Dietmar Vestweber
Adhesion and signaling molecules
controlling the transmigration of
leukocytes through endothelium
Author’s address
Dietmar Vestweber
Max Planck Institute of Molecular Biomedicine,
Münster, Germany.
Summary: Migration of leukocytes into tissue is a key element of innate
and adaptive immunity. While the capturing of leukocytes to the blood
vessel wall is well understood, little is known about the mechanisms
underlying the actual transmigration of leukocytes through the vessel wall
(diapedesis). Even a basic question such as whether leukocytes migrate
through openings between adjacent endothelial cells (junctional pathway)
or through single endothelial cells (transcellular pathway) is still a matter of
intensive debate. It is generally accepted that both pathways exist; however,
whether they are of equal physiological significance is unclear. Several
endothelial adhesion and signaling molecules have been identified, most of
them at endothelial cell contacts, which participate in leukocyte diapedesis.
A concept is evolving suggesting that transendothelial migration of
leukocytes is a stepwise process. Blocking or eliminating some of the
different adhesion and signaling proteins results in very different effects,
such as trapping of leukocytes above endothelial cell contacts, in between
endothelial cells, or between the endothelium and the underlying
basement membrane. Other proteins are involved in the opening of
endothelial cell contacts and yet others in their maintenance providing the
barrier for extravasating leukocytes. The various molecular players and the
functional steps involved in diapedesis are discussed.
Correspondence to:
Dietmar Vestweber
Max Planck Institute of Molecular Biomedicine
Röntgenstr. 20
D-48149 Münster, Germany
Tel.: þ 49 251 70365 210
Fax: þ 49 251 70365 299
E-mail: [email protected]
Keywords: leukocyte extravasation, diapedesis, transendothelial migration, leukocyte
trafficking
Introduction
Immunological Reviews 2007
Vol. 218: 178–196
Printed in Singapore. All rights reserved
ª 2007 The Author
Journal compilation ª 2007 Blackwell Munksgaard
Immunological Reviews
0105-2896
178
The endothelium regulates innate and adaptive immune
responses by controlling the extravasation of leukocytes from
the blood into tissues. To actively carry out immune defense,
neutrophils, monocytes, and antigen-experienced lymphocytes
enter tissues at any site of the body where an infection or injury
stimulates an inflammatory reaction. During the process of
immune surveillance, lymphocytes continuously recirculate
between the blood and lymphoid tissue to seek out their
cognate antigen. The latter process is called ‘lymphocyte
homing’ and occurs in lymph nodes at specialized sites of
the vascular bed, the high endothelial venules (HEVs). In
contrast, inflamed tissue is entered by leukocytes through
Vestweber Transmigration of leukocytes through endothelium
non-specialized postcapillary venules that have been transiently
activated by inflammatory stimuli to express the molecular
machinery that is required to capture leukocytes and attract
them into tissue.
Activated endothelium in inflamed tissue as well as the
specialized endothelium of HEVs in lymph nodes recruit
leukocytes, using a number of well-studied adhesion molecules
and chemoattractants. These molecules function in a multistep
cascade (1, 2) that is initiated by the selectins, a small group of
three carbohydrate-binding cell adhesion molecules that are
able to capture leukocytes from the rapidly flowing blood
stream (3). The binding characteristics of the selectins and their
ligands are specialized for capturing flowing leukocytes to the
endothelium. This still transient interaction slows down
leukocytes, resulting in a rolling movement on the vessel wall.
Rolling leukocytes ‘sense’ chemotactic factors such as chemokines presented on the endothelial surface, which bind to their
respective G-protein-coupled receptors on the leukocyte
surface. Signaling by these receptors leads to the activation of
leukocyte integrins, improving their affinity and/or avidity for
ligands on the endothelium, which are usually members of the
immunoglobulin superfamily (IgSF). This multistep model has
been further refined by studies focusing on the mechanisms that
promote the transition of the rolling movement to the firm
adhesion. Whereas P-selectin and its main ligand P-selectin
glycoprotein ligand 1 are specialized in the initiation of
capturing (4), E-selectin stabilizes the rolling and is responsible
for slowing it down (5, 6). A low-affinity conformation of the
integrin leukocyte function-associated antigen 1 (LFA-1) seems
to be able to take over from this state and is able to support
rolling (7). The fully activated integrin then transforms the
rolling movement to the arrested state (8).
These sophisticated studies illustrate that the process of
leukocyte capturing and chemoattraction is very well studied,
and the major adhesion molecules and chemokines, the order
by which they interact, and the principle mechanisms are well
known. In contrast, the actual diapedesis process, the transmigration of leukocytes through the endothelial cell layer and
the underlying basement membrane, is still a fascinating
unexplained phenomenon with many more questions than
answers. Even principal questions such as whether leukocytes
move through the contacts between endothelial cells (junctional pathway) or through single endothelial cells (transcellular pathway) are subject of intense debates (9–12).
During recent years, several new endothelial membrane
proteins, most of them located at endothelial cell contacts, have
been shown to be involved in leukocyte diapedesis (Table 1).
Although we are still far from understanding how all these
different molecules function in concert, for several of these
proteins, interesting mechanistic details have just recently been
shown. Evidence has been presented defining different steps in
the diapedesis process. Furthermore, the various membrane
proteins participating in the diapedesis process can be
subdivided into different categories: those that function for
myeloid cells and lymphocytes and those that exclusively
mediate extravasation of myeloid cells. Another criterion by
which they can be distinguished is their dependence on certain
cytokines. They also differ in their subcellular localization, with
some being expressed at tight or adherens junctions, others all
along the endothelial contacts, and a few on the apical cell
surface (Fig. 1).
Among the various endothelial membrane proteins that have
been shown in recent years to participate in leukocyte
diapedesis, the endothelial-specific cadherin VE-cadherin is
Table 1. Adhesion receptors shown to participate in transendothelial migration of leukocytes
Molecule
PECAM-1
CD99
JAM-A
JAM-B*
JAM-Cy
ESAM
PVR
VE-cadherin
ICAM-1
ICAM-2
Superfamily
IgSF
Unique
IgSF
IgSF
IgSF
IgSF
IgSF
Cadherin
IgSF
IgSF
Ligands
Homophilic, Homophilic,
Homophilic,
Homophilic,
Homophilic Homophilic LFA-1
JAM-C, VLA-4 JAM-B, Mac-1 Homophilic, DNAM-1 fibrin
b2-integrins LFA-1
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
n.d.
þ
þ
þ
þ
þ
þ
þ
þ
þ
forms a barrier
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
n.d.
n.d.
þ
þ
n.d.
þ
þ
n.d.
n.d.
þ
þ
þ
þ
Distribution
Endothelium
Neutrophils
Monocytes
Lymphocytes
Mediates transmigration
of neutrophils
Monocytes
Lymphocytes
*A systematic expression analysis on leukocytes was only reported in one publication, showing that JAM-B is not expressed on human peripheral blood
cells (150). Although it is likely that JAM-B could be involved in leukocyte extravasation, direct evidence is still lacking.
yJAM-C has not been detected on peripheral blood leukocytes of the mouse, but was detected on human monocytes and on a subpopulation of B
lymphocytes and on activated T cells. n.d., not determined
Immunological Reviews 218/2007
179
Vestweber Transmigration of leukocytes through endothelium
Fig. 1. Adhesion receptors involved in the
diapedesis process. The junctional and the
non-junctional adhesion receptors at endothelial cell contacts are depicted between
different cells for optical clarity. The nectinrelated PVR was classified as adherens junction protein because of its similarity to the
nectins. The two receptor-type tyrosine
phosphatases VE-PTP and RPTP are indeed
able to affect the function of VE-cadherin;
whether they are indeed involved in the
leukocyte diapedesis process has not yet been
shown.
unique because it is the only one that does not support but
rather blocks leukocyte extravasation. It represents a barrier for
extravasating leukocytes because antibodies against VE-cadherin support leukocyte extravasation. In addition, VE-cadherin
moves away from endothelial cell contacts at sites where
leukocytes transmigrate, implying that homophilic VE-cadherin
interactions probably need to be dissociated to allow leukocytes
to pass. In contrast, all other membrane proteins involved in
diapedesis support the process because antibodies against these
proteins or elimination by gene disruption slows down or
inhibits diapedesis. Some of these proteins are found at tight
junctions: junctional adhesion molecule (JAM)-A, JAM-B, JAMC, and the endothelial selective adhesion molecule (ESAM).
