1 Department of Physiology, University of Pennsylvania School of Medicine, B-400
Richards/6085, 3700 Hamilton Walk, Philadelphia, PA 19104, USA
2 Department of Pharmacology and Medicine, Pulmonary, University of Pennsylvania
School of Medicine, B-400 Richards/6085, 3700 Hamilton Walk, Philadelphia, PA
19104, USA
3 Department of Critical Care Division, University of Pennsylvania School of
Medicine, B-400 Richards/6085, 3700 Hamilton Walk, Philadelphia, PA 19104,
USA
4 Institute for Environmental Medicine, University of Pennsylvania School of
Medicine, B-400 Richards/6085, 3700 Hamilton Walk, Philadelphia, PA 19104,
USA
* Authors for correspondence (e-mail: mkoval{at}mail.med.upenn.edu, muzykant{at}mail.med.upenn.edu)
Accepted 14 February 2003
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: HUVEC, Vascular endothelium, Cell adhesion, Macropinocytosis, Endocytosis
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
It is becoming apparent that both PECAM-1 and ICAM-1 may serve as plasma
membrane receptors to mediate internalization of natural ligands by different
types of cells. For instance, coxsackieviruses and rhinoviruses bind ICAM-1
and are internalized (Shafren et al.,
1997a), although other coreceptors may be involved in this process
(Shafren et al., 1997b
). HIV
is internalized into brain endothelial cells by a pathway that is analogous to
macropinocytosis into endocytic vesicles that also contain ICAM-1
(Liu et al., 2002
). A pathway
related to macropinocytosis has also been implicated in the clearance of
apoptotic cell fragments by epithelial cells
(Fiorentini et al., 2001
). The
notion that endothelial cells might help scavenge apoptotic cells is
underscored by the observation that PECAM-1 is required for the binding of
malaria-infected red blood cells to human umbilical vein endothelial cells
(HUVEC) in culture (Treutiger et al.,
1997
), although these particles were too large to be endocytosed.
In a recent study, PECAM-1 expressed by macrophages was found to play an
important role in cellular recognition and uptake: apoptotic cells binding to
macrophage PECAM-1 were efficiently phagocytosed, whereas live cells activated
a signaling cascade through macrophage PECAM-1 to weaken their engagement to
macrophages and enable their release (Brown
et al., 2002
). Cells, apoptotic fragments and viruses binding to
ICAM-1 and PECAM-1 are multivalent, complex and, in the case of live cells,
active partipicants in cellcell interactions, making it difficult to
discern roles for specific plasma membrane proteins as potential receptors. By
contrast, anti-ICAM-1 and anti-PECAM-1 conjugates, although multivalent, will
primarily engage only the cell adhesion molecule of interest, which makes them
useful probes for examining specific internalization pathways mediated by
ICAM-1 or PECAM-1.
There are multiple pathways for ligand internalization involving vesicles
100-300 nm in diameter, including clathrin-mediated endocytosis and the
clathrin-independent caveolae-mediated pathway
(Mukherjee et al., 1997;
Nichols and Lippincott-Schwartz,
2001
). Each of these endocytic mechanisms differs in sensitivity
to pharmacological agents, which enables the mechanism of ligand
internalization to be determined. Caveolae-mediated endocytosis is a
particularly important pathway in endothelial cells, where ligands such as
albumin (Minshall et al.,
2000
) and orosomucoid
(Predescu et al., 1998
) are
internalized via receptors clustered into caveolae and subsequently
transcytosed across the endothelial barrier
(McIntosh et al., 2002
). There
are also clathrin-independent pathways distinct from caveolar endocytosis,
which mediate uptake of glycosylphosphatidylinositol (GPI)-anchored proteins,
such as the folate receptor (Mayor et al.,
1998
) and diptheria toxin receptor
(Skretting et al., 1999
).
The regulation of ICAM-1 and PECAM-1 internalization by endothelial cells is not well understood at present. In particular, whether ICAM-1 and PECAM-1 are internalized by similar pathways is not known. In this study, we defined some key elements regulating the internalization of anti-ICAM-1 or anti-PECAM-1 conjugates by endothelial cells. In each case, clustering of the CAM was required for efficient internalization. Given that anti-ICAM-1 and anti-PECAM-1 conjugates did not colocalize with known endocytic coat proteins and from the analysis of the signaling pathways that regulate the uptake of anti-ICAM-1 and anti-PECAM-1 conjugates, our data suggests that that endothelial cells internalize clustered ICAM-1 and PECAM-1 using a novel endocytic pathway.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell culture
Pooled human umbilical vein endothelial cells (HUVEC) from Clonetics (San
Diego, CA) were maintained in M199 medium (GibcoBRL, Grand Island, NY)
supplemented with 15% fetal bovine serum (FBS), 2 mM glutamine, 15 µg/ml
endothelial cell growth supplement (ECGS), 100 µg /ml heparin, 100 U/ml
penicillin and 100 µg/ml streptomycin. EAhy926 cells from an
endothelial-like hybrid cell line generated from HUVEC and A549 cells
(Edgell et al., 1983) were
cultured in DMEM medium (GibcoBRL) supplemented with 10% FBS, glutamine and
antibiotics. Cultures were maintained at 37°C, 5% CO2 and 95%
relative humidity in 1% gelatin-coated tissue culture plastic. HUVEC were used
between passage 4 and 5. When seeded for experiments, the cells were cultured
onto 12 mm2 gelatin-coated coverslips in 24-well plates in the
absence of antibiotics and then treated with tumor necrosis factor-
(TNF-
) for at least 16 hours.
Preparation of immunobeads and immunoconjugates
Fluorescent microspheres were coated with either anti-ICAM-1, anti-PECAM-1
or control murine IgG by incubation at room temperature (RT) for 1 hour as
previously described (Wiewrodt et al.,
2002). The coated microspheres (immunobeads) were centrifuged to
remove unbound antibodies, then resuspended in 1% bovine serum albumin-PBS and
microsonicated for 20 seconds at low power. The effective immunobead diameter
was determined by dynamic light scattering (DLS) using BI-90Plus particle size
analyzer with BI-9000AT Digital auto-correlator (Brookhaven Instruments,
Brookhaven, NY) as previously described
(Wiewrodt et al., 2002
). This
immunobead protocol yielded uniform preparations with particle diameters
ranging from 180 to 250 nm. For anti-ICAM-1 immunoconjugates, anti-ICAM-1 was
biotinylated and complexed to streptavidin (90% unlabeled, 10% rhodamine
labeled) in a manner equivalent to anti-PECAM-1 immunoconjugates as previously
described (Wiewrodt et al.,
2002
). The ratio of biotinylated-ICAM-1 to streptavidin was varied
to generate immunoconjugates either smaller than 500 nm or larger than 1000
nm, as determined by DLS.
