 (2000)
different tyrosine and
148. Mamdouh, Z., X. Chen, L. M. Pierini, F. R. Maxfield
& W. A. Muller: Targeted recycling of PECAM from
endothelial surface-connected
diapedesis. Nature 421, 748-53 (2003)
compartments during
149. Schenkel, A. R., Z. Mamdouh, X. Chen, R. M.
Liebman & W. A. Muller: CD99 plays a major role in the
migration of monocytes through endothelial junctions. Nat
Immunol 3, 143-50 (2002)
Abbreviations: ERM: ezrin-radixin-moesin proteins;
ESAM: endothelial cell-selective adhesion molecule;
ICAM-1: intercellular adhesion
junctional adhesion molecule;
function-associated antigen-1;
endothelial adhesion molecule-1; PSGL-1: P-selectin
glycoprotein ligand-1; TEM: transendothelial migration;
VCAM-1: vascular cell adhesion molecule-1; VLA-4: very-
late antigen-4.
molecule-1;
LFA-1:
PECAM-1:
JAM:
lymphocyte
platelet
Key Words: Adhesion, Cytoskeleton, Docking structure
Endothelium, Leukocyte,
Transendothelial migration, Review
Rolling, Tethering,
Send correspondence to: Dr. Francisco Sánchez-Madrid:
Servicio de Inmunología, Hopital de la Princesa,
Universidad Autónoma de Madrid, C/Diego de León 62,
28006 Madrid, Spain. Tel.: 34-91-3092115. Fax: 34-91-
5202374. E-mail: fsanchez.hlpr@salud.madrid.org