Another potential adherens junction protein involved in
diapedesis is related to the nectin family and is called the
poliovirus receptor (PVR). Proteins found all along the contacts
of endothelial cells are the platelet endothelial adhesion
molecule 1 (PECAM-1), CD99, and the intercellular adhesion
molecule 2 (ICAM-2), although none of them is completely
absent from the apical surface. ICAM-1 is a major player in
leukocyte extravasation. It is essential for adhesion of basically
any type of leukocyte to the apical surface of endothelium and
probably also involved in the transmigration step. In contrast,
vascular cell adhesion molecule 1 (VCAM-1), which also
mediates adhesion of monocytes and lymphocytes to endothelium, is probably only indirectly involved in diapedesis by
signaling mechanisms.
This review focuses on our current knowledge of the
diapedesis of leukocytes through the vessel wall. It begins with
a short survey of some of the more historical studies and of the
most recent results that have rekindled the rather old debate on
the transcellular versus the paracellular pathway. A detailed
discussion of the currently known endothelial cell surface and
signaling molecules known to be involved in the diapedesis
process follows. The review ends by comparing the various
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Immunological Reviews 218/2007
known players, stressing the functional differences of the
various membrane proteins that may allow for classification
into different categories and for assigning some to different
steps of the process.
Do leukocytes move through endothelial cell contacts or
through endothelial cells?
Since the very first days of studying leukocyte extravasation,
evidence for the two possible pathways, either through the
junctional route or through the body of an endothelial cell, has
been reported. Ultrastructural analysis of in vivo extravasating
leukocytes performed in the 1960s and 1970s generated
evidence supporting both pathways. On investigating blood
vessels in inflamed tissue, polymorphonuclear leukocytes were
found to migrate through or close to junctions (13), although
some were also found to be surrounded by endothelial
cytoplasm, at a distance from intact junctions (13, 14). Even
studies that analyzed serial sections came to different conclusions. Two such studies from the 1960s suggested that
leukocytes would mostly passage through interendothelial
junctions of inflamed venules (15, 16), whereas a more recent
in vivo study reported that N-formyl-methionyl-leucyl-phenylalanine (fMLP)-attracted neutrophils extravasated through the
endothelial body near intact junctions (17).
The constitutive migration of lymphocytes into lymph
nodes, which occurs in the HEVs, was suggested to proceed
directly through the cell body of high endothelial cells (18),
whereas granulocytes were described in the same type of vessels
in inflamed lymph nodes as moving through endothelial cell
contacts (18). The migration of lymphocytes through the
endothelial cytoplasm was confirmed by another study (19),
while others reported that lymphocytes move through the
extracellular space between endothelial cells, leaving the
intracellular lumen of endothelial cells intact (20).
Vestweber Transmigration of leukocytes through endothelium
The conclusion of all these ultrastructural studies is that each
of the two pathways exists in vivo. Because these studies are quite
laborious and only serial sections through the whole body of
a single transmigrating leukocyte are able to clearly show which
of the two pathways is used, it is almost impossible by this
technique to determine whether both pathways are of equal
physiological relevance or whether one of the two pathways
may be the dominant route. It is possible that the relative
contribution of each pathway may depend on the type of blood
vessel, the type of tissue, the leukocyte-recruiting stimulus, or
the type of leukocyte.
Quantitative real-time imaging studies showed recently that
both pathways can be observed in vitro when leukocytes
transmigrate through endothelial cell monolayers. However,
each of these studies found that only a minority of leukocytes
used the transcellular route through the endothelium. Allowing
human neutrophils, monocytes, and lymphocytes to migrate
through the monolayer of human umbilical vein endothelial
cells (HUVECs), which were growing on cover slips coated with
various matrix proteins, showed that only 7% of monocytes, 5%
of neutrophils, and 11% of the lymphocytes were able to move
through the monolayer on a transcellular route (21). The
remainder of the leukocytes transmigrated at interendothelial
cell contacts.
This finding was confirmed in another report analyzing
leukocyte transendothelial migration through HUVECs under
flow, which described a minor but under certain conditions
increasing contribution of the transcellular pathway (22).
While others described a stimulatory influence of flow on
transcellular diapedesis (23), flow did not influence the
tendency of leukocytes to use the transcellular path in this
report. Whereas almost none of the migrating neutrophils were
found to take the transcellular route through 4 h tumor necrosis
factor-a (TNF-a)-activated HUVECs, about 15% took this route
through 24-h-activated endothelial cells. In addition, overexpression of ICAM-1 in transfected immortalized HUVECs led
to an increased relative contribution (up to 50%) of the
transcellular pathway to diapedesis. T cells were found not to
use this pathway (22). In another report, about 10% of
transmigrating lymphocytes were found to use the transcellular
pathway through HUVECs. ICAM-1 was suggested to participate in this process (24). As a first hint for a possible
mechanism, antibody-triggered cross-linking of apical ICAM1 as well as docking of T lymphocytes induced the translocation
of apical ICAM-1 to caveolin-rich membrane domains,
resulting in transcytosis of ICAM-1 and of the lymphocytes to
the endothelial basal membrane. Downregulation of caveolin
expression in HUVECs blocked transcellular migration of
T cells, although a reduction of the overall transendothelial
migration was too weak to be detectable. This finding
highlights that the transcellular pathway only represents a minor
contribution to the overall migration process or that the
leukocytes could easily switch to the junctional pathway.
Surprisingly, Nieminen et al. (25) found that the majority of
peripheral blood mononuclear cells (mainly T and B cells) used
the transcellular pathway, whereas neutrophils were found to
use the junctional route. It may well be that slight variations of
the assay conditions affect the choice of leukocytes between the
different pathways.
It has been suggested that the migration through endothelial
junctions occurs preferentially at specialized sites, the so-called
tricellular contacts, where three endothelial cells touch each
other (26), although this idea has not been confirmed in all
in vitro studies (27). In epithelium, tricellular contacts are
structures with unique cell contact proteins, such as tricellulin,
an occludin-related membrane protein that is strongly enriched
at such special sites of cell contacts (28). Muller (10) pointed
out that extended overlaps of endothelial cell edges at tricellular
contacts could easily create a topology that would explain how
a leukocyte migrating through endothelial junctions might
appear in tissue sections, as if it would be migrating directly
through endothelial cytoplasm, with endothelial junctions
intact adjacent to the transmigrating leukocyte (10).
A major question in future studies will be to determine
whether both pathways are of equal physiological relevance, or
whether the junctional pathway is the major one, as suggested
by most of the in vitro results. At present, several endothelial
membrane proteins that participate in the transendothelial
migration of leukocytes in vitro and in vivo have been localized at
endothelial cell contacts. The significant contribution of these
adhesion receptors to the extravasation of leukocytes in vivo is
a strong argument for the relevance of the junctional pathway
under physiological conditions.
Platelet endothelial cell adhesion molecule-1
The transmembrane protein at interendothelial cell contacts that
was suggested first to participate in transendothelial migration
of leukocytes was PECAM-1 (CD31). It belongs to the IgSF,
contains six Ig domains, can mediate homophilic interactions,
and is not confined to any type of junctional structures (29). In
addition to endothelial cells, PECAM-1 is expressed on platelets,
neutrophils, monocytes, and particular subsets of T cells.
Pretreating neutrophils and monocytes with antibodies
against PECAM-1 blocks transendothelial migration of these
leukocytes (30). Blocking endothelial cell PECAM-1 with these
Immunological Reviews 218/2007
181
Vestweber Transmigration of leukocytes through endothelium
antibodies had the same effect. Antibodies against PECAM-1 as
well as a PECAM–Fc fusion protein could block neutrophil
extravasation in vivo in mice and rats (31, 32). Although
disruption of the PECAM-1 gene in the mouse strain C57/Bl6
had only a minor effect on neutrophil extravasation in
a peritonitis model (33), breeding these mice with a different
mouse strain showed that the lack of PECAM-1 caused a dramatic
reduction in the recruitment of neutrophils into inflamed
peritoneum (34).
The mechanism by which PECAM-1 participates in leukocyte
extravasation is not yet known in detail. Early in vitro studies with
endothelial cells grown on a collagen gel suggested that PECAM1 acts at two different steps of the diapedesis process (35)
(Fig. 2). The first step was shown in a study with antibodies
against the first two PECAM-1 Ig domains, which blocked the
migration of monocytes through the endothelial cell layer,
resulting in the accumulation of leukocytes on the apical surface
of the endothelial cells. The second step seemed to require the
domain six because antibodies against this domain blocked the
migration of monocytes into the collagen gel, resulting in
monocytes being stuck between endothelial cells and the
collagen matrix (Fig. 2).
