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* Ludwig Institute for Cancer Research, Royal Free and University College School of Medicine, and
Department of Biochemistry and Molecular Biology, University College London, London, United Kingdom; and
Experimental Medicine, GlaxoSmithKline, Addenbrooke’s Center for Clinical Investigation, Addenbrooke’s Hospital, Cambridge, United Kingdom
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2 integrin LFA-1 (CD11a/CD18), which is
expressed on all leukocytes (3, 4). Recently, ICAM-2 has
also been reported to bind to another ligand, dendritic cell
(DC)3-specific
ICAM-grabbing nonintegrin, a C-type lectin expressed only on DCs
(5).
In resting endothelial cells, ICAM-1 is expressed at very
low levels whereas ICAM-2 is expressed abundantly. Proinflammatory
cytokines such as TNF-
induce a large increase in ICAM-1 levels
(1) and a concomitant decrease in ICAM-2 levels on the
surface of endothelial cells (6). ICAM-1 is essential for
stable adhesion and transmigration of leukocytes in nearly all types of
inflammatory response (1), and ICAM-1-deficient mice have
impaired inflammatory and immune responses (7). In
contrast, in vitro studies indicate that ICAM-2 contributes to
leukocyte transendothelial migration under noninflammatory conditions
and therefore could play a role in leukocyte recirculation (8, 9). Although lymphocyte homing to lymph nodes was apparently
unimpaired in ICAM-2-deficient mice (10), it
has recently been reported that ICAM-2 plays an important role in
mediating DC transmigration across resting endothelium
(5), and thus it may contribute to the tissue recruitment
of a subset of hemopoietic cell types.
As well as being critical for leukocyte adhesion, ICAM-1 can activate signaling pathways. Ab-induced cross-linking of ICAM-1 on rat brain-derived endothelial cell lines has been reported to induce the activation of the Src tyrosine kinase and phosphorylation of the cytoskeletally associated proteins focal adhesion kinase, paxillin, p130Cas and cortactin, as well as the activation of the small GTPase Rho (11, 12, 13). In the same cell lines, ICAM-1 cross-linking induced actin cytoskeletal reorganization, which was shown to involve Rho, intracellular calcium signaling, and p38 mitogen-activated protein kinase activation (12, 14). Rho mediates the assembly of stress fibers and associated focal contacts in many cell types, including endothelial cells (12, 15, 16), and induces contractility in HUVECs leading to the formation of intercellular gaps which could aid the transmigration of leukocytes (16). In addition, Rho is required for monocyte adhesion to HUVECs, and mediates clustering of ICAM-1 underneath the monocytes (17). Furthermore, Rho can activate gene expression in coordination with Ras activation, by activating the serum response element (SRE) promoter sequence (18). In contrast to ICAM-1, a possible signaling function of ICAM-2 in endothelial cells has not been investigated. However, in T cells, it has recently been reported that ICAM-2 can activate the serine/threonine kinase Akt/protein kinase B (19).
The cytoplasmic tails of ICAM-1 and ICAM-2 could be responsible for transducing signals in the cell. Despite their short length (ICAM-1 has 28 aa and ICAM-2 has 26 aa), both ICAM-1 and ICAM-2 cytoplasmic tails can interact with ezrin in vitro (20). Ezrin is a member of the ERM (ezrin, radixin, and moesin) family, which can function as a linker between the plasma membrane and the actin cytoskeleton (21), and play an important role in lamellipodium extension (22), cell-cell adhesion (23), microvillus assembly, and cytokinesis (23). Ab-cross-linked ICAM-1 coclusters with ERM proteins in endothelial cells (17). The N-terminal domain of ERM proteins (N-ERMAD) can interact with both phosphoinositides and a number of proteins, while the C-terminal domain of ERM proteins binds to filamentous actin (F-actin). There is evidence that Rho activation occurs downstream of ERMs as N-ERMADs can interact with RhoGDI, which normally holds Rho in an inactive GDP-bound complex in the cytoplasm. N-ERMAD binding to RhoGDI facilitates exchange of GDP for GTP on Rho, presumably because Rho is released from RhoGDI (24). Also, more recently, the ICAM-1 cytosolic tail has been shown to interact with the Src homology 2 domain containing tyrosine phosphatase-2 (SHP-2) in a phosphotyrosine-dependent manner (25).