Binding and uptake of anti-ICAM-1 and anti-PECAM-1 immunobeads
Confluent HUVEC or EAhy926 cells were pre-incubated overnight with 250
units of TNF-. By flow cytometry, TNF-
treatment increased
ICAM-1 expression by HUVEC and EAhy926 cells
10-fold and had little
effect on PECAM-1 expression, which is consistent with previously published
results (Delisser and Albeda, 1998). The cells were then washed in serum-free
medium and incubated in 1% BSA-medium containing a 1:10 dilution of either
uncoated microspheres, or immunobeads coated with control murine IgG,
anti-ICAM-1 or anti-PECAM-1. The cells were incubated with immunobead
preparations for different time periods at 4°C or 37°C, washed in
medium and fixed with 2% paraformaldehyde at RT. To distinguish between
surface-bound or internalized immunobeads, nonpermeabilized fixed cells were
counterstained for 30 minutes at RT with Texas Red (TxR)-conjugated goat
anti-mouse IgG to produce double-labeled, yellow particles. The cells were
washed in PBS, mounted onto slides with Mowiol and analyzed by fluorescence
microscopy. Alexa Fluor 594-labeled cholera toxin B (FL-cholera toxin)
counterstained with goat anti-cholera toxin + fluorescein rabbit anti-goat IgG
was used as a control for caveolae-mediated uptake. TxR-labeled transferrin
counterstained with goat anti-transferrin + fluorescein rabbit anti-goat IgG
was used as a control for clathrin-mediated endocytosis.
To identify compartments containing internalized immunobeads, HUVEC monolayers were incubated with immunobeads for 1 hour at 4°C to allow surface binding, washed, then incubated at 37°C for different time periods to permit endocytosis. The cells were fixed, permeabilized and incubated with rabbit polyclonal anti-human caveolin-1, followed by incubation with goat anti-rabbit IgG conjugated to Alexa Fluor 350. Colocalization with clathrin heavy chain was done in a comparable manner, using TRITC-conjugates anti-clathrin.
For microscopy, samples mounted onto glass slides were observed using an
Olympus IX70 inverted fluorescence microscope, 40x or 60x PlanApo
objectives and filters optimized for fluorescent immunobeads (excitation
BP460-490 nm, dichroic DM505 nm, emission BA515-550 nm), TxR fluorescence
(excitation BP530-550 nm, dichroic DM570 nm, emission BA590-800+ nm) and Alexa
Fluor 450 (excitation BP360-370 nm, dichroicDM400 nm, emission BA420-460 nm)
(Chroma Technology, Brattleboro, VT). Separate images for each fluorescence
channel were acquired using a Hamamatsu Orca-1 CCD camera. The images were
then merged and analyzed with ImagePro 3.0 imaging software (Media
Cybernetics, Silver Spring, MD) as previously described
(Wiewrodt et al., 2002). For
quantitation, merged images of cells labeled with immunobeads were scored
automatically for total green fluorescent particles and noninternalized
immunobeads (double-labeled yellow particles). Uptake was calculated as the
percentage of internalized immunobeads with respect to the total number of
cell-associated immunobeads. Statistical significance was determined by
Student's t test.
Mechanisms of ICAM-1- and PECAM-1-mediated uptake
Mammalian pcDNA3 expression vectors encoding for 6-His-tagged versions of
human dynamin-2 [wild-type and dominant-negative forms (K44A), (PH*)] were
gifts from Drs S. Schmid (Scripps Research Institute, La Jolla, CA)
(Altschuler et al., 1998) and
M. Lemmon (U Penn School of Medicine, Philadelphia, PA)
(Lee et al., 1999
). EAhy926
endothelial cells were transfected using Lipofectin (GibcoBRL) complexed to
1.5 µg DNA/dish encoding either dynamin-2, dynamin-2(K44A) or
dynamin-2(PH*). Each construct includes a 6-His amino terminus tag to
distinguish it from endogenous dynamin-2. Twelve hours after transfection, the
cells were stimulated with TNF-
, incubated for 36 hours and then
anti-ICAM-1 or anti-PECAM-1 uptake was determined by double labeling as
described above. Following labeling of surface-bound material, the cells were
permeabilized with 0.2% Triton X-100 and then immunostained using 5 µg/ml
rabbit anti-6-His-Tag and goat anti-rabbit IgG conjugated to Alexa Fluor 350
to identify cells expressing recombinant dynamin-2.
For studies using pharmacological inhibitors, TNF--stimulated HUVEC
or EAhy926 were pre-incubated for 30 minutes at 37°C in the presence of
one of the following agents: 50 µM monodansyl-cadaverine (MDC), 1 µg/ml
filipin, 50 µM genistein, 3 mM amiloride, 25 µM monensin, 0.5 mM
cytochalasin D, 0.1 µM latrunculin A, 20 µM nocodazole, 5 µM
bisindolyl-maleimide-1 (BIM-1), 10 µM 1-(5-isoquiniline
sulphonyl)-2-methylpiperazine (H7), 0.1 µM phorbol 12-myristate 13-acetate
(PMA), 10 µM radicicol, 10 µM Y-27346 or 0.5 µM wortmannin
(Barreiro et al., 2002
;
Fujimoto et al., 2000
;
Parton et al., 1994
;
Racoosin and Swanson, 1989
;
Sahai and Marshall, 2002
;
Schlegel et al., 1982
;
Schnitzer et al., 1994
;
Swanson, 1989
;
Torgersen et al., 2001
;
Watanabe et al., 2001
;
West et al., 1989
). Molecular
targets for selected inhibitors are shown in
Fig. 10. The concentration of
each agent was selected using literature values and was optimized
qualitatively by fluorescence microscopy (not shown). Also, we examined the
effectiveness of each agent using suitable controls (e.g.