The first step was confirmed in vivo with a PECAM–Fc fusion
protein containing the first Ig domain of PECAM-1, which
caused accumulation of leukocytes on the luminal side of
the endothelium (32), although no electron microscopy or
intravital microscopy were performed in this study. The second
step was shown in vivo, showing accumulation of neutrophils
between endothelial cells and the basement membrane upon
blocking PECAM-1 with polyclonal antibodies in rat mesenteric
venules (36). Trapping of neutrophils between the endothelium and the basement membrane was also found in PECAM-1deficient mice (33).
It is still unknown whether PECAM-1 functions during
diapedesis as an adhesion molecule or as a signaling receptor. Its
function as a receptor has been thoroughly investigated (37).
PECAM-1 belongs to a subfamily of the IgSF that contain
immunoreceptor tyrosine-based inhibitory motifs (ITIMs) (38,
39). When members of the Ig–ITIM family are stimulated, they
become phosphorylated on distinct tyrosine residues located
within their cytoplasmic ITIM, resulting in the recruitment of
Src-homology (SH)-2-domain-containing intracellular lipid
and protein tyrosine phosphatases, such as SHIP, SHP-1, or
SHP-2. These enzymes, once recruited to their cytoplasmic
anchors and activated, are then able to affect a wide range of
cellular events by dephosphorylating their appropriate substrates, thereby inhibiting tyrosine kinase-mediated signaling,
proliferation, and cellular activation.
PECAM-1 carries two ITIMs and is able to bind to SHP-2 (40,
41), SHP-1 (42, 43), and SHIP (44). Cellular activation events
that lead to PECAM-1 tyrosine phosphorylation include shear
(45) and aggregation of the IgE receptor (46) or of PECAM-1
Fig. 2. Different steps of the diapedesis process. On the basis of
blocking the function of PECAM-1 and CD99, three consecutive
steps of the diapedesis process have been defined, depicted here
from left to right. Step 1: Blocking the first N-terminal Ig domain
of PECAM-1 has been reported to arrest leukocytes on the apical
surface of endothelial cells (32, 35). Step 2: Blocking CD99
resulted in vitro in arresting monocytes between endothelial cells
(140). Step 3: The lack of PECAM-1 as well as antibodies against
PECAM-1 cause an accumulation of myeloid cells between the basal
side of endothelial cells and the basement membrane (35, 36, 52).
It is important to note that only step 3 has been demonstrated so
far in vivo.
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Immunological Reviews 218/2007
Vestweber Transmigration of leukocytes through endothelium
itself (47). Monoclonal antibodies against PECAM-1 can cause
activation of b1-integrins (48), b2-integrins (49–51), and b3integrins (47). It has been speculated that this activation could
be based on antibody-mediated sequestration of the inhibitory
receptor PECAM-1 away from activating receptors, resulting in
activated integrins (37).
An interesting suggestion is that the expression of integrin
a6b1 becomes upregulated on the surface of transmigrating
neutrophils and that this process requires the presence of
PECAM-1 on endothelium as well as on the neutrophils (52).
The same report showed that antibodies against the integrin
a6b1 trapped neutrophils between endothelium and basement
membrane, similarly as observed with PECAM-1 deficiency.
Thus, PECAM-1 may act by inducing a6b1 on neutrophils, and
this interaction may be needed for migration on or through the
basement membrane. In agreement, it was found in an in vitro
study using a human mesothelioma cell line that PECAM-1 is
needed for signaling on the leukocyte side rather than on the
endothelial side during transendothelial migration of leukocytes (53). Provided the mesothelioma cells are a valid model
for endothelial cells, this finding is quite important because
PECAM-1 is a very efficient receptor on endothelial cells in other
settings (54, 55); yet none of these signaling functions would
seem to be needed during transmigration.
The idea that PECAM-1 is needed as a receptor on neutrophils
to activate these cells is also in line with another finding.
PECAM-1 deficiency was found to reduce leukocyte extravasation, only if inflammation was induced with interleukin (IL)-1b
but not if TNF-a was used (56). Surprisingly, mice that carried
the PECAM-1 gene disruption on another genetic background
(FVB/n mice) did not show this IL-1b specificity of the PECAM1 function (34). Another study showed that anti-PECAM-1
antibodies only could block IL-1b-triggered but not fMLPtriggered leukocyte extravasation in rats (36). In line with this
study, only IL-1b-triggered but not IL-8- or leukotriene-B4triggered neutrophil transmigration through a layer of
endothelial cells could be blocked with antibodies against
PECAM-1. The inhibitory effect on neutrophil extravasation
in vivo with antibodies against the integrin a6b1 showed the same
cytokine dependency as was described for PECAM-1. Because
TNF-a, leukotriene-B4, IL-8, and fMLP are all potent activators
of leukocytes, whereas IL-1b exclusively activates the endothelium, it is intriguing to speculate that PECAM-1 is needed as
a leukocyte activation signal, yet is dispensable if leukocyte
activation has already been triggered by inflammatory mediators. While this is indeed possible, novel findings (see below)
suggest that ICAM-2 and JAM-A show the same cytokinespecific role in leukocyte extravasation as PECAM-1, although
they are ligands of leukocyte integrins rather than stimulators of
integrin activation. Thus, further studies are necessary to fully
understand the basis of the cytokine specificity of the function
of PECAM-1 and that of other endothelial contact proteins.
Tight junction-associated endothelial membrane proteins
Junctional adhesion molecule-A
JAM-A was originally discovered in the mouse as a cell surface
protein at endothelial and epithelial cell contacts and on platelets
(57) as well as on leukocytes (58, 59). In epithelial cells, it was
found to be closely associated with tight junctions (60). Like
PECAM-1, JAM-A is a member of the IgSF, containing two Ig
domains. A monoclonal antibody against JAM-A was found to
block the migration of monocytes through the monolayer of
cultured endothelial cells and to inhibit monocyte accumulation
in a chemokine-triggered air pouch inflammation model in vivo
(57). The same antibody inhibited recruitment of monocytes
and neutrophils to the cerebrospinal fluid in a short-term TNFa-triggered meningitis model (61), whereas no effect was seen
with this antibody in a meningitis model triggered by bacteria
or viruses (62).
Two types of binding partners for JAM-A have been reported.
JAM-A is able to form homophilic interactions (63) by the Nterminus of the two Ig domains (64). In addition, human JAMA was found to bind by its second Ig domain to the leukocyte
integrin LFA-1, and this interaction was reported to be involved
in lymphocyte migration through human endothelial cell layers
in vitro (65). However, phorbol myristate acetate-activated
neutrophils that redistributed LFA-1 in a ring-like structure at
the site of transmigration through TNF-a-activated endothelial
cells caused coclustering of endothelial ICAM-1 but not of
endothelial JAM-A at the site of transmigration (66).
Besides the proposed function of JAM-A in leukocyte
extravasation, this protein seems to be important in the process
of epithelial cell contact formation. Monoclonal as well as
polyclonal antibodies were found to inhibit recovery of
transepithelial resistance after disruption of cell contacts by
calcium depletion (59, 67). However, the same antibodies that
could block the function of JAM-A in epithelial cell contact
formation did not inhibit neutrophil migration through
monolayers of cultured human endothelial cells (59). The
function of JAM-A in cell contact formation between epithelial
and possibly endothelial cells may well be connected to its
association with an intracellular complex of signaling molecules
comprising protease-activated receptor-3 (PAR-3), atypical
protein kinase C (PKC), Cdc42, and PAR-6 (60, 68). This
complex has been described as an important prerequisite for cell
Immunological Reviews 218/2007
183
Vestweber Transmigration of leukocytes through endothelium
polarity. Besides PAR-3, JAM-A binds also to other PDZ-domain
proteins, as has been reviewed (69).
It is still unclear as to how JAM-A participates in leukocyte
extravasation. Various studies with JAM-A-deficient mice
confirmed that JAM-A is involved in leukocyte extravasation.
However, JAM-A seemed to be necessary on different types of
cells, depending on the type of the inflammation model
analyzed. Two studies even suggested that it is exclusively JAMA on myeloid leukocytes that is involved in the diapedesis step,
whereas JAM-A on endothelial cells was found to be dispensable, as shown with mice selectively deficient for JAM-A on
endothelial cells (70, 71). It is the motility of leukocytes that
seems to be affected by the lack of JAM-A. Unexpectedly,
dendritic cells lacking JAM-A migrate faster through lymphatic
endothelial cell layers in vitro and migrate more efficiently into
lymph node tissue in vivo (70), whereas neutrophils lacking
JAM-A are slowed down, resulting in reduced neutrophil
recruitment into inflamed peritoneum (71). In contrast to these
two studies, participation of endothelial JAM-A in neutrophil
extravasation was clearly shown in a liver model for ischemia–
reperfusion injury (72). Thus, JAM-A seems to function in very
different ways and only in some cases does endothelial JAM-A
seem to be involved in the diapedesis process. Importantly,
leukocyte JAM-A seems to affect leukocyte motility independent
of endothelial JAM-A or other endothelial binding partners.