In this study, we compare the effects of ICAM-1 and -2 cross-linking in primary human umbilical cord endothelial cells upon cytoskeletal arrangements and gene expression. We show that although ICAM-1, ICAM-2, and moesin all localize in microvilli in unstimulated cells, only ICAM-1 cross-linking coclusters moesin, activates RhoA, and causes stress fiber formation, whereas ICAM-2 cross-linking does not affect the actin cytoskeleton. Furthermore, ICAM-1 cross-linking induces rhoA and c-fos gene expression. Thus, we provide a mechanistic basis for the difference between the roles of ICAM-1 and -2 in leukocyte transmigration.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Reagents were obtained from the following sources: medium 199
modified Earle’s salt solution and HBSS (Life Technologies, Paisley,
U.K.); Nutridoma NS (Boehringer Mannheim, Lewes, U.K.); pooled
HUVECs and endothelial growth medium (EGM)-2 (Clonetics, San
Diego, CA); human fibronectin, heparin, endothelial cell growth
supplement, tetramethylrhodamine isothiocyanate
(TRITC)-phalloidin, HEPES, collagenase type II, aprotinin, leupeptin,
and other lysis buffer components, unless otherwise stated
(Sigma-Aldrich, Gillingham, U.K.); TNF- (Insight Biotechnology,
Wembley, U.K.); mouse monoclonal anti-ICAM-1 Ab (clone
BBIG-I1; R&D Systems, Abingdon, U.K.); mouse monoclonal anti-ICAM-2
Ab (clone CBR-IC2/2; Alexis Biochemicals, Nottingham, U.K.); goat
polyclonal anti-moesin Ab (C-15; Sigma-Aldrich); Alexa 488-labeled
goat anti-mouse IgG and Alexa 546-labeled goat anti-mouse IgG
Abs (Molecular Probes, Leiden, The Netherlands); FITC- and
TRITC-labeled goat anti-mouse and goat anti-rabbit Abs
(Southern Biotechnology Associates, Birmingham, AL); Cy5-conjugated
goat anti-rat Ab (Jackson ImmunoResearch Laboratories, West Grove,
PA); rabbit polyclonal anti-myosin II Ab (Biogenesis, Poole, U.K.);
mouse monoclonal anti-RhoA Ab (Santa Cruz Biotechnology, Santa
Cruz, CA); HRP-conjugated goat anti-mouse Ab (Bio-Rad, Hemel
Hempstead, U.K.); and Fugene 6 (Boehringer Mannheim). Rabbit polyclonal
anti-moesin Ab was generously provided by Dr. P. Mangeat
(Université Montpellier II, Montpellier, France); rat monoclonal
anti-COOH (Thr-558) C-terminally phosphorylated ERM proteins
(anti-CPERM; 2975) were generously provided by Dr. S.
Tsukita (Kyoto University Faculty of Medicine, Kyoto, Japan). Integrin
peptide P6 was generously provided by Dr. S. Hart (Institute of Child
Health, London, U.K.). Lightcycler apparatus and PCR reagents
were obtained from Roche (Basel, Switzerland), primers for
rhoA gene expression analysis were obtained from Life
Technologies, and primers for c-fos gene expression analysis
were obtained from Sigma-Genosys (Pampisford, U.K.).
Cell culture
HUVECs were isolated from umbilical cords using 0.1% collagenase type II. The cells were cultured in TC Nunclon flasks in medium 199 modified Earle’s salt solution containing 1.25 g/L NaHCO3 and Glutamax and supplemented with 20% FCS, 100 µg/ml endothelial cell growth supplement, 1% Nutridoma NS, and 100 µg/ml heparin. Alternatively, HUVECs from Clonetics were grown in EGM-2. Cells were cultured at 37°C in humidified air containing 5% CO2. For experiments, cells were used between one and three passages.