Fig. 4). Potassium depletion
was done by pre-incubating the cells for 15 minutes in potassium depletion
buffer (0.14 M NaCl, 2 mM CaCl2, 1 mg/ml glucose, 20 mM HEPES, pH
7.4) diluted 1:1 with water to make it hypotonic
(Koval et al., 1998
). After
treatment, the cells were incubated with immunobeads, cholera toxin or
transferrin at 37°C, in the presence of K+-depletion buffer or
the given inhibitors, and then fixed and double labeled for surface-bound
material as described above.
|
|
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
On average, 125 immunobeads/cell were internalized after a 1 hour
incubation at 37°C. Fig. 1G
shows that uptake of anti-ICAM-1 immunobeads was more rapid (
10 minutes
half time for uptake) than that of anti-PECAM-1 immunobeads (
20 minutes
half time for uptake). Similar results were obtained for anti-ICAM-1 and
anti-PECAM-1 immunobeads internalized by naïve HUVEC, indicating that
TNF-
had little effect on the mechanism of internalization. This also
suggests that the mechanism for internalization is not sensitive to the
surface density of ICAM-1, as virtually all of the anti-ICAM-1 immunobeads
were internalized after 30 minutes at 37°C, despite the difference in
binding in the absence (42±15 beads/cell) or presence of TNF-
(165±54 beads/cell). Also, as found for anti-PECAM-1, monomeric
anti-ICAM-1 was not internalized (S.M., R.W. and A.T. et al.,
unpublished).
Because dynamin-2 is frequently involved in vesicle-mediated
internalization and phagocytosis
(Altschuler et al., 1998;
Gold et al., 1999
;
Henley et al., 1998
;
Lee et al., 1999
), we examined
the role for dynamin-2 in uptake of anti-ICAM-1 immunobeads. Given the low
transfection rate of HUVEC, we used the endothelial-like cell line, EAhy926,
which showed a 40% transfection efficiency using Lipofectin. EAhy926 cells
internalized anti-ICAM-1 immunobeads in a manner comparable to HUVEC
(Fig. 2). These cells were
transiently transfected to express amino-terminal 6-His-tagged forms of either
wild-type or dominant-negative dynamin-2 (K44A or PH*). Expression of
recombinant proteins was identified by immunofluorescence, using an antibody
that recognizes the 6-His epitope (Fig.
2). Cells transfected with wild-type dynamin-2 showed no effect on
the uptake of anti-ICAM-1 immunobeads compared with control cells. By
contrast, cells expressing ether dynamin-2(K44A) or dynamin-2(PH*) showed less
anti-ICAM-1 immunobead uptake than control cells, suggesting that uptake of
these immunobeads required dynamin-2 (Fig.
2). This was not due to a net decrease in immunobead binding,
which was equivalent for nontransfected EAhy926 cells (23±3 beads/cell)
and EAhy926 cells expressing wild-type (24±16 beads/cell) or mutant
dynamin-2 [25±4 beads/cell (PH*), 14±5 beads/cell (K44A)].
|
We also found that the dominant-negative dynamin-2 constructs inhibited the
uptake of Alexa Fluor 594-conjugated cholera toxin (FL-cholera toxin, S.M.,
R.W. and A.T. et al., unpublished) that is internalized by caveolae-mediated
endocytosis (Schnitzer et al.,
1994). However, few, if any, anti-ICAM-1 immunobeads colocalized
with caveolin-1-positive structures, regardless of whether the immunobeads
were bound to the plasma membrane or internalized by HUVEC
(Fig. 3). By contrast,
FL-cholera toxin showed extensive colocalization with caveolin, which is
consistent with previously published reports
(Puri et al., 2001
). Newly
internalized anti-ICAM-1 immunobeads also did not colocalize with clathrin. In
fact, there was a low level of clathrin immunofluorescence shown by HUVEC,
consistent with a less-dominant role for clathrin-coated pits in endocytosis
than caveolae-mediated pathways in endothelial cells
(Schubert et al., 2001
). Thus,
despite being a dynamin-dependent process, anti-ICAM-1 conjugate uptake by
endothelial cells was unlikely to be through caveolae- or clathrin-coated
vesicles.
|
We therefore used a series of pharmacological inhibitors to further
characterize internalization of anti-ICAM-1 and anti-PECAM-1 conjugates by
HUVEC. The specificity of different inhibitors was confirmed using fluorescent
transferrin and cholera toxin as controls for clathrin-mediated and caveolar
endocytosis, respectively (Fig.
4). As shown in Fig.
5, inhibitors of clathrin-mediated transferrin endocytosis (MDC,
potassium depletion) did not inhibit the uptake of anti-ICAM-1 or anti-PECAM-1
immunobeads. Furthermore, inhibitors of caveolae-dependent cholera toxin
uptake (filipin, genestein) were not effective at inhibiting anti-ICAM-1 or
anti-PECAM-1 immunobead internalization. Because uptake of anti-ICAM-1 and
anti-PECAM-1 immunobeads appeared to be through a unique internalization
pathway, we examined the effect of other inhibitors on immunobead endocytosis.
Previous work has indicated that amiloride, an inhibitor of the sodium/proton
pump, can inhibit macropinocytosis by dendritic cells
(West et al., 1989). Amiloride
had little effect on internalization of FL-cholera toxin or transferrin,
suggesting that it did not inhibit caveolae- or clathrin-mediated endocytosis
(Fig. 4). However, amiloride
inhibited uptake of anti-ICAM-1 and anti-PECAM-1 immunobeads by HUVEC
(55±15% and 60±9%, respectively) and by EAhy926 cells
(34±9% and 24±3% inhibition, respectively. Anti-ICAM-1
immunobead binding was equivalent for control (125±21 beads/cell) and
amiloride-treated HUVEC (154±37 beads/cell), suggesting that amiloride
did not decrease ICAM-1 surface expression. TNF-
stimulation was not
required, given that amiloride inhibited anti-PECAM-1 immunobead uptake by
naïve HUVEC by 50.5±6.2%. Also, anti-ICAM-1 immunobead uptake by
amiloride-treated HUVEC remained inhibited during a 3 hour (43.8±2.9%)
and 5 hour incubation (50.6±5.2%), suggesting that amiloride altered
the extent of immunobead uptake, rather than uptake kinetics. Furthermore,
this was not likely to be due to an effect on ion homeostasis, given that the
ionophore monensin had little, if any, effect on anti-ICAM-1 and anti-PECAM-1
immunobead uptake (Fig. 5).
|
Because protein kinase C (PKC) has been reported to play a pivotal role in
macropinocytosis and phagocytosis by macrophages
(Araki et al., 1996;
Larsen et al., 2000
;
Swanson, 1989
), we tested the
effect of PKC inhibitors on immunobead internalization by HUVEC. As shown in
Fig. 6, the PKC inhibitors
BIM-1 and H-7 inhibited immunobead uptake by
30% and
60%,
respectively. H-7 treatment also inhibited the uptake of anti-ICAM-1 and
anti-PECAM-1 by EAhy926 cells by 55±11% and 48±7%, respectively.