JAM-B and JAM-C
The two JAM-A-related proteins JAM-B and JAM-C are also
expressed at endothelial cell contacts (73). However, in contrast
to JAM-A, the tight junction localization is less clear. JAM-B is
not restricted to the apical part of lateral cell contacts in
transfected madin darby canine kidney (MDCK) cells (73),
whereas JAM-C is enriched at such sites (74). However,
ultrastructural analysis showed that JAM-C is a component of
desmosomes in intestinal epithelial cells (75).
A role for JAM-C in leukocyte extravasation is suggested by
experiments showing that a soluble JAM-C–Fc fusion protein
inhibits neutrophil extravasation in vivo (76) and neutrophil
migration through epithelial cell layers (75). Accumulation of
leukocytes in alveoli during acute pulmonary inflammation was
partially blocked in mice, using neutralizing antibodies against
JAM-C (77).
Each of the three JAM proteins seems able to interact with
leukocyte integrins (78). In addition, they bind (although
weakly) in a homophilic fashion, and JAM-B was found to bind
very efficiently to JAM-C (79). These interactions provide
numerous ways by which these molecules could participate in
leukocyte extravasation. In vitro studies showed that antibodies
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Immunological Reviews 218/2007
against JAM-C are able to dissociate JAM-C/JAM-B heterodimers at endothelial cell contacts and render JAM-C accessible
for the integrin Mac-1 on leukocytes (80).
An alternative function for JAM-C was recently suggested by
the finding that its upregulation in cultured endothelial cells
counteracted the adhesive function of VE-cadherin, and downregulation of its expression by RNA interference enhanced VEcadherin-mediated adhesion (81) (Fig. 3). Whether this finding
is related to leukocyte diapedesis is still speculative but certainly
worth analyzing.
Endothelial selective adhesion molecule
ESAM was found by a differential cDNA hybridization approach
as a gene expressed in endothelial cells that coded for a cell
adhesion molecule (82). Parallel to these studies, ESAM was
identified as an endothelial junction protein, by screening
monoclonal antibodies for staining of endothelial cell contacts.
On the basis of purifying and sequencing the protein, the
corresponding gene was cloned (83).
Although related to the JAM family members, ESAM differs
considerably in structure, cytosolic binding partners (84, 85), and
tissue distribution. ESAM and JAM-A, JAM-B, and JAM-C are
members of the CTX subset of the IgSF, whose members are
characterized by one V-like and one C2-like extracellular Ig domain
(86). ESAM belongs to a different subgroup of the CTX family than
the three JAMs. In addition, ESAM contains a type-I PDZ-domainbinding motif, whereas the three JAMs harbor a type-II motif.
Consequently, ESAM binds to different types of PDZ-domain
proteins than the JAMs. JAM-A, JAM-B, and JAM-C directly bind to
the cell polarity PDZ-domain proteins PAR-3 (60, 68, 84), ZO-1,
and others. ESAM does not bind to PAR-3 and ZO-1; instead, it
binds to the tight junction-associated PDZ-domain protein MAGI-1
(membrane-associated guanylate kinase with inverted domain
structure 1) (85). The cytoskeleton-associated binding partners,
such as synaptopodin and a-actinin 4, suggest that MAGI-1 might
be implicated in actin cytoskeleton dynamics. These differences in
the specificity for various scaffolding proteins strongly suggest
different signaling potentials for ESAM and the JAMs.
In contrast to the JAMs, expression of ESAM is strictly limited
to endothelial cells and platelets, not leukocytes or epithelia
(83). On platelets, ESAM is only expressed upon activation; on
endothelial cells, it is strictly restricted to tight junctions, as was
shown by immunogold labeling (83). Ectopic expression in
transfected epithelial cells showed a strict limitation of its
subcellular distribution to the apical ZO-1-containing areas of
lateral contacts.
Analyzing gene-deficient mice, it was found that ESAM is
involved in tumor angiogenesis (87), although no obvious
Vestweber Transmigration of leukocytes through endothelium
Fig. 3. Speculative model for potential mechanisms involved in the
regulation of the opening of endothelial junctions. Three potential
mechanisms that could be involved in the opening of endothelial cell
contacts during leukocyte diapedesis are depicted. The first is based on
the fact that the receptor-type tyrosine phosphatase VE-PTP supports the
function of VE-cadherin and dissociates from VE-cadherin upon
docking of neutrophils to the apical endothelial cell surface. It is an
attractive hypothesis that the binding of neutrophils to an endothelial
receptor might trigger the dissociation of VE-PTP from VE-cadherin and
thereby induce the opening of endothelial cell junctions. The second
mechanism is based on results with ESAM/ mice, showing that the
lack of ESAM slows down neutrophil extravasation as well as the
VEGF-induced opening of endothelial cell contacts. Thus, ESAM seems
to be involved in signaling mechanisms that trigger the opening of
endothelial cell junctions. Because knocking down ESAM in endothelial
cells leads to lower levels of activated Rho and because Rho has been
shown to destabilize endothelial tight junctions, Rho could be involved
in mechanisms that link ESAM to the destabilization of endothelial
junctions. The third mechanism refers to recent results that showed that
JAM-C can counteract the adhesive function of VE-cadherin and that the
GTPase Rap-1 was required for this effect. Whether this mechanism is
related to leukocyte extravasation has not yet been studied.
defects during embryonal angiogenesis have been observed.
Analyzing a possible function of ESAM in leukocyte extravasation, it was found that despite using various monoclonal and
polyclonal antibodies against ESAM in different inflammation
models, it was not possible to block the migration of activated
T cells into inflamed skin or of neutrophils into inflamed
peritoneum (88). However, ESAM-deficient mice had a clear
delay in neutrophil extravasation at early time points (2 h) after
stimulation with either thioglycollate or cytokines such as TNF-a
or IL-1b. Leukocyte extravasation was delayed at the diapedesis
step, as was observed by intravital microscopy of cytokinestimulated cremaster tissue (88). The fact that eliminating ESAM
inhibited neutrophil diapedesis, independent of the type of
cytokine or inflammatory stimulus, represents an important
difference in the function of PECAM-1 and ICAM-2. Blocking the
latter two with antibodies (in the C57Bl6 background) reduces
neutrophil extravasation only in IL-1b-stimulated but not in
TNF-a- or thioglycollate-stimulated tissue.
As for the other adhesion receptors at endothelial cell
contacts, the detailed mechanism by which ESAM participates in
leukocyte extravasation is not yet known. However, some
aspects of this mechanism could be clarified. The first concerned
a possible involvement of ESAM on platelets. Because platelets
are the only other cell type besides endothelial cells that express
ESAM and because platelets are known to contribute to
neutrophil extravasation, it was tested whether the depletion
of platelets would still affect neutrophil recruitment into
inflamed peritoneum in ESAM-deficient mice. Indeed, platelet
depletion led to a clear reduction of neutrophil extravasation in
wildtype mice; however, the same reduction was observed
when platelets were depleted in ESAM-deficient mice. Thus,
platelets contribute to neutrophil recruitment independent of
whether or not they express ESAM (88). It follows that only
ESAM at endothelial tight junctions is involved in neutrophil
diapedesis.
Another question was whether the lack of ESAM would affect
vascular permeability. The steady-state permeability for the
plasma protein-adsorbed dye Evans blue was unaltered in blood
vessels of ESAM-deficient mice. However, the vascular
endothelial growth factor (VEGF)-triggered increase of vascular
Immunological Reviews 218/2007
185
Vestweber Transmigration of leukocytes through endothelium
permeability in the skin was dramatically delayed in ESAM/
mice, suggesting that ESAM is involved in the signaling pathway
that connects VEGF signaling to the opening of endothelial
junctions (88). The studies measured the leak of plasma
protein-adsorbed Evans blue dye and were not affected by
changes in the blood flow in the analyzed VEGF-injected skin
area, as determined by measuring the quantity of hemoglobin
in this skin area by non-invasive white-light spectroscopy (88).
Thus, ESAM must be involved in signaling mechanisms that
transmit the VEGF stimulus to the opening of endothelial
junctions.