For immunofluorescence experiments, cells were grown on glass coverslips coated with 10 µg/ml human fibronectin until confluent. For biochemical experiments, cells were grown on 6- and 10-cm dishes coated with human fibronectin until confluent. To obtain quiescent-starved cells, the culture medium was replaced by medium containing 10% FCS, but no heparin or other growth factors. Cells were incubated in this medium for no longer than 24 h. Cells grown in EGM-2 were starved in 1% FCS. No differences between HUVEC responses were observed between cells grown in different media.
Receptor clustering and immunofluorescence
To induce receptor clustering, either mouse monoclonal
anti-ICAM-2 Ab was added to starved cells at a final concentration
of 10 µg/ml for 60 min, or mouse monoclonal anti-ICAM-1/ICAM-2
were added to cells that had been stimulated for 24 h with 10
ng/ml TNF-. After incubation with primary Abs, TNF- and the
primary Abs were removed from the cell medium and 10 µg/ml of Alexa
488-labeled goat anti-mouse Ab was added to the cells for between
15 and 60 min. Cells were then washed three times in TBS (10 mM Tris,
pH 7.5, 150 mM NaCl) containing 0.25% BSA, fixed in 4%
paraformaldehyde for 20 min at room temperature, permeabilized for 6
min with 0.2% Triton X-100, and then incubated with 1 µg/ml
TRITC-phalloidin for 45 min to stain actin filaments, or for 45 min
with rabbit polyclonal anti-moesin Ab diluted 1/200, or
rabbit polyclonal anti-myosin II Ab diluted 1/25, followed by the
appropriate TRITC-labeled secondary Ab. The specimens were mounted in
moviol. To observe responses in cells without receptor clustering,
cells were incubated with primary Abs as before for 60 min. These cells
were then washed, fixed, and stained with the secondary Ab for 45 min.
Staining for intracellular epitopes was then conducted as described
above. All incubations were conducted in TBS containing 0.25%
BSA.
To use the phosphospecific anti-CPERM Ab, a different fixation procedure was used as previously described (38). In brief, after receptor clustering, cells were fixed in 10% TCA in distilled water at 4°C for 20 min. All further staining was conducted in TBS containing 0.25% BSA. Cells were incubated with rat monoclonal anti-CPERM Ab (297S) for 45 min, followed by Cy5-conjugated goat anti-rat Ab for 45 min.
Confocal laser scanning microscopy was conducted with an LSM 510 (Zeiss, Welwyn Garden City, U.K.) mounted over an affinity corrected Axioplan microscope (Zeiss). Image files were collected as a matrix of 1024 x 1024 pixels describing the average of eight frames scanned at 0.062 Hz where FITC, TRITC, and Cy5 were excited at 488, 543, and 633 nm and visualized with 540 ± 25, 608± 32, and 690 ± 30 nm bandpass filters, respectively, where the levels of interchannel cross-talk were insignificant.
DNA constructs and transfection of HUVECs
pCDM8-ICAM-1 (human) has been previously described (26). The cDNA encoding human ICAM-2 (3) was subcloned into pcDNA3 using EcoRV and NotI restriction sites. HUVECs were transfected using a mixture of P6 integrin peptide and Lipofectin (Life Technologies) as previously described (27). Briefly, P6 integrin-targeting peptide, lipofectin, and plasmid DNA were allowed to form a complex, which was incubated with subconfluent cells in Optimem for 4 h. Cells were then washed in Optimem and grown to confluence in their normal growth medium (medium 199 with supplements).
RhoA activity assay
GST-rhotekin Rho-binding domain (TRBD; a gift from Dr. M.
Schwartz, Scripps Institute, San Diego, CA) was expressed in
Escherichia coli and purified as previously described
(28). TNF--activated HUVECs (on 10-cm dishes) were
subject to Ab-induced ICAM-1/ICAM-2 clustering as described above.