HUVEC pretreated with 0.1 µM PMA for 30 minutes (conditions that stimulate
PKC activity) showed a high level of anti-ICAM-1 and anti-PECAM-1
internalization (>95%), and the total level of immunobead uptake by HUVEC
was stimulated nearly twofold. Anti-ICAM-1 immunobead binding was equivalent
for control (125±21 beads/cell) and BIM-1-treated HUVEC (140±7
beads/cell), suggesting that BIM-1 did not decrease ICAM-1 surface expression.
Stimulation by TNF-
was not required, since inhibiting PK-C activity
also inhibited uptake of anti-PECAM-1 immunobeads by naïve HUVEC
(47.5±9.8%) and uptake of anti-PECAM-1 immunobeads by naïve HUVEC
was enhanced 1.3-fold by PMA. Also, anti-ICAM-1 immunobead uptake by
BIM-1-treated HUVEC remained inhibited during a 3 hour (39.6±4.2%) and
5 hour incubation (42.9±5.2%), suggesting that BIM-1 altered the extent
of immunobead uptake, rather than uptake kinetics. Taken together, these
results are consistent with internalization of anti-ICAM-1 and anti-PECAM-1
immunobeads by a PKC-dependent pathway. However, since H-7 may also interfere
with actin-based contractility (Volberg et
al., 1994
), this effect may also contribute to the inhibition of
uptake.
|
In fact, the formation of F actin stress fibers is frequently associated
with ICAM-1 crosslinking (Thompson et al.,
2002; Wang and Doerschuk,
2002
). Therefore, we examined the effect of anti-ICAM-1
immunobeads on the formation of actin stress fibers by HUVEC. As shown in
Fig. 7, stress fibers were
rapidly induced by anti-ICAM-1 immunobead binding. Immunobeads appeared to
align along actin stress fibers before internalization; this is shown most
prominently by the blue labeled immunobeads in
Fig. 7C,D. Vesicles containing
internalized immunobeads continued to be associated with stress fibers after
internalization and remained associated with actin during a 3 hour incubation.
Few, if any, anti-ICAM-1 immunobeads induced formation of an actin coat
(phagocytic cup) at the site of internalization, which is a hallmark of
phagocytosis and macropinocytosis (Grimmer
et al., 2002
; Lee and Knecht,
2002
). Note that this is probably not a problem with the detection
of actin coats, as a previous study found that
20% of 0.2 µm beads
internalized by Fc receptors in macrophages were associated with actin coats
(Koval et al., 1998
).
|
Given the dramatic association of anti-ICAM-1 immunobeads with actin in
HUVEC, we also examined the cytoskeletal requirements for the uptake of
anti-ICAM-1 and anti-PECAM-1 immunobeads. In contrast to macropinocytosis by
macrophages (Racoosin and Swanson,
1992), microtubules were not required for the internalization of
anti-ICAM-1 immunobeads by HUVEC, since internalization was not inhibited by
nocodazole (Fig. 8). Also,
nocodazole did not significantly inhibit the uptake of anti-PECAM-1
immunobeads (12±9% inhibition). Cytochalasin D, which caps short actin
filaments, had little effect on the uptake of anti-ICAM-1 immunobeads
(18±7% inhibition) or anti-PECAM-1 immunobeads (8±8%
inhibition). However, the more effective actin depolymerizing agent,
latrunculin, inhibited anti-ICAM-1 immunobead uptake
(Fig. 8) and uptake of
anti-PECAM-1 immunobeads (69±15% inhibition).
|
We also examined inhibitors that affect kinases known to play a role in
regulating actin organization. In contrast to macropinocytosis by phagocytes
(Araki et al., 1996;
West et al., 2000
), wortmanin
had little, if any, effect on uptake of anti-ICAM-1 immunobeads by HUVEC,
suggesting that PI3-kinases were not involved in immunobead uptake. However,
as shown in Fig. 8, uptake of
anti-ICAM-1 immunobeads was inhibited by the Src kinase inhibitor radicicol
and the ROCK inhibitor Y27632, consistent with the notion that both the PKC
pathway and Rho pathway regulate actin cytoskeletal rearrangements required
for internalizaton of clustered ICAM-1. Four inhibitors of anti-ICAM-1
immunobead internalization latrunculin, amiloride, radicicol and
Y27632 all inhibit the formation of actin stress fibers induced by
anti-ICAM-1 immunobead binding (Fig.
9), underscoring the correlation of uptake by HUVEC with actin
mobilization.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
On the basis of amiloride sensitivity and PKC dependence, internalization
of clustered anti-ICAM-1 and anti-PECAM-1 seemed to be related to
macropinocytosis (Lamaze and Schmid,
1995; Nichols and
Lippincott-Schwartz, 2001
;
Orth et al., 2002
;
Swanson and Watts, 1995
), a
pathway that is not typically associated with endothelial cells. Nonetheless,
CAM-mediated endocytosis was distinct from `classical' macropinocytosis, on
the basis of several criteria (Table
1). For example, in contrast to the dynamin-2 requirement we
observed for uptake of anti-ICAM-1 immunobeads by EAhy926 endothelial cells,
the K44A dominant-negative dynamin-2 did not inhibit macropinocytosis by
fibroblasts (Orth et al.,
2002
). Although dynamin is required for endocytosis
(Altschuler et al., 1998
;
Henley et al., 1998
;
Lee et al., 1999
) and
phagocytosis (Gold et al.,
1999
), CAM-mediated endocytosis differed in other ways from these
processes. For instance, anti-ICAM-1 immunobeads did not colocalize with
either clathrin or caveolin (Fig.
4). Also, uptake of anti-ICAM-1 immunobeads did not require PI3K
activity, which is needed for phagocytosis
(Araki et al., 1996
;
Cox et al., 1999
), as well as
macropinocytosis (West et al.,
2000
).
The uptake of anti-ICAM-1 and anti-PECAM-1 conjugates required clustering
of cell adhesion molecules (Muzykantov et
al., 1999; Wiewrodt et al.,
2002
). ICAM-1 clustering has been found to stimulate multiple
intracellular signaling pathways (Adamson
et al., 1999
; Etienne et al.,
1998
), including a PKC signaling pathway that results in the
phosphorylation of cytoskeletal and focal adhesion proteins, thereby enabling
actin filament rearrangement
(Etienne-Manneville et al.,
2000
). Consistent with this, anti-ICAM immunobeads induced actin
stress fiber formation and were associated with stress fibers before
internalization (Fig. 7). Both
Src kinase and ROCK activity are required for CAM-mediated endocytosis and
these inhibitors also inhibited stress fiber formation induced by anti-ICAM-1
immunobeads (Fig. 9). ROCK
activity also enables remodeling of F-actin attached to adherence junctions
and controls their stability (Sahai and
Marshall, 2002
). This also provides a potential link between
ICAM-1 or PECAM-1 clustering, Src kinase, PKC and dynamin-2
(Fig. 10), as dynamins are
downstream targets for Src kinase (Ahn et
al., 2002
) and PKC (Powell et
al., 2000
).