Although this signaling mechanism is not yet known in
detail, it is interesting that knocking down the expression of
ESAM in cultured endothelial cells leads to reduced levels of the
guanosine triphosphatase (GTPase) Rho. Transendothelial
migration of monocytes and neutrophils has been reported to
require active Rho and Rho kinase (89, 90). In addition, Rho
was found to mediate the opening of endothelial tight junctions
triggered by the chemokine monocyte chemotactic protein 1
(91). The same group showed recently that Rho acted in this
process by Rho kinase and PKCa and that increased endothelial
permeability was accompanied by Ser/Thr phosphorylation of
occludin and claudin 5 (92). Phosphorylation of occludin and
claudin 5 was also achieved by coculturing endothelial cells
with monocytes, and this phosphorylation as well as transendothelial migration of monocytes was blocked by inhibiting
Rho and Rho kinase (93). Collectively, these reports suggest that
activation of Rho is linked to the opening and destabilization of
endothelial tight junctions. This assumption combined with the
in vitro results showing reduced levels of activated endothelial
Rho upon inhibiting the expression of ESAM suggests that the
inhibition of neutrophil extravasation, which was observed in
ESAM-deficient mice, could at least partly be because of
insufficient levels of activated Rho required for the opening of
endothelial junctions (Fig. 3). In this context, it is intriguing
that the PDZ-domain protein MAGI, which was found to
associate with ESAM, is able to bind the RhoA-specific
nucleotide exchange factor mNET1 (94).
The lack of ESAM only delayed neutrophil extravasation into
inflamed peritoneum but not the recruitment of activated T cells
into inflamed skin (88). To exclude that this different result was
because of tissue differences or differences in the timing of
extravasation of the two different types of leukocytes,
a modified peritonitis experiment was performed by recruiting
neutrophils and lymphocytes with a mixture of IL-1b and the
chemokine CCL19, which were both injected into the
peritoneum. This experimental setup allowed the recruitment
of T cells together with neutrophils at the same time into the
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Immunological Reviews 218/2007
same tissue. Even under these conditions, the recruitment of
neutrophils but not of lymphocytes was delayed. Thus, by
whatever mechanism ESAM participates in the opening of
endothelial cell contacts during neutrophil extravasation, this
mechanism is not required for lymphocytes.
The permeability experiments suggest that ESAM is generally
involved in signaling mechanisms that control and mediate the
opening of endothelial contacts. The fact that the lack of ESAM
slows down VEGF-triggered opening of endothelial contacts
and slows down leukocyte diapedesis, in combination with the
fact that ESAM is located at tight junctions, very strongly argues
for ESAM’s participation in the junctional pathway of leukocyte
extravasation.
Adherens junction proteins: VE-cadherin and the
nectin PVR
VE-cadherin
Among the known surface proteins that are located between
endothelial cells and are able to mediate homophilic interactions, VE-cadherin is the only cell adhesion molecule known
to be essential for the maintenance of interendothelial cell
contacts. In analogy to the importance of E-cadherin for the
integrity of epithelial cell layers, it was found that antibodies
against human VE-cadherin dissociate the contacts of endothelial cells in culture (95). VE-cadherin is expressed at adherens
junctions and associates, like most other cadherins, with the
cytoplasmic catenins. b-catenin and placoglobin (g-catenin)
connect VE-cadherin to a-catenin, an important connection for
the stability of cadherin-mediated adhesions. It is thought that
this complex is needed for the interaction with the cytoskeleton,
although a direct, stable interaction with the actin cytoskeleton
has been questioned recently (96).
VE-cadherin-mediated endothelial cell contacts represent
a barrier for extravasating leukocytes in vivo because a monoclonal antibody against VE-cadherin administered intravenously in mice accelerated neutrophil migration into the
inflamed peritoneum (97). The importance of VE-cadherin for
the integrity of the blood vessel endothelium in vivo was
confirmed with another antibody that induced a concentrationand time-dependent increase in vascular permeability in heart
and lungs (98). In agreement with this finding, real-time
imaging of a VE-cadherin–green fluorescence protein fusion
protein in cultured HUVEC showed that this protein is removed
from cell contact sites during leukocyte transmigration and
reappears afterward, possibly by diffusion in the plane of the
membrane (99).
Vestweber Transmigration of leukocytes through endothelium
It is not yet known how VE-cadherin-mediated interendothelial cell contacts are opened during leukocyte extravasation.
According to an attractive hypothesis, the docking of leukocytes
to endothelial receptors triggers signals that lead to the decrease
of VE-cadherin-mediated adhesive forces. It was shown that
cross-linking of VCAM-1 (see below) leads to activation of
Rac1, which in turn leads to the production of reactive oxygen
species (ROS) and supports the opening of interendothelial cell
contacts and the transendothelial migration of leukocytes (100,
101). Furthermore, it was proposed that the effect of ROS could
be mediated by ROS-activated proline-rich tyrosine kinase 2
(Pyk2) that phosphorylates b-catenin and thereby affects the
function of VE-cadherin (102).
Several reports have correlated changes in tyrosine phosphorylation of cadherin/catenin complexes with modulation of
cadherin-mediated adhesion (103). Such correlations were also
found for VE-cadherin. Increased confluence of endothelial cells
in culture was accompanied by a decrease in VE-cadherin/
catenin tyrosine phosphorylation and a decrease of staining of
adherens junctions by anti-phosphotyrosine antibodies (104).
Thrombin stimulation of human endothelial cells promotes the
dissociation of the phosphatase SHP-2 from b-catenin, which
correlates with an increase in catenin phosphorylation (105).
Furthermore, stimulation of HUVECs with VEGF induced
tyrosine phosphorylation of VE-cadherin and the associated
catenins (except a-catenin) and increased endothelial permeability (106). Inhibition of expression of the receptor protein
tyrosine phosphatase-m (RPTP-m) in human lung microvascular
cells increased permeability for serum albumin in vitro, although
specific effects were only seen after 3 days of blocking the
expression of RPTP-m (107). The phosphorylation of the two
tyrosine residues at position 658 and 731 in the cytoplasmic tail
of human VE-cadherin was reported to inhibit the binding to
the catenins p120 and b-catenin and to reduce the barrier
function of VE-cadherin in transfected cells (108).
The endothelial-specific RPTP, called VE-PTP, was found to
associate specifically and selectively with VE-cadherin (109).
VE-PTP had originally been identified as an endothelial-specific
VE-PTP (110) that is highly homologous to human HPTP-b and
that associates with the tyrosine kinase receptor Tie-2. The
association of VE-PTP with VE-cadherin is fundamentally
different from other RPTP/cadherin interactions because it is
not mediated by a catenin or by cytoplasmic cadherin domains
but instead by the most membrane proximal, extracellular
domains of VE-cadherin and VE-PTP. Expression of VE-PTP in
triple-transfected Chinese hamster ovary (CHO) cells reversed
the tyrosine phosphorylation of VE-cadherin elicited by VEGFreceptor 2 (VEGFR-2). Expression of VE-PTP under an inducible
promoter in CHO cells, transfected with VE-cadherin and
VEGFR-2, increased VE-cadherin-mediated barrier integrity of
a cellular monolayer. Thus, VE-PTP is a transmembrane-binding
partner of VE-cadherin that associates through an extracellular
domain, reverses VEGFR-2-mediated phosphorylation of VEcadherin, and influences cell layer permeability. The highly
specific interaction of VE-PTP with VE-cadherin further
supports the hypothesis that tyrosine phosphorylation of the
VE-cadherin/catenin complex or of factors in its vicinity is
involved in the regulation of VE-cadherin-mediated cell
contacts.
Indeed, knocking down VE-PTP expression in endothelial
cells leads to increased permeability across the endothelial cell
layer as well as to increased transendothelial migration of
neutrophils (A. Gamp et al., manuscript in preparation). In
addition, the docking of neutrophils to the apical surface of
endothelial cells triggers the dissociation of VE-cadherin from
VE-PTP (A. Gamp et al.). Thus, VE-PTP is required for the
adhesive function of VE-cadherin and the maintenance of
endothelial cell contacts and is likely to participate in neutrophil
transmigration (Fig. 3).
Other mechanisms that could control VE-cadherin-mediated
endothelial cell contacts rely on the cyclic adenosine monophosphate-triggered activation of the guanine nucleotide
exchange factor Epac1 that in turn activates the small GTPase
Rap-1, which supports the function of VE-cadherin (111, 112).
