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*
Physiology Program, Harvard School of Public Health, Boston, MA 02115; and
Department of Pediatrics, Rainbow Babies and Children’s Hospital, Case Western Reserve University, Cleveland, OH 44106
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-treated pulmonary microvascular ECs induced cytoskeletal
remodeling in ECs that required ICAM-1 ligation and oxidant production
and was mimicked by cross-linking ICAM-1. In this study, we examined
the role of ICAM-1-induced signaling pathways in mediating actin
cytoskeletal remodeling. Cross-linking ICAM-1 induced alterations in
ICAM-1 distribution, as well as the filamentous actin rearrangements
and stiffening of ECs shown previously. ICAM-1 cross-linking induced
phosphorylation of the p38 mitogen-activated protein kinase (MAPK) that
was inhibited by allopurinol and also induced an increase in the
activity of the p38 MAPK that was inhibited by SB203580. However,
SB203580 had no effect on oxidant production in ECs or ICAM-1
clustering. ICAM-1 cross-linking also induced phosphorylation of heat
shock protein 27, an actin-binding protein that may be involved in
filamentous actin polymerization. The time course of heat shock protein
27 phosphorylation paralleled that of p38 MAPK phosphorylation and was
completely inhibited by SB203580. In addition, SB203580 blocked the EC
stiffening response induced by either neutrophil adherence or ICAM-1
cross-linking. Moreover, pretreatment of ECs with SB203580 reduced
neutrophil migration toward EC junctions. Taken together, these data
demonstrate that activation of p38 MAPK, mediated by xanthine
oxidase-generated oxidant production, is required for cytoskeletal
remodeling in ECs induced by ICAM-1 cross-linking or neutrophil
adherence. These cytoskeletal changes in ECs may in turn modulate
neutrophil migration toward EC junctions. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2 integrins with endothelial ICAM-1, is often
a prerequisite for neutrophil transmigration across endothelium in most
inflammatory responses (1, 2). Neutrophil adhesion to
endothelial cells
(ECs)4 results in
activation of ECs, as demonstrated by studies showing increased
intracellular Ca2+, phosphorylation of myosin
light chain kinase, and filamentous actin (F-actin) stress fiber
formation in ECs upon neutrophil adherence (3, 4, 5, 6).
Inhibition of these events prevents neutrophil transendothelial
migration, suggesting that EC activation upon neutrophil adherence may
be essential for subsequent neutrophil transmigration
(3, 4, 5, 6). Ligation of EC adhesion molecules may initiate downstream signaling events that result in EC activation during neutrophil adhesion. This hypothesis is supported by recent studies demonstrating that EC adhesion molecules, including ICAM-1, are capable of transducing signals (4, 7, 8, 9, 10, 11, 12). Cross-linking ICAM-1 in brain EC lines or venular ECs induces increases in intracellular Ca2+, and activation of p60src, Rho, and protein kinase C (8, 9, 10, 13, 14). These signaling pathways act upon several actin-associated proteins including cortactin, focal adhesion kinase, paxillin, and p130cas, which in turn induce changes in the actin cytoskeleton of ECs (8, 9). In addition, cross-linking ICAM-1 induces transcription of VCAM-1 and ICAM-1 through activation of transcription factors including AP-1 and extracellular signal-regualated kinase-1 (13). These studies demonstrate that ICAM-1-induced signaling events result in changes in ECs, including cytoskeletal rearrangement and gene transcription, that are likely to modulate leukocyte migration during inflammatory responses.
Activation of the mitogen-activated protein kinase (MAPK) pathways
plays important roles in regulating cell growth, differentiation, and
cell responses to environmental stress (15). One member of
the MAPK family, p38 MAPK, is activated by various cellular stresses
including heat, oxidative stress, and cytokines (16, 17, 18).
There are at least four members of the p38 MAPK identified: p38
(19, 20), p38 (21), p38
(22), and p38
(23, 24). Activated and
isoforms of p38 MAPK in turn phosphorylate two kinases,
MAPK-activated protein kinase (MAPKAP)-2 and -3 (18, 25).