Control dishes were treated with primary or secondary Abs alone, or
were given fresh medium before lysis. Cells were lysed in lysis
buffer (50 mM Tris, pH 7.3, 1% Triton X-100, 0.5% sodium
deoxycholate, 0.1% SDS, 500 mM NaCl, 10 mM
MgCl2, 10 µg/ml each of leupeptin and
aprotinin, 1 mM PMSF). Lysates were incubated with 20 µg of GST-TRBD
beads for 1 h at 4°C. Beads were washed in buffer B (50 mM Tris
pH 7.3, 1% Triton X-100, 150 mM NaCl, 10 mM
MgCl2, 10 µg/ml each of aprotinin and
leupeptin, 100 µM PMSF). Proteins retained on the GST-TRBD beads were
resolved by 13% SDS-PAGE. Bound RhoA was detected by Western blotting
using mouse monoclonal anti-RhoA Ab. Quantification of relative
RhoA activity was performed using the photodensitometry program
Quantity One (Bio-Rad). The amount of activated RhoA was
normalized to the amount of total RhoA in the corresponding lysate for
each treatment. The significance of any difference in RhoA activity
after ICAM clustering was determined by Student’s t
test.
Gene expression analysis
TNF--activated HUVECs (10 ng/ml, 24 h) were stimulated
with either ICAM-1 or -2 cross-linking Abs for either 60 or 90 min.
Total RNA was isolated using the Qiagen RNeasy kit (Valencia, CA).
First strand-cDNA synthesis was performed on the total RNA preparations
to provide a cDNA template for gene expression analysis.
Qualitative gene expression was performed using the Lightcycler system. Primers for real-time PCR were designed close to the 3' end of rhoA and c-fos and were as follows: rhoA 5'-ATGTGCCCACAGTGTTTGAGAAC; rhoA 3'-CGTTGGGACAGAAATGCTTGACT; c-fos 5'-TCACCCTGCCTCTCCTCAAT; c-fos 3'-GCTGCATAGAAGGACCCAGATAG.
Standard curves were generated using rhoA cDNA as a template and the rhoA primers. Therefore, samples were analyzed for gene copy numbers as compared with the rhoA standard curve. In the case of samples analyzed for c-fos, the standard curve was still made from rhoA transcripts so this analysis could only be qualitative. Normalization of the samples was conducted by assessing the GAPDH mRNA content in each sample against a GAPDH standard curve. For qualitative analysis, all 0-min timepoint samples were taken as the reference point, with changes induced by stimulation represented as changes relative to the 0-min timepoint. Results were obtained from two separate stimulations.
RhoA protein levels were also determined after ICAM-1 and -2 cross-linking. After cross-linking, cells were lysed in 2x Laemmli sample buffer. Proteins were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membrane followed by immunoblotting for RhoA. Blots shown are representative of three separate experiments.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ICAMs have been shown to interact with ERM proteins in vitro and
using exogenously expressed proteins. Endothelial cells express ICAM-2
constitutively and can be induced to express high levels of ICAM-1 by
inflammatory stimuli. Therefore, immunofluorescence techniques were
used to investigate the subcellular localization of ICAMs and ERM
proteins in primary endothelial cells. In resting HUVECs, ICAM-2
colocalized with moesin at intercellular junctions and at the apical
surface (Fig. 1
, a–c).
Interestingly, colocalization also occurred in distinct
microvillus-like structures on the apical surface. These structures
also contained F-actin (Fig. 1, d–f) and are therefore
likely to be microvilli. Colocalization of moesin and ICAM-2 was also
observed in TNF--activated HUVECs (data not shown). ICAM-1 was not
expressed significantly in resting, unstimulated HUVECs (data not
shown; Ref. 1). After 24 h of TNF- stimulation (10
ng/ml), ICAM-1 expression was highly induced and colocalized with
moesin at intercellular junctions and in microvillus-like structures
with moesin (Fig. 1, g–i), similar to the distribution of
ICAM-2. These results indicate that leukocyte-binding receptors ICAM-1
and -2 are optimally positioned on endothelial cell microvilli for
interaction with 2 integrins.
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It has previously been shown that Ab-induced ICAM-1 clustering
induces the activation of Rho and the formation of stress fibers in rat
brain-derived microvascular endothelial cell lines (13).