Given that ESAM affects the opening of endothelial cell contacts,
it is intriguing that MAGI-1, the PDZ protein that binds to ESAM
(85), is required for Rap-1 activation and for the enhancement
of VE-cadherin-mediated cell contacts (113). How Rap-1
improves the adhesive function of the cadherin is not known,
but it has been proposed that for E-cadherin, Rap-1 is necessary
for proper targeting of E-cadherin to maturing cell contacts
(114).
PVR/CD155, a nectin-related protein
PVR was originally found as the human poliovirus receptor and
is related to the nectins that form a subset of four IgSF members,
each containing three Ig domains. Nectins are widely
distributed, are capable of mediating homophilic and heterophilic interactions, and are involved in the regulation of
epithelial or neuronal cell contacts (115). Recently, PVR and
nectin 2 were found expressed at cell contacts of primary
endothelial cells. Both proteins had been described before to
function on tumor cells as ligands for the natural killer (NK) cell
surface protein DNA accessory molecule 1 (DNAM-1)/CD226
(116). The latter is a 65-kDa member of the IgSF, containing
two V-like Ig domains. DNAM-1 is found on a subset of
Immunological Reviews 218/2007
187
Vestweber Transmigration of leukocytes through endothelium
B lymphocytes, on all T lymphocytes, NK cells, monocytes, and
platelets.
A soluble recombinant Fc-fusion protein of DNAM-1 was
shown to bind to endothelial cell contacts, and this binding was
blocked by antibodies against PVR but not by antibodies against
nectin 2, arguing for the interaction of PVR with DNAM-1 at
endothelial cell contacts. Direct binding of recombinant PVR–Fc
to immobilized DNAM-1-Fc was shown. Antibodies against
PVR and against DNAM-1 blocked the migration of human
monocytes through the monolayer of HUVECs, arresting the
monocytes above interendothelial junctions. This study was
very carefully controlled. The inhibitory effects were seen with
complete antibody as well as with Fab fragments, excluding
possible side effects of the Fc parts of the antibody. Anti-PVR
antibodies blocked when selectively incubated only with
endothelial cells; likewise, the anti-DNAM-1 antibodies blocked
on the leukocyte side. No effect of the anti-PVR antibodies on
endothelial cell layer integrity or permeability was observed.
This study suggests that DNAM-1 on monocytes participates in
their migration through an endothelial cell layer by binding to
endothelial PVR.
The mechanism of action of this very interesting novel pair of
adhesion and/or signaling molecules in leukocyte diapedesis is
not yet known. DNAM-1 is known to associate with the integrin
LFA-1 on T cells, and its signaling function requires the integrin
LFA-1 (117). Whether the interaction of PVR with DNAM-1 has
in turn any effect on LFA-1 is not known. The arrest of
monocytes right above interendothelial junctions is remarkable.
Thus, it looks as if blocking the DNAM-1/PVR-mediated step
arrests leukocytes at the same stage as antibodies against the first
two N-terminal Ig domains of PECAM-1 (35).
Major ligands for leukocyte integrins: ICAM-1, ICAM-2,
and VCAM-1
ICAM-1 and ICAM-2 are endothelial ligands for the leukocyte
b2-integrin LFA-1 (118, 119). ICAM-1 has been intensively
analyzed as one of the most important adhesive receptors on
endothelial cells for the firm adhesion of leukocytes (2). ICAM1 is constitutively expressed on endothelium, and it is further
upregulated by inflammatory cytokines. In contrast, ICAM-2 is
constitutively expressed and not inducible.
Both ICAMs participate in the docking of leukocytes to the
endothelium, and it is likely that they are both also relevant for
leukocyte diapedesis. Participation of ICAM-1 in diapedesis was
suggested in a very early report that described the expression of
ICAM-1 on the apical surface as well as all along the endothelial
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Immunological Reviews 218/2007
cell contact (120). In contrast, the same study showed that
VCAM-1, a major ligand for the b1-integrin very late antigen
(VLA)-4, was only found on the apical cell surface. In vitro
studies with mouse endothelioma cells suggested that ICAM-1
and ICAM-2 participated in the transendothelial migration of T
cells (121), although it is difficult to distinguish in such assays
between a role of the ICAMs in adhesion and diapedesis. Using
monolayers of HUVECs and real-time imaging, LFA-1 on
neutrophils was found to rapidly redistribute to form a ring-like
structure that coclustered with endothelial ICAM-1 as the
neutrophil transmigrated (66). A role for ICAM-1 and ICAM-2
in the diapedesis step was questioned in another in vitro study
that described the exclusive involvement of both ICAMs in the
attachment of monocytes to the apical endothelial surface and
the subsequent migration to endothelial cell contacts, a step that
was called ‘locomotion’ (122).
ICAM-1 is so far the only membrane protein that has been
suggested to participate in the junctional as well as the
transcellular diapedesis step. Upregulation of ICAM-1 in
a transformed HUVEC line increased the usually low contribution of the transcellular pathway (up to 50% of transmigrating
cells) (22). The study by Millan et al. (24) showed that ICAM-1
became recruited to caveola- and F-actin-rich sites of
lymphocyte uptake.
ICAM-1 has been described to transmit signals into
endothelial cells. Cross-linking with antibodies or interaction
with T cells induced tyrosine phosphorylation of the actinbinding protein cortactin (123) as well as activation of Rho
(124, 125). In subsequent studies, it was shown that PLCg1 and
PKC and Src were involved in tyrosine phosphorylation of
cortactin (126). Signaling by ICAM-1 is essential for the
transendothelial migration of leukocytes because the cytoplasmic tail of ICAM-1 was shown to be absolutely required for the
participation of ICAM-1 in transmigration (127, 128). In
addition to ICAM-1, E-selectin cross-linking induces cortactin
tyrosine phosphorylation, and both adhesion receptors associate with cortactin (129). Downregulation of cortactin in
HUVECs with small interfering RNA inhibited transendothelial
migration of neutrophils in vitro, and expression of a mutant of
mouse cortactin, in which the tyrosine phosphorylation sites
were mutated to phenylalanine, failed to rescue neutrophil
transmigration (130). Because neutrophil adhesion to endothelium was not affected, it is likely that cortactin is relevant for
the diapedesis step. Cortactin and its tyrosine phosphorylation
were also required for the clustering of ICAM-1 around
transmigrating neutrophils (131). Whether these signaling
and redistribution events are involved in the transcellular or in
the junctional migration of leukocytes are unknown.
Vestweber Transmigration of leukocytes through endothelium
ICAM-1 is well documented as being relevant for leukocyte
extravasation, based on numerous antibody studies as well as on
the analysis of gene-deficient mice (132). The lack of induced
expression of ICAM-2 and the minor role as endothelial LFA-1
ligand for the attachment of leukocytes are probably the reasons
why ICAM-2 did not attract much attention for many years in
the leukocyte extravasation field. An in vitro study with mouse
endothelioma cells reported that ICAM-2 was involved in the
transmigration of leukocytes, although it seemed to be less
important than ICAM-1 (121, 133). ICAM-2 gene-deficient
mice were reported first to show a delay in the recruitment of
eosinophils into the airway lumen and a prolonged presence of
eosinophils in an asthma model (134). An antibody study
comparing the roles of ICAM-1 and ICAM-2 in leukocyte
extravasation in vivo showed that ICAM-1 and ICAM-2 have
redundant functions in lymphocyte recirculation through
lymph nodes, but ICAM-1 is unique in supporting migration
of lymphocytes into inflamed sites and trapping within the lung
(135).
Using intravital microscopy, it was recently shown that
ICAM-2 participates in the diapedesis of neutrophils into
inflamed tissue (136). Antibodies against ICAM-2 as well as
gene deficiency impaired neutrophil recruitment into inflamed
peritoneum and in the cytokine-stimulated cremaster muscle.
Intravital microscopy of the cremaster showed that leukocyte
extravasation was reduced, but rolling and adhesion within
venules of the cremaster were unaffected. Like for PECAM-1, it
was found that blocking of ICAM-2 or the lack of ICAM-2
impaired neutrophil extravasation, if IL-1b was used as
inflammatory stimulus but not if thioglycollate (peritonitis)
or TNF-a (cremaster) was applied. Thus, PECAM-1 and ICAM-2
share the same cytokine selectivity.
In addition, this study (136) is the first that compared two
adhesion mechanisms in leukocyte diapedesis in vivo. It was
found that a monoclonal antibody against ICAM-2 strongly
inhibited neutrophil recruitment into inflamed peritoneum
beyond the inhibitory effect found in PECAM-1-deficient mice,
suggesting that the proteins act at different steps in the
diapedesis process. Blocking ICAM-2 reduced neutrophil
extravasation in the peritonitis model and in the cremaster
model to the same low levels in wildtype and in PECAM-1deficient mice. Thus, PECAM-1 had no role in ICAM-2independent leukocyte transmigration. These results suggest
that ICAM-2 and PECAM-1 act in the same molecular pathway,
possibly at sequential steps.