Dynamin, via interactions with endophilin and profilin, helps recruit actin
to sites of endocytic activity (Farsad et
al., 2001; Witke et al.,
1998
). Proteins in the ezrin-radixin-moesin (ERM) family are also
good candidates to link internalization of ICAM-1 and PECAM-1 to the actin
cytoskeleton (Bretscher et al.,
1997
; Cao et al.,
1999
). For instance, ezrin binds directly to the C-terminus of
ICAM-1 (Heiska et al., 1998
).
Another actin binding protein,
-actinin, has also been shown to bind to
the C-terminus of ICAM-1 (Carpen et al.,
1992
). Perhaps signaling induced by ICAM-1 or PECAM-1 clustering
can indirectly recruit ERM proteins to the plasma membrane. For instance,
phosphorylation of ERM proteins by a ROCK-dependent pathway can help recruit
them to the plasma membrane (Hirao et al.,
1996
). Intriguingly, ROCK activity has been associated with
complement receptor-mediated phagocytosis, but is not required for Fc
receptor-mediated endocytosis (Olazabal et
al., 2002
). ROCK has also been shown to phosphorylate sodium
proton exchangers (NHE) to enhance binding of ERM proteins
(Denker et al., 2000
). Both
processes might correspond to the ROCK requirement for uptake of clustered
ICAM-1 or PECAM-1 (Fig. 10).
This is further suggested by the ability of amiloride to inhibit uptake of
clustered ICAM-1 and PECAM-1, since amiloride can disrupt the association of
ERM proteins with NHE, an effect that is independent of ion channel activity
(Denker et al., 2000
;
Putney et al., 2002
).
Another major distinction from macropinocytosis and phagocytosis is that
uptake of anti-ICAM-1 and anti-PECAM-1 conjugates larger than 500 nm in
diameter was poor (Fig. 1)
(Wiewrodt et al., 2002). Also,
anti-ICAM-1 immunobeads smaller than 500 nm diameter did not induce the
formation of an actin cup or coat (Fig.
7), which is typically induced by larger particles internalized by
phagocytosis (Koval et al.,
1998
), suggesting that formation of an actin coat is crucial for
internalization of larger particles. Although the mechanisms that cells use to
control the size threshold for internalization is not known at present, given
that ICAM-1 and PECAM-1 primarily regulate cellcell contacts, a small
size threshold for internalization may be a means by which endothelial cells
avoid engulfing other cells.
Consistent with a size threshold for ICAM-1-mediated internalization, an
ICAM-1 enriched structure is formed at the contact site between lymphocytes
and HUVEC, where the endothelial cell appears to partially engulf the
lymphocyte (Barreiro et al.,
2002). Furthermore, the lymphocyte-endothelial cell docking
structure requires ROCK activity, but not phosphatidylinositol 3-kinase (PI-3
kinase) (Barreiro et al.,
2002
), comparable to our observations for anti-ICAM-1 and
anti-PECAM-1 immunobeads (Fig.
9). Whether plasma membrane internalization is part of the
mechanism required to maintain this docking structure is not known at present.
One possibility is that CAM-mediated endocytosis might help to remodel
cellcell junctions as leukocytes migrate along endothelial cells. If
so, this might be analogous to the turnover of gap junctions, which is
mediated by the engulfment of cellcell junctions sites to create
endocytic vesicles in the 200-500 nm diameter size range
(Gaietta et al., 2002
;
Jordan et al., 2001
).
The size threshold for internalization of clustered ICAM-1 and PECAM-1
might also enable endothelial cells to distinguish small apoptotic fragments
from intact cells bound to the endothelial cell surface, such as other
endothelial or blood cells (Barreiro et
al., 2002; DeLisser and
Albelda, 1998
; Johnson-Leger
et al., 2000
; Worthylake and
Burridge, 2001
). The notion that endothelial cells could also
scavenge apoptotic fragments via a pathway comparable to CAM-mediated
endocytosis is appealing (Brown et al.,
2002
; Treutiger et al.,
1997
); however, whether this is the case remains to be
determined.
Understanding the mechanisms that regulate uptake of anti-ICAM-1 and
anti-PECAM-1 conjugates will probably help to extend the utility of these
agents as the basis for endothelium-specific drug-targeting vehicles
(Li et al., 2000;
Muzykantov et al., 1999
;
Scherpereel et al., 2002
;
Scherpereel et al., 2001
;
Wiewrodt et al., 2002
). For
instance, inhibitors of conjugate uptake might help to increase their
stability by reducing the extent of delivery to lysosomes and other
degradative compartments. Animal studies combining the agents used in this
work with the administration of pharmacologically active, enzyme-carrying
anti-ICAM or anti-PECAM conjugates will be used to determine the feasibility
of this approach.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Adamson, P., Etienne, S., Couraud, P. O., Calder, V. and
Greenwood, J. (1999). Lymphocyte migration through brain
endothelial cell monolayers involves signaling through endothelial ICAM-1 via
a rho-dependent pathway. J. Immunol.
162,2964
-2973.
Ahn, S., Kim, J., Lucaveche, C. L., Reedy, M. C., Luttrell, L.
M., Lefkowitz, R. J. and Daaka, Y. (2002). Src-dependent
tyrosine phosphorylation regulates dynamin self-assembly and ligand-induced
endocytosis of the epidermal growth factor receptor. J. Biol.
Chem. 277,26642
-26651.
Altschuler, Y., Barbas, S. M., Terlecky, L. J., Tang, K., Hardy,
S., Mostov, K. E. and Schmid, S. L. (1998). Redundant and
distinct functions for dynamin-1 and dynamin-2 isoforms. J. Cell
Biol. 143,1871
-1881.
Araki, N., Johnson, M. T. and Swanson, J. A. (1996). A role for phosphoinositide 3-kinase in the completion of macropinocytosis and phagocytosis by macrophages. J. Cell Biol. 135,1249 -1260.[Abstract]
Barreiro, O., Yanez-Mo, M., Serrador, J. M., Montoya, M. C.,
Vicente-Manzanares, M., Tejedor, R., Furthmayr, H. and Sanchez-Madrid, F.