One of the physiological substrates of these two kinases is the small
heat shock protein 27 (Hsp 27) (18, 25). Phosphorylation
of Hsp 27 appears to enhance F-actin polymerization and stabilize the
F-actin network in response to oxidative and other stresses
(26, 27, 28).
We have previously reported that neutrophil adherence to
TNF--treated human pulmonary microvascular ECs induces actin
cytoskeletal remodeling in ECs, as demonstrated by actin rearrangement
in ECs and an actin cytoskeleton-dependent increase in the apparent
stiffness of ECs, measured using magnetic twisting cytometry
(29). These changes in the actin cytoskeleton are
inhibited by an anti-ICAM-1 Ab and are mimicked by ICAM-1
cross-linking. In addition, ligation of ICAM-1 by neutrophils induces
oxidant production in ECs, which is required for the cytoskeletal
remodeling (30). Moreover, the cytoskeletal changes in ECs
require production of phosphatidylinositols, because pretreatment of
ECs with a phosphatidylinositol-binding peptide inhibits the
cytoskeletal changes (29). These studies suggest that
ligation of ICAM-1 induces cascades of signaling events in ECs, some of
which lead to the cytoskeletal changes. The studies presented here
examined the roles of reactive oxygen species and p38 MAPK in the
cytoskeletal remodeling.
We hypothesized that activation of p38 MAPK was required for the cytoskeletal remodeling in ECs induced by ICAM-1 ligation. The data demonstrate that activation of p38 MAPK, mediated by xanthine oxidase-generated oxidant production, is essential for the cytoskeletal remodeling in ECs induced by ICAM-1 cross-linking or neutrophil adherence. These cytoskeletal changes in ECs may in turn modulate neutrophil migration toward EC junctions.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Allopurinol was obtained from Sigma (St. Louis, MO), and
2',7'-dichlorofluorescein diacetate was obtained from Molecular Probes
(Eugene, OR). Recombinant human TNF- was obtained from R&D Systems
(Minneapolis, MN), murine anti-human ICAM-1 Ab (clone 6.5B5)
from Dako (Carpinteria, CA), goat F(ab')2 of IgG
raised against the murine IgG Fc portion from Organon Teknika (Durham,
NC), and murine anti-human 1-integrin Ab
(clone P5D2) from Chemicon (Temecula, CA), murine IgG (clone MOPC-21)
was obtained from BD PharMingen (San Diego, CA),
H3[32P]O4
from NEN (Boston, MA), murine anti-human Hsp 27 Ab (clone G3.1)
from StressGen Biotechnologies (Victoria, BC, Canada), and rabbit
anti-human p38 MAPK and rabbit anti-phosphorylated p38 MAPK Ab
from New England Biolabs (Beverly, MA). Finally, HRP-conjugated
anti-rabbit and anti-mouse Abs were obtained from Santa Cruz
Biotechnology (Santa Cruz, CA).
Isolation of human neutrophils
Blood was drawn from healthy humans by venipuncture after informed consent was obtained. Human neutrophils were isolated using Histopaque density gradients (Sigma) according to the manufacturer’s protocol. The purity of isolated neutrophils was >95%.
Cultivation of human pulmonary microvascular ECs
Human pulmonary microvascular ECs were obtained from Clonetics
(Walkersville, MD), and plated onto fibronectin-coated culture dishes
according to the manufacturer’s protocol. ECs were used between
passage 6 and 10. All experiments were performed using cells 3–6 days
after they reached confluence. These cells can be induced to
up-regulate ICAM-1 expression upon TNF- stimulation (30, 31).
Experimental protocol
ECs treated with buffer or TNF- for 24 h were studied.