Therefore, we investigated the effects of ICAM-1 and -2 Ab-induced
cross-linking on the actin cytoskeleton of HUVECs. Ab-cross-linking of
ICAM-1 for 60 min on HUVECs induced the formation of F-actin bundles
which aligned parallel to the elongated axis of the cell (compare Fig. 2, b and d). ICAM-1
cross-linking also induced the assembly of myosin II-containing
filaments (data not shown), confirming that the newly formed F-actin
structures were stress fibers, and this was accompanied by the
appearance of intercellular gaps, indicative of increased
contractility.
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c), as previously described
(17). The majority of these large ICAM-1 clusters were on
the cell surface and not internalized (data not shown). As we showed
that ICAM-1 colocalized with moesin in microvilli (Fig. 1), we
investigated the effects of Ab-induced ICAM-1 cross-linking on the
localization of moesin. ICAM-1 cross-linking caused a dramatic
redistribution of moesin, so that moesin colocalized with ICAM-1
clusters (Fig. 2, e and f), consistent with our
previous report of ERM colocalization with cross-linked ICAM-1
(17).
To determine whether TNF- signaling is required for ICAM-1 to induce
stress fiber assembly, we transfected HUVECs with exogenous ICAM-1 so
that they could be cross-linked on resting, unstimulated cells. Under
these conditions, ICAM-1 cross-linking was able to stimulate the
formation of stress fibers (Fig. 2, g and h),
indicating that TNF- prestimulation is not required for this
response.
In contrast to ICAM-1, Ab cross-linking of ICAM-2 for 60 min on resting
HUVECs only induced the formation of small clusters of ICAM-2 and had
no effect on the arrangement of F-actin (Fig. 3, a–d). Myosin II staining
also confirmed that no increase in stress fibers occurred in response
to ICAM-2 cross-linking (data not shown). TNF- treatment for 24
h induced a small increase in stress fibers relative to quiescent
HUVECs (compare Fig. 3, b and f), but ICAM-2
cross-linking after TNF- stimulation had no effect upon the number
or organization of F-actin cables (Fig. 3, e–h).
Surprisingly, moesin did not cocluster with ICAM-2 (Fig. 3, i and j), although it colocalized with moesin in
microvilli before clustering (Fig. 1, a–c).
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As stress fiber formation is dependent on RhoA (15),
we assayed the effects of ICAM-1 and -2 cross-linking upon RhoA
activity using the TRBD to affinity-precipitate endogenous GTP-bound
RhoA from endothelial cell lysates (28). RhoA was
activated between 15 and 30 min after ICAM-1 clustering and remained
activated at 1 h (Fig. 4). RhoA
activity showed a 2.5-fold increase as compared with resting levels at
this time point. ICAM-2 clustering, however, had no effect on RhoA
activity. This data confirms results from immunofluorescence
experiments in Figs. 2 and 3, and indicates that the effects of ICAM-1
and -2 cross-linking on the endothelial cytoskeleton are dramatically
different.
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RhoA has been shown to stimulate transcription of the
c-fos gene through activation of the SRE (29).
As ICAM-1 cross-linking activated RhoA, we tested whether
c-fos expression was affected by ICAM-1 or -2 cross-linking.
ICAM-1 cross-linking induced a dramatic 6-fold increase in
c-fos mRNA levels (Fig. 5a), returning to resting
levels after 90 min of ICAM-1 cross-linking. In contrast, 60 min of
ICAM-2 cross-linking induced a relatively small 1.4-fold increase in
c-fos mRNA levels. It is possible that this small increase
was caused by addition of Abs alone, rather than a specific consequence
of ICAM-2-induced signaling pathways.
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b). At 90 min
after ICAM-1 cross-linking, a 1.5-fold increase in rhoA mRNA
levels was observed as compared with resting levels. This increase in
mRNA was paralleled by an increase in RhoA protein levels after ICAM-1
cross-linking (Fig. 5c). Quantification of the increase in
protein levels by photodensitometry showed that at 135 min of ICAM-1
cross-linking, the level of RhoA protein levels was increased by
1.5-fold compared with resting levels. In contrast, ICAM-2
cross-linking did not induce an increase in rhoA mRNA or
protein levels. In fact, at 90 min of ICAM-2 cross-linking, there was a
small decrease in rhoA mRNA levels (Fig. 5b).