(2002). Dynamic interaction of VCAM-1 and ICAM-1 with moesin and
ezrin in a novel endothelial docking structure for adherent leukocytes.
J. Cell Biol. 157,1233
-1245.
Bretscher, A., Reczek, D. and Berryman, M.
(1997). Ezrin: a protein requiring conformational activation to
link microfilaments to the plasma membrane in the assembly of cell surface
structures. J. Cell Sci.
110,3011
-3018.
Brown, S., Heinisch, I., Ross, E., Shaw, K., Buckley, C. D. and Savill, J. (2002). Apoptosis disables CD31-mediated cell detachment from phagocytes promoting binding and engulfment. Nature 418,200 -203.[CrossRef][Medline]
Cao, T. T., Deacon, H. W., Reczek, D., Bretscher, A. and von Zastrow, M. (1999). A kinase-regulated PDZ-domain interaction controls endocytic sorting of the beta2-adrenergic receptor. Nature 401,286 -290.[CrossRef][Medline]
Caron, E. and Hall, A. (2001). Phagocytosis. In Endocytosis (ed. M. Marsh), pp.58 -77. Oxford: Oxford University Press.
Carpen, O., Pallai, P., Staunton, D. E. and Springer, T. A. (1992). Association of intercellular adhesion molecule-1 (ICAM-1) with actin-containing cytoskeleton and alpha-actinin. J. Cell Biol. 118,1223 -1234.[Abstract]
Cox, D., Tseng, C. C., Bjekic, G. and Greenberg, S.
(1999). A requirement for phosphatidylinositol 3-kinase in
pseudopod extension. J. Biol. Chem.
274,1240
-1247.
Danilov, S. M., Gavrilyuk, V. D., Franke, F. E., Pauls, K.,
Harshaw, D. W., McDonald, T. D., Miletich, D. J. and Muzykantov, V. R.
(2001). Lung uptake of antibodies to endothelial antigens: key
determinants of vascular immunotargeting. Am. J. Physiol. Lung
Cell. Mol. Physiol. 280,L1335
-1347.
DeLisser, H. M. and Albelda, S. M. (1998). The
function of cell adhesion molecules in lung inflammation: more questions than
answers. Am. J. Respir. Cell Mol. Biol.
19,533
-536.
Denker, S. P., Huang, D. C., Orlowski, J., Furthmayr, H. and Barber, D. L. (2000). Direct binding of the NaH exchanger NHE1 to ERM proteins regulates the cortical cytoskeleton and cell shape independently of H(+) translocation. Mol. Cell 6,1425 -1436.[Medline]
Edgell, C. J., McDonald, C. C. and Graham, J. B. (1983). Permanent cell line expressing human factor VIII-related antigen established by hybridization. Proc. Natl. Acad. Sci. USA 80,3734 -3737.[Abstract]
Etienne, S., Adamson, P., Greenwood, J., Strosberg, A. D.,
Cazaubon, S. and Couraud, P. O. (1998). ICAM-1 signaling
pathways associated with Rho activation in microvascular brain endothelial
cells. J. Immunol. 161,5755
-5761.
Etienne-Manneville, S., Manneville, J. B., Adamson, P.,
Wilbourn, B., Greenwood, J. and Couraud, P. O. (2000).
ICAM-1-coupled cytoskeletal rearrangements and transendothelial lymphocyte
migration involve intracellular calcium signaling in brain endothelial cell
lines. J. Immunol. 165,3375
-3383.
Farsad, K., Ringstad, N., Takei, K., Floyd, S. R., Rose, K. and
De Camilli, P. (2001). Generation of high curvature membranes
mediated by direct endophilin bilayer interactions. J. Cell
Biol. 155,193
-200.
Fiorentini, C., Falzano, L., Fabbri, A., Stringaro, A., Logozzi,
M., Travaglione, S., Contamin, S., Arancia, G., Malorni, W. and Fais, S.
(2001). Activation of rho GTPases by cytotoxic necrotizing factor
1 induces macropinocytosis and scavenging activity in epithelial cells.
Mol. Biol. Cell 12,2061
-2073.
Fujimoto, L. M., Roth, R., Heuser, J. E. and Schmid, S. L. (2000). Actin assembly plays a variable, but not obligatory role in receptor-mediated endocytosis in mammalian cells. Traffic 1,161 -171.[CrossRef][Medline]
Gaietta, G., Deerinck, T. J., Adams, S. R., Bouwer, J., Tour,
O., Laird, D. W., Sosinsky, G. E., Tsien, R. Y. and Ellisman, M. H.
(2002). Multicolor and electron microscopic imaging of connexin
trafficking. Science
296,503
-507.
Gold, E. S., Underhill, D. M., Morrissette, N. S., Guo, J.,
McNiven, M. A. and Aderem, A. (1999). Dynamin 2 is required
for phagocytosis in macrophages. J. Exp. Med.
190,1849
-1856.
Grimmer, S., Van Deurs, B. and Sandvig, K.
(2002). Membrane ruffling and macropinocytosis in A431 cells
require cholesterol. J. Cell Sci.
115,2953
-2962.
Hansen, S. H., Sandvig, K. and van Deurs, B. (1993). Clathrin and HA2 adaptors: effects of potassium depletion, hypertonic medium, and cytosol acidification. J. Cell Biol. 121,61 -72.[Abstract]
Heiska, L., Alfthan, K., Gronholm, M., Vilja, P., Vaheri, A. and
Carpen, O. (1998). Association of ezrin with intercellular
adhesion molecule-1 and -2 (ICAM-1 and ICAM-2). Regulation by
phosphatidylinositol 4,5-bisphosphate. J. Biol. Chem.
273,21893
-21900.
Henley, J. R., Krueger, E. W., Oswald, B. J. and McNiven, M.
A. (1998). Dynamin-mediated internalization of caveolae.
J. Cell Biol. 141,85
-99.
Hirao, M., Sato, N., Kondo, T., Yonemura, S., Monden, M., Sasaki, T., Takai, Y. and Tsukita, S. (1996). Regulation mechanism of ERM (ezrin/radixin/moesin) protein/plasma membrane association: possible involvement of phosphatidylinositol turnover and Rho-dependent signaling pathway. J. Cell Biol. 135, 37-51.[Abstract]
Jacobson, B. S., Stolz, D. B. and Schnitzer, J. E. (1996). Identification of endothelial cell-surface proteins as targets for diagnosis and treatment of disease. Nat. Med. 2,482 -484.[CrossRef][Medline]
Johnson-Leger, C., Aurrand-Lions, M. and Imhof, B. A.