Cells were cultured in 96-well plates with 
40,000 cells/well. For
each of the experiments, an average response from the cells in a single
well was recorded, and the n values in this study refer to
the number of wells studied on separate days. After measuring the
baseline biomechanical properties or dichlorofluorescein (DCF)
fluorescence of ECs, neutrophils (neutrophil:EC = 1:1) or buffer
were added to ECs, and these parameters were measured after 2, 6, 10,
and 15 min of neutrophil adhesion. In many experiments, ECs were
pretreated with a control vehicle or SB203580, a p38 MAPK inhibitor,
and washed two times before the addition of neutrophils. SB203580
belongs to a class of pyridinal imidazoles. It binds to the ATP-binding
pocket of the p38 MAPK and, therefore, inhibits its activity
(32). It specifically inhibits the activity of the and
the isoforms of p38 MAPK (reviewed in Ref. 33).
Biomechanical properties of ECs
The biomechanical properties of ECs were measured using magnetic
twisting cytometry (34, 35). This technique measures the
angular rotation of ferromagnetic beads bound to cells through specific
ligands upon application of a magnetic torque (stress). The degree of
angular rotation is inversely proportional to the stiffness of the
cells to which the ferromagnetic beads are bound. Ferromagnetic beads
coated with goat anti-mouse IgG Fc portions were obtained from
Spherotech (Libertyville, IL). These beads were incubated with a murine
Ab against human 1 integrin at a concentration
of 1 µg/106 beads for 30 min at 4°C, followed
by three washes in PBS. ECs treated with 20 ng/ml TNF- or buffer for
24 h at 37°C were washed twice with DMEM containing 5% FBS and
then incubated with anti-1 integrin
Ab-coated beads at 37°C for 30 min. The unbound beads were gently
washed off, and the well was placed in the magnetic twisting
cytometer. As previously described (29, 30, 34, 35), the bound beads were exposed to a brief (10 µs) but
strong (1000 gauss) magnetic field, which magnetizes the beads in the
horizontal direction. After 20 s, the beads were twisted by a much
weaker (30 gauss) but continuous (1 min) vertical magnetic field. This
twisting field was not strong enough to remagnetize the beads, but it
caused the beads to rotate. The magnitude of magnetic vector in the
horizontal direction (remnant magnetic field) was measured by an
in-line magnetometer. From this value, the average bead rotation
(angular strain) was calculated (34, 35). The rotational
stress was calculated by rotating the beads in a viscous standard. For
these beads, a twisting field of 10 gauss corresponded to an applied
torque at the start of the twist (initial stress) of 7
dyne/cm2. The specific torque (stress) on the
beads at the end of the 1 min twist (stress1 min)
was calculated using the initial stress times and the ratio of the
remnant field at the end of the 1-min twist and the remnant field at
time 0. The apparent stiffness was measured at 1 min of twist and was
defined as the ratio of stress1 min to the
angular strain at this time point.
ICAM-1 cross-linking
ICAM-1 was cross-linked as previously described
(13). Briefly, ECs treated with TNF- or buffer for
24 h were washed twice with culture medium and incubated with 15
µg/ml murine anti-human ICAM-1 Ab (clone 6.5B5) or murine IgG for
30 min. The cells were washed twice, and the baseline apparent
stiffness of ECs was measured. Goat F(ab')2 of
IgG raised against the murine IgG Fc portion was added at 50 µg/ml.
The apparent stiffness of ECs was measured 2–15 min later, or in some
experiments, the cell lysates were collected for immunoblot
analysis.
Visualization of ICAM-1 and F-actin distribution
ICAM-1 distribution before or after cross-linking with a
secondary Ab was visualized as described (11). ECs treated
with TNF- for 24 h were incubated with anti-ICAM-1 Ab or
mouse IgG for the negative controls for 30 min and washed. The cells
were either 1) incubated with fluorescein-conjugated goat
anti-mouse IgG (50 µg/ml) for 6–15 min and then fixed with 3.7%
paraformaldehyde or 2) fixed immediately, followed by incubation with
fluorescein-conjugated goat anti-mouse IgG (50 µg/ml) for 15 min.
These cells were then incubated with PBS containing
L-lysophosphatidyl choline (200 µg/ml) and
rhodamine-labeled phalloidin (3.3 x 10-7
M) for 1 h for F-actin visualization. The coverslips were mounted
on slides and examined using a Zeiss LSM 410 laser scanning confocal
microscope (Zeiss, Oberkochen, Germany).