VCAM-1 is not constitutively expressed on most endothelial
cells and is strongly upregulated by cytokines (137). It binds to
the b1-integrin VLA-4 on monocytes and lymphocytes and is
mainly involved in the inflammation-related leukocyte extravasation. In addition, VCAM-1 plays a minor role in lymphocyte
homing into primary lymphatic organs, which could only be
shown in LFA-1-deficient mice (138). As mentioned above,
VCAM-1 was not found at regions of the endothelial plasma
membrane lining the endothelial cell contacts, in contrast to
ICAM-1 (120). Thus, VCAM-1 is exclusively mediating the
attachment of leukocytes to the apical endothelial cell surface.
As with ICAM-1, this function is accompanied by signaling
events that contribute to the diapedesis step.
Antibody cross-linking of VCAM-1 leads to the activation of
Rac1, the production of ROS, and gap formation between
endothelial cells (101, 139). Cross-linking of ICAM-1 did not
increase endothelial cell layer permeability (139). ROS can
stimulate the Pyk2, and this kinase in turn induces phosphorylation of b-catenin, accompanied with enhanced endothelial
permeability and downregulated VE-cadherin function (102).
CD99
CD99 is one of the most recently identified molecular players in
leukocyte extravasation. It was known for years as a pan
leukocyte protein, before it was identified on the surface of
endothelial cells. Antibodies against CD99 as well as Fab
fragments blocked the transmigration of monocytes through
the monolayer of HUVECs in vitro, independent of whether the
antibodies had been incubated with the leukocytes or the
endothelial cells (140). Adhesion to endothelial cells was not
affected, suggesting that CD99 acts in the diapedesis step.
Comparison of the inhibitory effect of anti-PECAM-1 and antiCD99 antibodies showed that CD99 functions downstream of
the first of the two steps for which PECAM-1 was reported to be
responsible (Fig. 2). This analysis was performed with antiPECAM-1 antibodies that bind the first two N-terminal domains
of PECAM-1 and were known to arrest monocytes above
endothelial junctions. In contrast, anti-CD99 antibodies trapped
monocytes in such a way that a part of the cell was still
detectable above the endothelium and that another part was
between endothelium and the underlying collagen matrix
(140). The second step of leukocyte arrest that can be achieved
with other anti-PECAM-1 antibodies and that traps leukocytes
between endothelium and the underlying matrix has not yet
been compared with the inhibitory effect of anti-CD99
antibodies.
Cloning of mouse CD99 allowed for analysis of the relevance
of CD99 for the extravasation of leukocytes in vivo (141). Antibodies against CD99 as well as F(ab#)2 fragments inhibited lymphocyte migration through monolayers of mouse endothelial
Immunological Reviews 218/2007
189
Vestweber Transmigration of leukocytes through endothelium
cells in vitro, but this treatment did not interfere with
adhesion between leukocytes and endothelial cells. The same
antibodies inhibited the recruitment of antigen-specific, in
vivo-activated T cells into inflamed areas of the skin. Even
edema formation was inhibited, pointing toward a crucial
role for CD99 in the inflammatory process (141). Recently,
a role for CD99 in in vivo leukocyte trafficking was confirmed
by showing that anti-human CD99 antibodies could block
the recruitment of xenotransplanted CD34þ human hematopoietic progenitors into mouse bone marrow (142).
As for PECAM-1, the mechanism by which CD99 mediates
leukocyte extravasation is not known in detail. It is possible that
CD99 functions as a homophilic adhesion molecule in
diapedesis because human as well as mouse CD99 are able to
support homotypic cell aggregation of transfected cells, which
is blocked by antibodies against CD99 (140, 141). In addition,
CD99 on the leukocyte and on the endothelial side are involved
in diapedesis. However, the following findings may argue
against a function of CD99 as a homophilic adhesion molecule.
First, if CD99 indeed mediated homophilic interactions, one
would expect that monomeric Fab fragments of inhibitory
antibodies should block the adhesive function. This was indeed
the case for antibodies against human CD99 but not for
antibodies against mouse CD99. Second, CD99-transfected cells
did not bind to CD99–Fc (141). Third, the structure of CD99 is
rather unusual for a homophilic cell adhesion molecule. It is
small with an extracellular part containing only about 100
amino acids, and it is highly O-glycosylated. Thus, the spatial
characteristics are not favorable for accessibility on the cell
surface or for the presentation of high-affinity interaction sites.
Of course, this does not rule out that homophilic binding might
occur with low affinity or might require the proper spatial
presentation or clustering of CD99 on the cell surface.
Various functional effects have been attributed to CD99 on
leukocytes based on ligation of the antigen with antibodies.
Aggregation of thymocytes and Jurkat T cells, but not of
peripheral primary T cells, triggered by antibodies against CD99
has been reported (143). This aggregation was independent of
b1- and b2-integrins. In contrast, ligation of CD99 on activated
but not on naive peripheral T cells was shown to activate the
integrin a4b1 (144). In this report, activation of the b2-integrin
LFA-1 by CD99 cross-linking was excluded. In contrast, super
cross-linking of CD99 with primary and secondary antibodies
for more than 4 h resulted in activation of LFA-1/ICAM-1mediated homotypic cell aggregation of the B-lymphoblastoid
cell line IM-9 (145).
Activation of leukocyte integrins by anti-CD99 antibodies,
which would then possibly induce leukocyte sticking to the
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Immunological Reviews 218/2007
endothelial surface, could be excluded as a simple explanation
for the inhibitory effect of anti-CD99 antibodies in transmigration assays. First, blocking of CD99 only on the
endothelial side was sufficient to inhibit leukocyte transmigration, in the human and in the mouse system. Second, antiCD99 antibodies neither inhibited nor enhanced leukocyte
adhesion to monolayers of endothelial cells. Third, mouse
lymphocyte adhesion to ICAM-1-transfected CHO cells was not
enhanced by anti-CD99 antibodies (141).
It is remarkable that the same antibodies that inhibited
recruitment of in vivo-activated T cells into sites of inflammation
did not inhibit the homing of naive T cells into lymph nodes,
although these lymphocytes as well as HEVs in lymph nodes
were both strongly positive for CD99 (141). Thus, the mere
presence of CD99 is not sufficient to mediate leukocyte
endothelial interactions. This finding may argue for the
necessity of specific posttranslational modifications for the
function of CD99 or, more likely, for the necessity of certain
signaling partners that need to be in place or in the right
activation state to be able to transmit CD99-triggered effects on
diapedesis. These results also indicate that diapedesis of naive
lymphocytes relies on different molecular mechanisms than
diapedesis of activated lymphocytes.
Comparison of the different molecular players in
diapedesis and novel mechanistic insights
Within the last few years, many new endothelial membrane
proteins have been found to participate in leukocyte diapedesis,
and for most of them, their in vivo significance for leukocyte
extravasation has recently been shown. The situation in the
diapedesis field is reminiscent of the late 1980s and early 1990s,
when more and more endothelial adhesion molecules were
identified that mediated leukocyte capturing and adhesion. As
in those days, the aim today is to sort out the way by which the
various membrane proteins act in concert to support the
transmigration of leukocytes through the vessel wall.
There are at least three (and probably more) major reasons
why a multitude of different membrane proteins is required
for leukocyte extravasation. The first is based on the fact that
leukocyte diapedesis occurs in several steps. PECAM-1 seems
to act in more than one of these steps. Depending on the
anti-PECAM-1 antibodies used, they either arrest leukocytes
above endothelial junctions (35) or they block the transmigration across the basement membrane arresting leukocytes
underneath endothelial cells (33, 35, 36). CD99 was the first
endothelial membrane protein shown to act at yet another step.
Antibodies blocked the transmigration of monocytes through
Vestweber Transmigration of leukocytes through endothelium
the endothelial monolayer, with a part of the leukocyte still
above the endothelial cells and another part stuck underneath
the endothelial cells (140). The first of the two PECAM-1dependent steps as well as the step defined by CD99 are based on
in vitro studies. Although CD99 is clearly involved in vivo in
leukocyte extravasation, it is not yet clear where leukocytes
become arrested or slowed down during diapedesis in vivo.
ICAM-2 and PECAM-1 represent the first two adhesion
molecules directly compared in leukocyte extravasation in vivo.
Based on neutrophil extravasation studies in a peritonitis model
and a cremaster model, ICAM-2 and PECAM-1 were found to act
in the same molecular pathway possibly at sequential steps.