(2000). The parting of the endothelium: miracle, or simply a
junctional affair? J. Cell Sci.
113,921
-933.
Jordan, K., Chodock, R., Hand, A. R. and Laird, D. W.
(2001). The origin of annular junctions: a mechanism of gap
junction internalization. J. Cell Sci.
114,763
-773.
Koval, M., Preiter, K., Adles, C., Stahl, P. D. and Steinberg, T. H. (1998). Size of IgG-opsonized particles determines macrophage response during internalization. Exp. Cell Res. 242,265 -273.[CrossRef][Medline]
Lamaze, C. and Schmid, S. L. (1995). The emergence of clathrin-independent pinocytic pathways. Curr. Opin. Cell Biol. 7,573 -580.[CrossRef][Medline]
Larsen, E. C., DiGennaro, J. A., Saito, N., Mehta, S.,
Loegering, D. J., Mazurkiewicz, J. E. and Lennartz, M. R.
(2000). Differential requirement for classic and novel PKC
isoforms in respiratory burst and phagocytosis in RAW 264.7 cells.
J. Immunol. 165,2809
-2817.
Lee, E. and Knecht, D. A. (2002). Visualization of actin dynamics during macropinocytosis and exocytosis. Traffic 3,186 -192.[CrossRef][Medline]
Lee, A., Frank, D. W., Marks, M. S. and Lemmon, M. A. (1999). Dominant-negative inhibition of receptor-mediated endocytosis by a dynamin-1 mutant with a defective pleckstrin homology domain. Curr. Biol. 9,261 -264.[CrossRef][Medline]
Li, S., Tan, Y., Viroonchatapan, E., Pitt, B. R. and Huang,
L. (2000). Targeted gene delivery to pulmonary endothelium by
anti-PECAM antibody. Am. J. Physiol. Lung Cell. Mol.
Physiol. 278,L504
-511.
Liu, N. Q., Lossinsky, A. S., Popik, W., Li, X., Gujuluva, C.,
Kriederman, B., Roberts, J., Pushkarsky, T., Bukrinsky, M., Witte, M. et al.
(2002). Human immunodeficiency virus type 1 enters brain
microvascular endothelia by macropinocytosis dependent on lipid rafts and the
mitogen-activated protein kinase signaling pathway. J.
Virol. 76,6689
-6700.
Mayor, S., Sabharanjak, S. and Maxfield, F. R.
(1998). Cholesterol-dependent retention of GPI-anchored proteins
in endosomes. EMBO J.
17,4626
-4638.
McIntosh, D. P., Tan, X. Y., Oh, P. and Schnitzer, J. E.
(2002). Targeting endothelium and its dynamic caveolae for
tissue-specific transcytosis in vivo: a pathway to overcome cell barriers to
drug and gene delivery. Proc. Natl. Acad. Sci. USA
99,1996
-2001.
Minshall, R. D., Tiruppathi, C., Vogel, S. M., Niles, W. D.,
Gilchrist, A., Hamm, H. E. and Malik, A. B. (2000).
Endothelial cell-surface gp60 activates vesicle formation and trafficking via
G(i)-coupled Src kinase signaling pathway. J. Cell
Biol. 150,1057
-1070.
Mukherjee, S., Ghosh, R. N. and Maxfield, F. R.
(1997). Endocytosis. Physiol. Rev.
77,759
-803.
Muzykantov, V. R., Atochina, E. N., Ischiropoulos, H., Danilov,
S. M. and Fisher, A. B. (1996). Immunotargeting of
antioxidant enzyme to the pulmonary endothelium. Proc. Natl. Acad.
Sci. USA 93,5213
-5218.
Muzykantov, V. R., Christofidou-Solomidou, M., Balyasnikova, I.,
Harshaw, D. W., Schultz, L., Fisher, A. B. and Albelda, S. M.
(1999). Streptavidin facilitates internalization and pulmonary
targeting of an anti-endothelial cell antibody (platelet-endothelial cell
adhesion molecule 1): a strategy for vascular immunotargeting of drugs.
Proc. Natl. Acad. Sci. USA
96,2379
-2384.
Nichols, B. J. and Lippincott-Schwartz, J. (2001). Endocytosis without clathrin coats. Trends Cell Biol. 11,406 -412.[CrossRef][Medline]
Olazabal, I. M., Caron, E., May, R. C., Schilling, K., Knecht, D. A. and Machesky, L. M. (2002). Rho-kinase and myosin-II control phagocytic cup formation during CR, but not FcgammaR, phagocytosis. Curr. Biol. 12,1413 -1418.[CrossRef][Medline]
Orth, J. D., Krueger, E. W., Cao, H. and McNiven, M. A.
(2002). The large GTPase dynamin regulates actin comet formation
and movement in living cells. Proc. Natl. Acad. Sci.
USA 99,167
-172.
Parton, R. G., Joggerst, B. and Simons, K. (1994). Regulated internalization of caveolae. J. Cell Biol. 127,1199 -1215.[Abstract]
Powell, K. A., Valova, V. A., Malladi, C. S., Jensen, O. N.,
Larsen, M. R. and Robinson, P. J. (2000). Phosphorylation of
dynamin I on Ser-795 by protein kinase C blocks its association with
phospholipids. J. Biol. Chem.
275,11610
-11617.
Predescu, D., Predescu, S., McQuistan, T. and Palade, G. E.
(1998). Transcytosis of alpha1-acidic glycoprotein in the
continuous microvascular endothelium. Proc. Natl. Acad. Sci.
USA 95,6175
-6180.
Puri, V., Watanabe, R., Singh, R. D., Dominguez, M., Brown, J.
C., Wheatley, C. L., Marks, D. L. and Pagano, R. E. (2001).
Clathrin-dependent and -independent internalization of plasma membrane
sphingolipids initiates two Golgi targeting pathways. J. Cell
Biol. 154,535
-547.