To ensure that the clusters were localized on EC surface but not internalized, the cells were incubated with trypan blue to quench extracellular fluorescence, because viable cells exclude this dye. After cross-linking ICAM-1 with the fluorescein-conjugated secondary Ab for 6–15 min, the cells were washed, and the fluorescence was quantified using a plate reader. The cells were then incubated with 0.025% trypan blue for 10 min and washed thoroughly. The remaining fluorescence was determined. Because light microscopy demonstrated that trypan blue did not enter ECs, any decrease in fluorescence reflected quenching of extracellular staining.
Measurement of oxidant production in ECs
Oxidant production in ECs was measured as previously described
(30). ECs treated with 20 ng/ml TNF- or buffer for
24 h were incubated with 20 µM 2',7'-dichlorofluorescein
diacetate for 45 min at 37°C and washed twice with HBSS containing
1.2 mM Ca2+, 0.4 mM Mg2+,
and 5.5 mM glucose. After measuring the baseline DCF fluorescence,
neutrophils or buffer was added, and the DCF fluorescence was measured
2–20 min later.
Localization of neutrophils on EC monolayer
To examine the position of neutrophils adherent to 24-h
TNF--activated ECs with respect to the EC borders, the cells were
stained using silver nitrate as previously described to identify the
borders (36, 37). Briefly, ECs were treated with 20 ng/ml
TNF- for 24 h, followed by two washes using DMEM containing 5%
FBS. Neutrophils were then added to the well and allowed to adhere for
1–15 min. After two rapid washes, the cells were fixed with 0.5%
glutaraldehyde for 10 min at room temperature, washed twice with PBS,
and stained with 0.25% AgNO3 for 30s. After two
washes in PBS, the stain was developed under UV light for 5 min. The
cells were examined under a light microscope, and the percentage of
total adherent neutrophils that were present at EC borders was then
determined at each time point.
Detection of p38 MAPK and Hsp 27 by immunoblot
Total p38 MAPK, the phosphorylated p38 MAPK, and Hsp27 were
detected by immunoblot. Cultured ECs were treated with 20 ng/ml TNF-
for 24 h. ICAM-1 cross-linking was performed as described above,
and the cells were washed twice with ice-cold PBS. The cells were then
scraped off culture dishes in lysis buffer containing 1% SDS, 1%
Triton X-100, 1 mM EDTA, 1 mM EGTA, 0.2 mM PMSF, 5 µg/ml leupeptin,
and 1 µg/ml pepstatin in 10 mM Tris (pH 8.0). The cell lysate was
collected, and the protein content was quantified using the QuantiPro
BCA assay (Sigma). An equal amount of proteins (20–30 µg) was loaded
onto each lane, and proteins were separated by SDS-PAGE using 4–12%
gradient gels according to the manufacturer’s protocol (NOVEX, San
Diego, CA). After eletrophoresis, the proteins were electroblotted onto
polyvinylidene difluoride membranes using the NOVEX system. The
membranes were incubated in blocking buffer containing 0.1% Tween 20
and 5% nonfat dry milk in TBS, followed by incubation with a primary
Ab against either p38 MAPK, phosphorylated p38 MAPK, or Hsp 27. The
membranes were then incubated with the respective secondary Ab
conjugated with HRP, and the protein of interest was visualized by
enhanced chemiluminescence (Amersham, Piscataway, NJ). The image of the
chemiluminograph was scanned using a desk scanner (Scanjet 4;
Hewlett-Packard, Palo Alto, CA), and the integrated density was
quantified by densitometry using Scion Image PC software (Frederick,
MD).
Measurement of p38 MAPK activity
The activity of p38 MAPK was evaluated using a p38 MAPK assay
kit according to the manufacturer’s protocol (New England Biolabs).