There results suggest that activation of RhoA may feed back to
stimulate an increase in RhoA protein levels. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ICAM localization and ERM proteins
We have shown that both ICAM-2 in resting cells and ICAM-1 on activated cells localize with moesin in apical microvilli. This is consistent with previous reports showing the presence of CD44 and exogenously expressed ICAM-2 at microvilli in fibroblasts, although fibroblasts do not express endogenous ICAM-2. Positioning of leukocyte-binding receptors on endothelial cell microvilli is likely to facilitate leukocyte capture. We have observed a high level of C-terminal phosphorylation of ERM proteins in HUVECs (data not shown), indicating that they are predominantly in an active conformation and are therefore likely to link ICAMs to F-actin at microvilli.
ICAM-1 and RhoA activation
We have found that ICAM-1 cross-linking on primary endothelial
cells induces the formation of stress fibers and the activation of
RhoA, in accordance with previous results with a rat brain-derived
endothelial cell line (12). Although ICAM-1 is only
normally expressed in HUVECs following activation with cytokines such
as TNF-, cytokine stimulation is not required for ICAM-1 responses
as cross-linking of exogenously expressed ICAM-1 on unstimulated HUVECs
can still induce stress fiber formation. The level of ICAM-1 engaged by
Ab in transfected cells is considerably lower than that on
TNF--stimulated cells, and may be similar to the amount of ICAM-1
normally engaged by monocyte binding.
In contrast to ICAM-1, ICAM-2 does not activate RhoA, either in resting
HUVECs or in TNF--stimulated HUVECs. Ab-induced ICAM-2 clusters are
very small and do not aggregate into larger clusters unlike those
formed by ICAM-1. Both ICAMs can bind to the same ligand, the
2 integrin, LFA-1, but their short cytoplasmic
tails are markedly different in sequence and may therefore interact
with different signaling proteins (1, 3, 30). Several
proteins have been reported to interact with the ICAM-1 cytosolic tail
(20, 25, 31), and of these, the SHP-2 and ERM proteins
could be involved in ICAM-1 signal transduction. SHP-2 has recently
been shown to interact with a poorly conserved immunoreceptor
tyrosine-based inhibitory motif contained within the cytosolic
tail of ICAM-1, after ICAM-1-mediated adhesion of cells to fibrinogen
(25). SHP-2 has been reported to down-regulate RhoA
activity in fibroblasts and epithelial cells (32, 33).
ICAM-2 would not be expected to bind to SHP-2 as the sequence in the
ICAM-1 tail that binds SHP-2 is not conserved in ICAM-2. Alternatively,
ERM proteins could mediate RhoA activation downstream of ICAM-1
(34). Rho-GDI has been shown to interact with moesin, and
this interaction with ERM proteins causes a decrease in the ability of
Rho-GDI to inhibit nucleotide exchange on Rho (24),
leading to Rho activation. Results from this study show that moesin
coclustered with ICAM-1 rather than ICAM-2 and therefore it is possible
that an ICAM-1/moesin interaction is required for signaling to RhoA.
However, it is also likely that the function of ERM interaction with
ICAMs is to stabilize interaction with the actin cytoskeleton.
ICAM-1 induces gene expression
In this study, we show that ICAM-1 cross-linking in
TNF--activated HUVECs stimulates expression of c-fos and
rhoA. ICAM-1 cross-linking has previously been reported to
stimulate expression of the leukocyte adhesion molecule VCAM-1 and to
activate the transcription factor activator protein (AP)-1 in
resting endothelial cells (35), but ICAM-1-mediated
changes in transcription in TNF--stimulated cells, which are more
physiologically relevant as they express high levels of ICAM-1, have
not been investigated before. Active RhoA has been shown to induce gene
expression through activation of the SRE (29). In
particular, RhoA can enhance transcriptional activation of c-fos,
egr-1, and cox-2. Therefore, the ICAM-1-induced
stimulation of c-fos transcription correlates well with the
observed activation of RhoA. It is extremely interesting that ICAM-1
cross-linking induced up-regulation of both rhoA mRNA and
RhoA protein levels. This may constitute a novel positive feedback
response to RhoA activation, providing more RhoA for subsequent
activation in response to further leukocyte binding. Little is known
about how rhoA gene expression is regulated, although
recently it was found that inhibition of the Ets family transcription
factor Erg led to a decrease in rhoA expression in
endothelial cells (36). Therefore, it would be interesting
to investigate whether Erg contributes to the observed up-regulation of
rhoA mRNA in response to ICAM-1.
Differences in ICAM-1 and -2 signaling and their biological roles
ICAM-1 is well-characterized for its role in endothelial cells during inflammation, whereas endothelial ICAM-2 does not appear to be important in inflammation, and in fact, proinflammatory cytokine stimulation of HUVECs down-regulates ICAM-2 protein levels (6). ICAM-2 can, however, contribute to monocyte transmigration through unstimulated endothelial cell monolayers (8), and transmigration of different T cell populations across ICAM-1-deficient endothelium (9). In the light of this evidence, and because ICAM-2 binds to the leukocyte integrin LFA-1, it had been proposed that ICAM-2 is involved in leukocyte recirculation. Recently, however, ICAM-2 was shown to mediate DC trafficking through its interaction with the C-type lectin, DC-specific ICAM-grabbing nonintegrin (5). DCs are professional APCs that continuously survey the tissues for incoming foreign Ags. Immature DCs have to migrate from the blood into the periphery and this is not dependent upon inflammatory signals. Therefore, constitutive expression of ICAM-2 on endothelial cells may be important for the transmigration of immature DCs.
Our results showing that ICAM-1, but not ICAM-2, activates RhoA, induces actin reorganization, and stimulates gene expression fit with their different roles in leukocyte transmigration. Under inflammatory conditions, where ICAM-1 is involved, RhoA activation would contribute to the inflammatory response of endothelial cells through its effects in disrupting intercellular junctions, enhancing endothelial permeability (16, 37). Increased transcription of selected genes is likely to contribute to the progression and eventual resolution of the endothelial response to inflammation. During routine leukocyte trafficking, such as immature DC transmigration, these responses to RhoA would be detrimental as they would promote vascular leakage, and thus it makes sense that ICAM-2 does not activate Rho. Indeed, it may actively inhibit Rho signaling, as indicated by the decrease in rhoA mRNA levels following ICAM-2 cross-linking. Therefore, the role of endothelial cell signaling in leukocyte transmigration is likely to be very different in inflammatory compared with noninflammatory conditions.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Anne J. Ridley, Ludwig Institute for Cancer Research, 91 Riding House Street, London W1W 7PS, U.K. E-mail address: anne{at}ludwig.ucl.ac.uk
3 Abbreviations used in this paper: DC, dendritic cell; SRE, serum response element; ERM, ezrin, radixin, and moesin; N-ERMAD, N-terminal domain of ERM proteins; F-actin, filamentous actin; CPERM, C-terminally phosphorylated ERM protein; TRITC, tetramethylrhodamine isothiocyanate; EGM, endothelial growth medium; TRBD, rhotekin Rho-binding domain; GST-TRBD, TRBD bound to GST; SHP-2, Src homology 2 domain-containing tyrosine phosphatase-2.
Received for publication February 13, 2002. Accepted for publication May 7, 2002.
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 H.-M. Oh, S. Lee, B.-R. Na, H. Wee, S.-H. Kim, S.-C. Choi, K.-M. Lee, and C.-D. Jun RKIKK Motif in the Intracellular Domain Is Critical for Spatial and Dynamic Organization of ICAM-1: Functional Implication for the Leukocyte Adhesion and Transmigration Mol. Biol. Cell, June 1, 2007; 18(6): 2322 - 2335. [Abstract] [Full Text] [PDF]
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 X. Wang, C. Lau, S. Wiehler, A. Pow, T. Mazzulli, C. Gutierrez, D. Proud, and C.-W. Chow Syk Is Downstream of Intercellular Adhesion Molecule-1 and Mediates Human Rhinovirus Activation of p38 MAPK in Airway Epithelial Cells J. Immunol., November 15, 2006; 177(10): 6859 - 6870. [Abstract] [Full Text] [PDF]
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L. Yang, J. R. Kowalski, P. Yacono, M. Bajmoczi, S. K. Shaw, R. M. Froio, D. E. Golan, S. M. Thomas, and F. W. Luscinskas Endothelial Cell Cortactin Coordinates Intercellular Adhesion Molecule-1 Clustering and Actin Cytoskeleton Remodeling during Polymorphonuclear Leukocyte Adhesion and Transmigration J. Immunol., November 1, 2006; 177(9): 6440 - 6449. [Abstract] [Full Text] [PDF]
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 P. S. T. Yuen, S.-K. Jo, M. K. Holly, X. Hu, and R. A. Star Ischemic and nephrotoxic acute renal failure are distinguished by their broad transcriptomic responses Physiol Genomics, May 16, 2006; 25(3): 375 - 386. [Abstract] [Full Text] [PDF]
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 E. Cernuda-Morollon and A. J. Ridley Rho GTPases and Leukocyte Adhesion Receptor Expression and Function in Endothelial Cells Circ. Res., March 31, 2006; 98(6): 757 - 767. [Abstract] [Full Text] [PDF]
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 C. Suzuki, Y. Daigo, N. Ishikawa, T. Kato, S. Hayama, T. Ito, E. Tsuchiya, and Y. Nakamura ANLN Plays a Critical Role in Human Lung Carcinogenesis through the Activation of RHOA and by Involvement in the Phosphoinositide 3-Kinase/AKT Pathway Cancer Res., December 15, 2005; 65(24): 11314 - 11325. [Abstract] [Full Text] [PDF]
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M.-T. Huang, J. C. Mason, G. M. Birdsey, V. Amsellem, N. Gerwin, D. O. Haskard, A. J. Ridley, and A. M. Randi Endothelial intercellular adhesion molecule (ICAM)-2 regulates angiogenesis Blood, September 1, 2005; 106(5): 1636 - 1643. [Abstract] [Full Text] [PDF]
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 C. Rosette, R. B. Roth, P. Oeth, A. Braun, S. Kammerer, J. Ekblom, and M. F. Denissenko Role of ICAM1 in invasion of human breast cancer cells Carcinogenesis, May 1, 2005; 26(5): 943 - 950. [Abstract] [Full Text] [PDF]
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 S. Coupel, F. Leboeuf, G. Boulday, J.-P. Soulillou, and B. Charreau RhoA Activation Mediates Phosphatidylinositol 3-Kinase-Dependent Proliferation of Human Vascular Endothelial Cells: An Alloimmune Mechanism of Chronic Allograft Nephropathy J. Am. Soc. Nephrol., September 1, 2004; 15(9): 2429 - 2439. [Abstract] [Full Text] [PDF]
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C. V. Carman, C.-D. Jun, A. Salas, and T. A. Springer Endothelial Cells Proactively Form Microvilli-Like Membrane Projections upon Intercellular Adhesion Molecule 1 Engagement of Leukocyte LFA-1 J. Immunol., December 1, 2003; 171(11): 6135 - 6144. [Abstract] [Full Text] [PDF]
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R. Lyck, Y. Reiss, N. Gerwin, J. Greenwood, P. Adamson, and B. Engelhardt T-cell interaction with ICAM-1/ICAM-2 double-deficient brain endothelium in vitro: the cytoplasmic tail of endothelial ICAM-1 is necessary for transendothelial migration of T cells Blood, November 15, 2003; 102(10): 3675 - 3683. [Abstract] [Full Text] [PDF]
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 S. Muro, R. Wiewrodt, A. Thomas, L. Koniaris, S. M. Albelda, V. R. Muzykantov, and M. Koval A novel endocytic pathway induced by clustering endothelial ICAM-1 or PECAM-1 J. Cell Sci., April 15, 2003; 116(8): 1599 - 1609. [Abstract] [Full Text] [PDF]
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B. Butler, M. P. Williams, and S. D. Blystone Ligand-dependent Activation of Integrin alpha vbeta 3 J. Biol. Chem., February 7, 2003; 278(7): 5264 - 5270. [Abstract] [Full Text] [PDF]
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