However, it should be noted that ultrastructural analysis of the
step of leukocyte arrest during leukocyte extravasation has only
been performed so far for PECAM-1, documenting the arrest
between endothelium and basement membrane. In vitro studies
suggest that blocking PVR with antibodies arrests leukocytes
above endothelial junctions, similar to the first postulated step
mediated by PECAM-1 (146).
The second reason why a multitude of membrane proteins is
needed for diapedesis is because of the fact that different types
of leukocytes use different endothelial adhesion molecules for
diapedesis. ICAM-1 and ICAM-2 are involved in the extravasation of neutrophils, monocytes, and lymphocytes. CD99
mediates transendothelial migration of monocytes in vitro and
lymphocyte recruitment into inflamed tissue. Other adhesion
molecules, such as PECAM-1 and ESAM, are selectively supporting the extravasation of only myeloid cells. ESAM is the only
endothelial membrane protein that has been analyzed for its
relevance in neutrophil and lymphocyte extravasation in the
same inflammation model. These experiments showed that the
lack of ESAM affected neutrophil but not lymphocyte
recruitment (88). Because ESAM seems to be involved in the
opening of endothelial cell contacts, these results suggest that
neutrophils and lymphocytes use different mechanisms to
overcome endothelial junctions.
The diapedesis of naive and activated lymphocytes seems also
to differ considerably. CD99 was only required for the
extravasation of activated lymphocytes, whereas naive lymphocytes transmigrated through the endothelium of HEVs independently of CD99, although CD99 is strongly expressed on
HEV and naive lymphocytes. Except for ICAM-1, ICAM-2, and
VCAM-1, the other endothelial surface proteins relevant for
leukocyte diapedesis have not yet been analyzed in lymphocyte
homing.
A third reason for the necessity of a complex repertoire of
adhesion molecules and receptors for leukocyte diapedesis is
based on the complexity of the process and the several different
functions that need to be supported during this process. First,
adhesion molecules are required that enable the leukocytes to
migrate through the endothelial cell contacts. PECAM-1 and
CD99 have been suggested to serve this purpose as homophilic
adhesion molecules. In addition, ICAM-2 at endothelial cell
contacts binding to the b2-integrin LFA-1 is likely to participate
as adhesion molecule in this transmigration process. Whether
ICAM-1 is involved in this step is difficult to test because it is
hard to distinguish experimentally between its adhesion and its
potential transmigration function. It is not yet known whether
some of the above or other adhesion molecules are also
responsible for the sealing between transmigrating leukocytes
and the lateral surface of endothelial cells.
Second, some endothelial membrane proteins may serve to
activate processes within leukocytes. Endothelial PECAM-1 was
suggested to mobilize the integrin a6b1 on neutrophils by
binding to neutrophil PECAM-1. This finding would explain
why blocking of PECAM-1 arrests neutrophils between
endothelium and the laminin-containing basement membrane.
It is also possible that endothelial PVR might activate leukocytes.
Another signaling function could generally affect the motility of
leukocytes. Indeed examples for this are known, as was recently
shown for neutrophils deficient for JAM-A (71) or deficient for
PECAM-1 (147). In both cases, neutrophil motility was
reduced.
Third, not all cell surface proteins involved in diapedesis
participate in leukocyte extravasation under the same inflammatory conditions. PECAM-1 and ICAM-2 were reported to be
dispensable for neutrophil recruitment in C57Bl/6 mice, if
inflammation was triggered with TNF-a or thioglycollate, but
these proteins were relevant if inflammation was triggered with
IL-1b. Selectivity for the inflammatory stimulus was also found
for PECAM-1 in rats (36), although it was not observed in other
mouse strains (34). In contrast, ESAM participated in neutrophil
diapedesis no matter which inflammatory mediator was used.
Thus, different pathways and repertoires of membrane
receptors may be relevant for the recruitment of neutrophils,
dependent on the type of inflammatory stimulus.
Fourth, in those cases where leukocytes overcome the
endothelial cell layer by transcytosis through endothelial cells,
endothelial membrane proteins need to serve as ‘receptors’ that
trigger the cytoskeleton rearrangements necessary inside the
endothelium to accommodate this process. The only known
candidate so far for such a process is ICAM-1 (24).
Fifth, the junctional pathway requires mechanisms that
regulate and trigger the opening of endothelial cell contacts. A
good candidate for this function is the endothelial tight junction
protein ESAM. On the one hand, the lack of ESAM impaired and
Immunological Reviews 218/2007
191
Vestweber Transmigration of leukocytes through endothelium
slowed down neutrophil extravasation in two different
inflammation models, and on the other hand, ESAM deficiency
impaired the opening of endothelial contacts triggered by the
growth factor VEGF (88). Thus, ESAM is likely to be involved in
signaling mechanisms that trigger the opening of endothelial
cell contacts. Recent in vitro studies suggest that JAM-C might be
a second endothelial junction protein that could be involved in
the regulation of endothelial cell contacts. Overexpression in
cultured endothelial cells reduced VE-cadherin adhesiveness,
and knocking down its expression by RNAi enhanced VEcadherin adhesiveness (81).
Sixth, in contrast to all the other endothelial membrane
proteins that support leukocyte diapedesis in different ways,
there must also be adhesion molecules, such as VE-cadherin,
that counteract leukocyte diapedesis because they maintain the
endothelial barrier. VE-cadherin indeed represents the major
adhesive mechanism between endothelial cells. The fact that VEcadherin ‘moves away’ from cell contacts at sites of transmigrating leukocytes has often been misinterpreted as evidence
that VE-cadherin would not be involved in the extravasation
process. Indeed, VE-cadherin is not involved once the
endothelial contacts are opened. However, the fact that VEcadherin moves away from sites of diapedesis strongly suggests
that in order to make this happen, the transinteractions of VEcadherin clusters need to be loosened. Although it has not yet
been shown, a failure in mechanisms involved in the opening of
VE-cadherin-mediated contacts would probably block the
diapedesis process. It is an attractive hypothesis that junctional
proteins such as ESAM and JAM-C and possibly the phosphatase
VE-PTP may be involved in leukocyte-triggered endothelial
signaling mechanisms that induce the opening of VE-cadherinmediated contacts (Fig. 3).
Concluding remarks
The many endothelial surface proteins that were recently found
to participate in leukocyte diapedesis illustrate that this process
has attracted much attention in the last few years. Except for
CD99 and PVR, gene-deficient mice are available for all
adhesion molecules and receptors that were discussed in this
review. They are all viable with the exception of VE-cadherin-,
VE-PTP-, and JAM-C-deficient mice. In combination with
function-blocking antibodies, these mice represent a good
repertoire of tools to unravel the principal mechanism of
diapedesis. Despite the recent progress, it is likely that not all
players have yet been identified.
In the future, two experimental strategies will hopefully
allow us to answer one of the fundamental questions in this
process: whether the junctional or the transcellular pathway is
of dominant importance and, if so, which one. First, we will
have to identify those molecules involved in the diapedesis
process that are unique for only one of the two pathways. ESAM,
VE-cadherin, and the JAMs are probably good candidates
because they are strictly limited to tight and adherens junctions,
and therefore it is likely that they are exclusively involved in the
junctional pathway. For most of the other endothelial
membrane proteins, this restriction is less clear because even
for ICAM-2, PECAM-1, and CD99 that are enriched at the lateral
endothelial cell surface, a weak expression on the apical surface
cannot be excluded. If targets can be defined that are selectively
involved in only one of the two pathways, it will be possible to
selectively block this pathway by pharmacological or genetic
means. This will allow us to determine the relevance of this
pathway in vivo, provided leukocytes would not immediately
switch to the alternative pathway.
The second experimental strategy needed to determine the
relative contribution of each pathway is intravital real-time
imaging of the extravasation process in combination with
reagents that block diapedesis at various steps. In this way,
cells can be caught in the act of moving through the vessel
wall. This approach already has resulted in very valuable and
exciting insights into this process. As was shown by Wang et al.
(148), neutrophils extravasating through venules of the
cytokine-stimulated cremaster were shown to do this
preferentially at endothelial junctions that were positioned
above thinner areas of the basement membrane and gaps
between pericytes (148). Another elegant intravital confocal
microscopy study of surgically inflamed cremaster venules
showed that 75% of firmly adherent leukocytes were arrested
overlapping an endothelial cell junction (149). Solving the
question of the prevailing physiological mechanism that
enables leukocytes to overcome the barrier of the blood vessel
wall will provide new targets for the interference with
inflammatory processes.
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