ECs were stimulated with 20 ng/ml TNF- for 24 h. ICAM-1
cross-linking was performed as described above, and the cells were
washed twice with ice-cold PBS. Cell lysis was performed according to
the manufacturer’s protocol, and the cell lysate containing 250 µg
protein/sample was preabsorbed with 30 µl protein A/G plus agarose
beads (Santa Cruz Biotechnology). Phosphorylated p38 MAPK was
immunoprecipitated, and the activity of p38 MAPK was evaluated using
exogenous activating transcription factor (ATF)-2 as a substrate
according to the manufacturer’s protocol (New England Biolabs).
Phosphorylation of ATF-2 was evaluated using an Ab that recognizes
phosphorylated ATF-2 at residue Thr71 by immunoblotting as
described above.
Detection of Hsp 27 phosphorylation
Hsp 27 phosphorylation was detected as previously described
(28). ECs cultured in phosphate-free medium were treated
with 20 ng/ml TNF- for 24 h. During the last 3 h of
treatment, the cells were incubated with 20 µCi/ml
H3[32P]O4
(NEN; 900 Ci/mmol). ICAM-1 was cross-linked as described above, and the
cells were washed twice with ice-cold PBS. The cells were lysed as
described above for detection of p38 MAPK and Hsp 27. The proteins were
separated by SDS-PAGE and transferred onto polyvinylidene difluride
membrane. After obtaining the autoradiograph of the membrane, the
membrane was subjected to immunoblot for Hsp 27 detection as described
above. The integrated density of the phosphorylated Hsp 27 was
quantified by densitometry and normalized by the integrated density of
the Hsp 27 band on the immunoblot.
Statistical analysis
Data were analyzed using the Student t test or one-way ANOVA. Values of p < 0.05 were considered significant. The data were expressed as the mean value ± SEM.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-treated ECs and F-actin
distribution
The biomechanical properties of ECs were evaluated by magnetic
twisting cytometry using anti-1 integrin
Ab-coated beads. ICAM-1 cross-linking resulted in changes in the
biomechanical properties of TNF--treated ECs. As shown in Fig. 1
, incubation of 24-h TNF--treated ECs
with anti-ICAM-1 Ab alone had no effect on the apparent stiffness
of ECs. However, cross-linking ICAM-1 with the secondary Ab increased
EC stiffness within 2 min. This increase persisted for at least 15 min.
This increase in EC stiffness upon ICAM-1 cross-linking has also been
shown in our previous studies, and this stiffening response mimicked
that induced by neutrophil adherence (30).
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). Clusters and thick
bundles of F-actin were observed after cross-linking ICAM-1 for 6 and
15 min when compared with ECs fixed before adding the cross-linking Ab
(Fig. 2a, c, and e). Cross-linking
ICAM-1 with a secondary Ab also resulted in ICAM-1 redistribution on EC
surface (Fig. 2, b, d, and f). In ECs
that were incubated with the anti-ICAM-1 Ab and then fixed, ICAM-1
was localized in small punctuate dots on the EC surface (Fig. 2b). Cross-linking ICAM-1 with a fluorescein-conjugated
secondary Ab for 6 and 15 min resulted in a redistribution of ICAM-1 to
form both small clusters and large aggregates that were no longer
punctuate (Fig. 2, d and f). These large
aggregates did not colocalize with F-actin clusters. No ICAM-1 staining
was observed in the controls in which the cells were incubated with the
secondary Ab only, without the anti-ICAM-1 Ab (data not shown).
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ICAM-1 cross-linking induced p38 MAPK activation that was oxidant-dependent
The p38 MAPK is activated by phosphorylation at residues Thr 180
and Tyr 182. The formation of activated p38 MAPK was examined using an
Ab that recognizes only p38 MAPK that is phosphorylated at these two
residues. As shown in Fig. 3, A and B, ICAM-1 cross-linking for 2 and 6 min
induced phosphorylation of p38 MAPK. Pretreatment of ECs for 30 min
with 0.3 mg/ml allopurinol, a xanthine oxidase inhibitor, prevented p38
MAPK phosphorylation upon ICAM-1 cross-linking (Fig. 3C).
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D). ICAM-1 cross-linking for 6 min
induced a 2.3 ± 0.2-fold increase in the monophosphorylated form
of ATF-2 in cells treated with control vehicles (Fig. 3D;
p < 0.05). Treatment with 25 µM SB203580 completely
inhibited this increase but had no effect on the baseline activity of
p38 MAPK (Fig. 3D). These data demonstrate that ICAM-1
cross-linking results in activation of p38 MAPK that is inhibited by
SB203580.
However, inhibition of p38 MAPK activation by SB203580 had no effect on
oxidant production in ECs upon neutrophil adherence. As shown in Fig. 4, neutrophil adherence to
TNF--treated ECs resulted in an increase in oxidant production, as
detected by an increase in DCF fluorescence. Pretreatment of ECs with
25 µM SB203580 for 30 min had no effect on this oxidant production.
Taken together, these data suggest that ICAM-1 cross-linking induced
p38 MAPK activation, that p38 MAPK activation occurred downstream of
oxidant production, and that p38 MAPK activation required xanthine
oxidase-generated oxidant production.
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To examine the roles of p38 MAPK in mediating the EC-stiffening
response induced by ICAM-1 cross-linking or neutrophil adherence, ECs
were pretreated with SB203580, a p38 MAPK inhibitor. As shown in Fig. 5, pretreatment of ECs with this p38 MAPK
inhibitor prevented the EC-stiffening response induced by either ICAM-1
cross-linking or neutrophil adherence, suggesting that activation of
P38 MAPK is essential for the EC-stiffening response upon ICAM-1
ligation. However, SB203580 had no effect on ICAM-1 redistribution
(data not shown), suggesting that activation of p38 MAPK was not
required for ICAM-1 redistribution induced by ICAM-1 cross-linking.
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To determine whether Hsp 27 is a downstream effector of p38 MAPK
activation upon ICAM-1 cross-linking, the effect of ICAM-1
cross-linking on Hsp 27 phosphorylation was measured. ICAM-1
cross-linking resulted in an increase in Hsp 27 phosphorylation that
persisted for at least 10 min (Fig. 6, A and B). To determine whether activation of p38
MAPK is essential for Hsp 27 phosphorylation, ECs were pretreated with
SB203580. Pretreatment with SB203580 completely inhibited Hsp 27
phosphorylation, suggesting that p38 MAPK activation is required
for Hsp 27 phosphorylation (Fig. 6, A and B).
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To determine the physiological roles of p38 MAPK activation in ECs
in mediating neutrophil migration on EC monolayer, the kinetics of
neutrophil migration toward EC borders was determined. There was a
time-dependent increase in the percentage of neutrophils found at the
EC borders between 1 and 5 min of neutrophil adherence (Fig. 7). After 5 min, this percentage remained
constant at 60%. Pretreatment of ECs with SB203580 attenuated this
response, suggesting that activation of p38 MAPK in ECs upon neutrophil
adherence played important roles in mediating neutrophil migration
toward EC junctions.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. The study
presented here addressed the signaling pathways though which these
changes in the actin cytoskeleton occurred. The data demonstrate that
ICAM-1 cross-linking induced a sequence of events including ICAM-1
redistribution, oxidant production, activation of p38 MAPK, and
phosphorylation of Hsp 27, and that activation of p38 MAPK is required
for actin cytoskeletal rearrangements to occur upon ICAM-1
cross-linking or neutrophil adherence. These ICAM-1-initiated events
are likely to play important roles in mediating neutrophil migration
toward EC borders. This study provides further evidence that ICAM-1 is capable of inducing signaling events in pulmonary microvascular ECs. Cross-linking ICAM-1 on these ECs induced phosphorylation of p38 MAPK, as well as an increase in the activity of p38 MAPK that was inhibited by SB203580. Interestingly, SB203580 inhibited the induced p38 MAPK activity but not the basal activity, suggesting that different isoforms of p38 MAPK may contribute to the induced and basal activity in these cells. Activation of p38 MAPK required cross-linking ICAM-1 via a secondary Ab, suggesting that the initiation of the signaling events may require ICAM-1 clustering. The results presented in this study demonstrate that cross-linking ICAM-1 indeed induced ICAM-1 redistribution, resulting in formation of small clusters and large aggregates of ICAM-1. ICAM-1 redistribution (clustering) induced by monocyte adhesion or cross-linking Abs has also been demonstrated on activated HUVECs (11). Interestingly, the patterns of ICAM-1 redistribution induced by the same cross-linking Abs observed on pulmonary microvascular ECs in this study appeared different from those reported on HUVECs, because ICAM-1 clusters were less discrete and large aggregates formed on pulmonary microvascular ECs (11). The mechanisms and the physiological functions of this difference between the two cell types remain to be determined. The formation of large ICAM-1 aggregates following ICAM-1 cross-linking suggests that the membrane cytoskeleton may play important roles in mediating ICAM-1 redistribution, although the molecular components of these aggregates have not been determined.
It is not apparent how ICAM-1 ligation initiates downstream signaling
events. The cytoplasmic tail of ICAM-1 does not have intrinsic kinase
activity or the Src homology domains that can recruit phosphorylated
proteins. Intercellular signaling induced by ICAM-1 may require
actin-associated proteins. The intracellular domain of ICAM-1 is linked
to actin-binding protein -actinin (38, 39), and ICAM-1
clusters induced by cross-linking colocalize with members of the ezrin,
radixin, and moesin family (11). Ezrin, radixin, and
moesin proteins have been implicated in activation of various signal
transduction pathways such as the phosphatidylinositol 3-kinase pathway
(40) and the Rho GTPase (41). In addition,
fibrinogen binding to ICAM-1 induces tyrosine phosphorylation of ICAM-1
itself, and association of ICAM-1 with Src homology domain 2-containing
protein tyrosine phosphatase-2 in human venular ECs
(42). Thus, ICAM-1 cross-linking-induced signaling events
may be initiated at the membrane-cytoskeletal interface and propagate
through cascades of signaling pathways leading to activation of
p38 MAPK.
Previous studies demonstrated that oxidant production in ECs induced by neutrophil adhesion was essential for the EC-stiffening response (30). This oxidant production was inhibited by an anti-ICAM-1 Ab, and the EC-stiffening response induced by ICAM-1 cross-linking was inhibited by allopurinol, suggesting that oxidant production may be involved in the signaling events induced by ICAM-1 ligation. The present study demonstrates that activation of p38 MAPK induced by ICAM-1 cross-linking was inhibited by allopurinol, whereas inhibition of p38 MAPK by SB203580 had no effect on oxidant production in ECs induced by neutrophil adherence. These data suggest that activation of p38 MAPK occurs downstream of intracellular oxidant generation. Intracellular oxidant production has been implicated in many signal transduction events induced by peptide growth factors or cytokines (43, 44, 45, 46, 47). Application of exogenous hydrogen peroxide induces activation of p38 MAPK in HUVECs (27, 28, 48). In addition, intracellular hydrogen peroxide production is essential for p38 MAPK activation upon angiotensin II stimulation in cultured smooth muscle cells (46).
Inhibition of p38 MAPK prevented the EC-stiffening response induced by ICAM-1 cross-linking or neutrophil adherence. It is unclear how activation of p38 MAPK induced the EC-stiffening response, although it is tempting to hypothesize that Hsp 27 may be involved in modulating the actin cytoskeleton rearrangement upon p38 MAPK activation. Activated p38 MAPK phosphorylates two kinases, MAPKAP-2 and -3 (18, 25). One of the physiological substrates of these two kinases is Hsp 27 (18, 25). In this study, ICAM-1 cross-linking also induced phosphorylation of Hsp 27, which was inhibited by a p38 MAPK inhibitor, SB203580. Phosphorylation of Hsp 27 in HUVECs upon p38 MAPK activation has been postulated to modulate F-actin rearrangement during oxidative stress induced by exogenous hydrogen peroxide (28). Hsp 27 is an actin-binding protein that is widely expressed in many cell types, and the level of its expression is particularly high in ECs (6 ng/µg total protein in cultured ECs) (28). Purified Hsp 27 inhibits actin polymerization (49), whereas phosphorylated Hsp 27 does not have this inhibitory effect (50). In addition, cell lines overexpressing wild-type Hsp 27 have more cortical F-actin compared with the controls and show a 2-fold increase in actin polymerization activity upon stimulation with thrombin or fibroblast growth factor (51). In contrast, cells overexpressing a phosphorylation-mutant form of Hsp 27 have the opposite phenotype (51). Moreover, the actin cytoskeleton of the cells overexpressing the wild-type, but not the phosphorylation-mutant, form of Hsp 27 are more resistant to disruption by cytochalasin D (26, 52). These studies suggest that Hsp 27 may be involved in modulating the F-actin cytoskeleton in a phosphorylation-dependent manner, although its role has not yet been tested directly.
Despite its complete inhibition of the EC-stiffening response induced by ICAM-1 cross-linking, SB203580 had no effect on ICAM-1 clustering. These data suggest that activation of p38 MAPK is not required for ICAM-1 clustering. A recent study by Wojciak-Stothard et al. demonstrated that activation of Rho, but not stress fiber formation, was required for ICAM-1 clustering induced by monocyte adhesion or ICAM-1 cross-linking (11). These data suggest that activation of p38 MAPK and subsequent cytoskeletal remodeling may occur downstream of ICAM-1 clustering.
Inhibition of p38 MAPK in ECs reduced neutrophil migration toward EC borders. These data suggest that signaling induced by ICAM-1 in ECs upon neutrophil adherence may be required for neutrophil migration on EC surface, and activation of p38 MAPK may be an essential regulatory element. It is unclear how activation of p38 MAPK in ECs may modulate neutrophil migration on EC surfaces. Activation of p38 MAPK and modulation of its downstream events induces changes in ECs, including actin cytoskeletal rearrangement. These changes may in turn modulate EC shape, surface characteristics, junctional functions, and/or signaling pathways in ECs that are essential for neutrophil emigration toward the EC junctions in inflammation.
In summary, this study demonstrates that ICAM-1 cross-linking induced p38 MAPK activation, which was required for cytoskeletal remodeling upon ICAM-1 ligation or neutrophil adherence. Phosphorylation of p38 required xanthine oxidase-derived reactive oxygen species. Hsp 27 was a downstream target of p38 MAPK pathway and may mediate actin cytoskeletal rearrangements. Activation of p38 MAPK in ECs upon neutrophil adherence may be essential for regulating neutrophil migration toward EC junctions, possibly through cytoskeleton-dependent mechanisms.
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Acknowledgments |
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Footnotes |
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2 Current address: Division of Integrative Biology, Department of Pediatrics, Case Western Reserve University, 11100 Euclid Avenue, Cleveland, OH 44106.
3 Address correspondence and reprint requests to Dr. Claire M. Doerschuk, Division of Integrative Biology, Department of Pediatrics, Case Western Reserve University, Room 8321, 11100 Euclid Avenue, Cleveland, OH 44106. E-mail address: cmd22{at}po.cwru.edu
4 Abbreviations used in this paper: EC, endothelial cell; F-actin, filamentous actin; MAPK, mitogen-activated protein kinase; MAPKAP, mitogen-activated protein kinase-activated protein kinase; Hsp 27, heat shock protein 27; DCF, dichlorofluorescein; ATF, activating transcription factor.
Received for publication December 12, 2000. Accepted for publication March 23, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) along with
anti-
4). 
, Anti-ICAM-1 Ab-pretreated ECs; 
,
mouse-IgG-pretreated ECs. *, p < 0.05 compared
with the baseline values. C, A representative immunoblot
showing the effect of allopurinol on p38 MAPK phosphorylation.
D, A representative immunoblot and densitometric
analysis showing the increases in p38 MAPK activity induced by ICAM-1
cross-linking and the effect of SB203580. Data were normalized by the
baseline values and expressed as means ± SEM
(n = 3 or 4).