Putney, L. K., Denker, S. P. and Barber, D. L. (2002). The changing face of the Na+/H+ exchanger, NHE1: structure, regulation, and cellular actions. Annu. Rev. Pharmacol. Toxicol. 42,527 -552.[CrossRef][Medline]
Racoosin, E. L. and Swanson, J. A. (1989). Macrophage colony-stimulating factor (rM-CSF) stimulates pinocytosis in bone marrow-derived macrophages. J. Exp. Med. 170,1635 -1648.[Abstract]
Racoosin, E. L. and Swanson, J. A. (1992). M-CSF-induced macropinocytosis increases solute endocytosis but not receptor-mediated endocytosis in mouse macrophages. J. Cell Sci. 102,867 -880.[Abstract]
Sahai, E. and Marshall, C. J. (2002). ROCK and Dia have opposing effects on adherens junctions downstream of Rho. Nat. Cell Biol. 4,408 -415.[CrossRef][Medline]
Scherpereel, A., Rome, J. J., Wiewrodt, R., Watkins, S. C.,
Harshaw, D. W., Alder, S., Christofidou-Solomidou, M., Haut, E., Murciano, J.
C., Nakada, M. et al. (2002). Platelet-endothelial cell
adhesion molecule-1-directed immunotargeting to cardiopulmonary vasculature.
J. Pharmacol. Exp. Ther.
300,777
-786.
Scherpereel, A., Wiewrodt, R., Christofidou-Solomidou, M.,
Gervais, R., Murciano, J. C., Albelda, S. M. and Muzykantov, V. R.
(2001). Cell-selective intracellular delivery of a foreign enzyme
to endothelium in vivo using vascular immunotargeting. FASEB
J. 15,416
-426.
Schlegel, R., Dickson, R. B., Willingham, M. C. and Pastan, I. H. (1982). Amantadine and dansylcadaverine inhibit vesicular stomatitis virus uptake and receptor-mediated endocytosis of alpha 2-macroglobulin. Proc. Natl. Acad. Sci. USA 79,2291 -2295.[Abstract]
Schnitzer, J. E., Oh, P., Pinney, E. and Allard, J. (1994). Filipin-sensitive caveolae-mediated transport in endothelium: reduced transcytosis, scavenger endocytosis, and capillary permeability of select macromolecules. J. Cell Biol. 127,1217 -1232.[Abstract]
Schubert, W., Frank, P. G., Razani, B., Park, D. S., Chow, C. W.
and Lisanti, M. P. (2001). Caveolae-deficient endothelial
cells show defects in the uptake and transport of albumin in vivo.
J. Biol. Chem. 276,48619
-48622.
Shafren, D. R., Dorahy, D. J., Greive, S. J., Burns, G. F. and Barry, R. D. (1997a). Mouse cells expressing human intercellular adhesion molecule-1 are susceptible to infection by coxsackievirus A21. J. Virol. 71,785 -789.[Abstract]
Shafren, D. R., Dorahy, D. J., Ingham, R. A., Burns, G. F. and Barry, R. D. (1997b). Coxsackievirus A21 binds to decay-accelerating factor but requires intercellular adhesion molecule 1 for cell entry. J. Virol. 71,4736 -4743.[Abstract]
Skretting, G., Torgersen, M. L., van Deurs, B. and Sandvig,
K. (1999). Endocytic mechanisms responsible for uptake of
GPI-linked diphtheria toxin receptor. J. Cell Sci.
112,3899
-3909.
Spragg, D. D., Alford, D. R., Greferath, R., Larsen, C. E., Lee,
K. D., Gurtner, G. C., Cybulsky, M. I., Tosi, P. F., Nicolau, C. and Gimbrone,
M. A., Jr (1997). Immunotargeting of liposomes to activated
vascular endothelial cells: a strategy for site-selective delivery in the
cardiovascular system. Proc. Natl. Acad. Sci. USA
94,8795
-8800.
Swanson, J. A. (1989). Phorbol esters stimulate macropinocytosis and solute flow through macrophages. J. Cell Sci. 94,135 -142.[Abstract]
Swanson, J. A. and Watts, C. (1995). Macropinocytosis. Trends Cell Biol. 5, 424-428.[CrossRef]
Thompson, P. W., Randi, A. M. and Ridley, A. J.
(2002). Intercellular adhesion molecule (ICAM)-1, but not ICAM-2,
activates RhoA and stimulates c-fos and rhoA transcription in endothelial
cells. J. Immunol. 169,1007
-1013.
Torgersen, M. L., Skretting, G., van Deurs, B. and Sandvig, K. (2001). Internalization of cholera toxin by different endocytic mechanisms. J. Cell Sci. 114,3737 -3747.[Medline]
Treutiger, C. J., Heddini, A., Fernandez, V., Muller, W. A. and Wahlgren, M. (1997). PECAM-1/CD31, an endothelial receptor for binding Plasmodium falciparum-infected erythrocytes. Nat. Med. 3,1405 -1408.[Medline]
Volberg, T., Geiger, B., Citi, S. and Bershadsky, A. D. (1994). Effect of protein kinase inhibitor H-7 on the contractility, integrity, and membrane anchorage of the microfilament system. Cell Motil. Cytoskeleton 29,321 -338.[Medline]
Wang, Q. and Doerschuk, C. M. (2002). The signaling pathways induced by neutrophil-endothelial cell adhesion. Antioxid. Redox Signal 4, 39-47.[CrossRef][Medline]
Watanabe, T., Pakala, R., Katagiri, T. and Benedict, C. R.
(2001). Synergistic effect of urotensin II with mildly oxidized
LDL on DNA synthesis in vascular smooth muscle cells.
Circulation 104,16
-18.
West, M. A., Bretscher, M. S. and Watts, C. (1989). Distinct endocytotic pathways in epidermal growth factor-stimulated human carcinoma A431 cells. J. Cell Biol. 109,2731 -2739.[Abstract]
West, M. A., Prescott, A. R., Eskelinen, E. L., Ridley, A. J. and Watts, C. (2000). Rac is required for constitutive macropinocytosis by dendritic cells but does not control its downregulation. Curr. Biol. 10,839 -848.[CrossRef][Medline]
Wiewrodt, R., Thomas, A. P., Cipelletti, L.,
Christofidou-Solomidou, M., Weitz, D. A., Feinstein, S. I., Schaffer, D.,
Albelda, S. M., Koval, M. and Muzykantov, V. R. (2002).
Size-dependent intracellular immunotargeting of therapeutic cargoes into
endothelial cells. Blood
99,912
-922.
Witke, W., Podtelejnikov, A. V., Di Nardo, A., Sutherland, J.
D., Gurniak, C. B., Dotti, C. and Mann, M. (1998). In mouse
brain profilin I and profilin II associate with regulators of the endocytic
pathway and actin assembly. EMBO J.
17,967
-976.
Worthylake, R. A. and Burridge, K. (2001). Leukocyte transendothelial migration: orchestrating the underlying molecular machinery. Curr. Opin. Cell Biol. 13,569 -577.[CrossRef][Medline]
Related articles in JCS: