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B-dependent leukocyte recruitment
Immunology Research Group, Departments of Physiology and Biophysics, University of Calgary, Calgary, Alberta, Canada T2N 4N1
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Thrombin-stimulated endothelium
synthesizes numerous adhesion molecules to recruit leukocytes; however,
it is unknown which intracellular pathways are responsible for this
event. A recent report from our laboratory has shown that thrombin
induces E-selectin expression and that blocking nuclear factor-
B
(NF-B) activity partially blocked both E-selectin expression (60%)
and leukocyte recruitment. In this study, we systematically assessed
the importance of p38 MAPK in thrombin-induced NF-B activation and
E-selectin-dependent leukocyte recruitment. Thrombin caused
phosphorylation of p38 MAPK, its substrate ATF-2, and JNK MAPK, but not
ERK MAPK. The p38 MAPK inhibitors, SKF86002 and SB-203580 only reduced
ATF-2 activity. We treated human umbilical vein endothelial cells with SKF86002, 1 h before thrombin stimulation, and noted inhibition of
NF-B mobilization and complete inhibition of leukocyte rolling and
adhesion in a laminar flow chamber. Significant inhibition of leukocyte
recruitment and E-selectin expression was also observed with SB-203580.
SKF86002 did not affect other systems, including tumor necrosis
factor-
-induced E-selectin-dependent leukocyte recruitment.
Moreover, thrombin-induced rapid mobilization of P-selectin from Weibel
Palade bodies was not p38 MAPK dependent. These data suggest that
thrombin induces p38 MAPK activation, which leads to NF-B
mobilization to the nucleus and causes the upregulation of E-selectin
and subsequent leukocyte recruitment.
endothelium; selectins; inflammation
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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LEUKOCYTE
RECRUITMENT from the bloodstream into inflammatory tissue sites
involves leukocyte tethering and rolling, followed by firm adhesion to
the endothelium and subsequent transmigration into the tissue. Briefly,
leukocyte tethering and rolling are mediated by interactions with
endothelial selectins (E- and P-selectin). Much work (2,
3) has been done to demonstrate that molecules like tumor
necrosis factor- (TNF-), interleukin-1, and lipopolysaccharides can upregulate E-selectin. E-selectin is synthesized de novo and expressed optimally on the endothelial cell surface 4-8 h after cytokine stimulation (2). More recently, we
(23) and others (13) have identified that
thrombin can also stimulate E-selectin expression leading to
significant leukocyte rolling and adhesion.
Thrombin, a multifunctional serine protease, is generated through the activation of the coagulation cascade, but its effects also clearly contribute to inflammation. Indeed, studies (7, 31) are emerging to suggest that the inhibition of thrombin reduces the inflammatory response. One proposed mechanism is related to the ability of thrombin to recruit leukocytes. For example, neutrophil recruitment is blocked by antithrombin III (ATIII), an endogenous inhibitor of thrombin, following ischemia-reperfusion (24). In addition, there is growing evidence that thrombin may contribute to atherosclerosis, rheumatoid arthritis, and other diseases wherein mononuclear leukocyte recruitment is a prominent feature (11, 21, 31). Therefore, understanding the mechanisms by which thrombin induces leukocyte recruitment may be very important to defining novel anti-inflammatory therapeutics.
Our understanding of the intracellular signaling pathways that link
thrombin and E-selectin-dependent leukocyte recruitment is incomplete.
Essentially all studies that have examined E-selectin synthesis have
used TNF- as a stimulus. TNF- stimulation has been shown to
activate two separate pathways to induce E-selectin expression. One
pathway involves activation of MKKK, leading to activation of MKK 4/7
and MKK 3/6 and subsequent activation of JNK and p38 MAPK, respectively
(6, 10). The activation of JNK and p38 MAPK leads to the
activation of the transcription factor AP-1 (26). TNF-
has also been shown to activate NF-B to induce transcription of
E-selectin (5). A recent report (14) from our
laboratory has shown that like TNF-, thrombin also activates NF-B
leading to E-selectin expression. However, blocking NF-B
mobilization to the nucleus with proteasome inhibitor MG-132 only
partially blocked E-selectin expression (60%) and leukocyte
recruitment, suggesting an NF-B-independent pathway (14). To our knowledge a role for p38 MAPK in
thrombin-induced E-selectin expression and leukocyte recruitment has
not been tested. The objective of our study was to systematically
assess the functional importance of p38 MAPK as a mediator of
E-selectin-dependent leukocyte recruitment. In addition, we compare the
results to E-selectin-dependent leukocyte recruitment induced by
TNF- and rapid nonnuclear P-selectin-dependent leukocyte recruitment
induced by thrombin.
Our data revealed an increase in both phosphorylation of p38 MAPK and
p38 MAPK activity (phosphorylated ATF-2) in response to thrombin.
Moreover, NF-B mobilization in response to thrombin can be blocked
with p38 MAPK inhibitors, suggesting that p38 is upstream of NF-B in
this particular intracellular pathway. Unexpectedly, there was complete
inhibition of thrombin-induced leukocyte recruitment in response to two
structurally different p38 MAPK inhibitors. This was specific for
thrombin because neither p38 inhibitor had any effect on
TNF--induced leukocyte recruitment.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Endothelium isolation. Human umbilical vein endothelial cells (HUVEC) were harvested and cultured from fresh human cords as previously described (23, 24). Briefly, fresh cords were perfused with sterile PBS. The cords were then filled with collagenase (320 U/ml in PBS, Worthington Biochemical) and incubated for 20 min in warm PBS. After incubation, the cords were gently massaged to facilitate the release of endothelial cells from the vessel walls. The digest from the cords was drained into centrifuge tubes containing heat-inactivated FBS and centrifuged for 8-10 min at 1,100 revolutions/min. The cell pellet was resuspended in medium 199 (M199; GIBCO-BRL), supplemented with 20% FBS, antibiotic cocktail, and glutamine. The cell suspension was then seeded in fibronectin-coated T25 flasks. Once the cells were confluent (3-5 days), trypsin-EDTA (GIBCO-BRL) was used to rapidly detach the endothelial cells, which were then plated onto fibronectin-coated glass coverslips. All endothelium was from first-passage HUVEC.
P38 MAPK assay and immunoblots. Cell lysates were prepared using the p38 MAPK assay kit with slight modifications (New England Biolabs; Mississauga, Ontario, Canada). Briefly, monolayers were washed once with PBS and incubated on ice for 5 min with 1× lysis buffer containing 1 mM phenylmethylsulfonyl fluoride (PMSF). Cells were scraped off the dish, transferred to microcentrifuge tubes, sonicated for 5 s, and kept on ice. Samples were microcentrifuged at 14,000 revolutions/min for 10 min at 4°C and the supernatants (cell lysate) were transferred to new tubes. Fifty microliters of the cell lysate was used for Western blotting to detect phospho-p38 MAPK, and 200 µl of the cell lysate was used in the immunoprecipitation to assess p38 MAPK activity.
Immunoprecipitation and kinase assay. Twenty microliters of immobilized phospho-p38 MAPK (Thr180/Tyr182) monoclonal antibody was added to 200 µl of cell lysate and incubated with gentle rocking overnight at 4°C. Samples were microcentrifuged at 14,000 revolutions/min for 30 s at 4°C. The pellet was washed twice with 1× lysis buffer and 1× kinase buffer and kept on ice. For the kinase assay, the pellet was suspended in 50 µl of 1× kinase buffer supplemented with 200 µM ATP and 2 µg ATF-2 fusion protein and incubated 30 min at 37°C. The reaction was terminated with 25 µl 2× SDS sample buffer [100 µl 20% SDS, 50 µl dithiothreitol (DTT), and 500 µl 2× sample buffer]. Samples were boiled for 5 min and resolved on 10% SDS-PAGE gel. Gels were transferred onto polyvinylidene difluoride membrane using a semidry apparatus. Membranes were blocked in 2% skim milk/Tween 20 Tris-based sodium (TTBS), washed once with TTBS, and incubated with phospho-ATF-2 or phospho-p38 antibody (1:1,000) in TTBS with gentle agitation overnight at 4°C. Membranes were washed with TTBS and incubated with horseradish peroxidase-conjugated anti-rabbit secondary antibody (1:2,000) and horseradish peroxidase-conjugated anti-biotin antibody (1:1,000) to detect biotinylated protein markers in 2% skim milk/TTBS at room temperature. After a secondary antibody was given, the membranes were washed with TTBS and proteins were detected using 1× LumiGLO (0.5 ml 20× LumiGLO, 0.5 ml 20× peroxide, and 9.0 ml water) with gentle agitation for 1 min at room temperature and exposed to X-ray film.
ELISA for cell surface adhesion molecule expression. Briefly, confluent HUVEC were seeded onto fibronectin-coated 48-well ELISA plates, treated with various stimuli, fixed with 1% formalin, and blocked with 1% BSA. The endothelial cells were labeled with 5 µg/ml of ES1 (E-selectin antibody). They were then washed and labeled with a peroxidase-labeled goat anti-mouse IgG (1 µg/ml, DAKO). After a final wash, a substrate system (TMB-One Step, DAKO) was added for color development and the color reaction was stopped with 0.18 M H2SO4. The plates were read at 450 nm.
Electrophoretic mobility shift assay.
After treatment with HUVEC, the cells were lysed and nuclear extracts
were prepared as described elsewhere with modifications (19,
25). Briefly, monolayers were incubated on ice for 15 min in
buffer (10 mM HEPES, 0.1 mM EDTA, 10 mM KCl, 1 M DTT, 0.33 M PMSF, 5 mg/ml of leupeptin, and 2.1 mg/ml of aprotinin, 0.6% Nonidet P-40).
The monolayers were harvested by scraping and the crude nuclei obtained
from lysis were collected by centrifugation (45 s at 14,000 revolutions/min) and resuspended in buffer C (20 mM HEPES, 5 mM EDTA, 0.42 M NaCl, 0.33 M PMSF, 1 M DTT, and 10% glycerol). The
nuclei were incubated on a rocking platform at 4°C for 30 min and
microcentrifuged for 10 min at 4°C. The resulting supernatants were
stored at 
70°C until needed. Protein concentrations were determined
with Bio-Rad protein assay (Bradford Assay).
B were synthesized on
a synthesizer (Oligo model 1000; Beckman), end labeled with
-[32P]ATP (3,000 Ci/mmol) (Amersham) and T4 DNA kinase
(New England Biolabs), and annealed by heating the oligonucleotides to
75°C for 10 min and were then slowly cooled to room temperature.
Unincorporated label was removed by gel filtration (Microspin G-25
columns; Pharmacia). Binding reaction mixtures contained 5 µl binding
buffer [50 mM Tris · HCl, 500 m M NaCl, 5 mM EDTA, 1 M MgCl2 (5 mM), 20% glycerol, 1 M DTT (5 mM)],
2-5 µg nuclear extract protein, 3.3 µg/µl poly(dI-dC) (2.95 mg/ml), and the final volume was brought to 27 µl with
ddH2O. The reaction mixture was kept at room temperature
for 15 min, followed by the addition of 100-200 fmol of
[32P]DNA and incubated at room temperature for 20 min.
Electrophoresis was performed on a 6% nondenaturing polyacrylamide gel
at 15 mA for 1 h in 0.5× Tris-borate EDTA. Gels were dried and
DNA protein complexes were analyzed by autoradiography. Competition
studies were performed by the addition of unlabeled double-stranded
oligonucleotides (20-fold in excess of labeled probe) to the binding
reaction mixtures.
Flow chamber assay. To study leukocyte-endothelial cell interactions under shear conditions in vitro, a whole blood flow chamber assay was used as described (27). Glass coverslips plated with confluent endothelial cells were mounted onto a polycarbonate chamber with parallel plate geometry. The flow chamber was placed onto the stage of an inverted microscope (Zeiss; Don Mills, Ontario, Canada), enclosed in a warm-air cabinet maintained at 37°C. Endothelial monolayers were visualized at ×200 magnification using phase-contrast microscopy. A syringe pump (Harvard Apparatus; S. Natick, MA) was used to draw whole blood over the endothelial monolayer. Whole blood was taken from healthy individuals and 30 U/ml of heparin (1,000 U/ml) was added to prevent coagulation. Heparin was shown not to affect leukocyte-endothelium interactions, whereas other anticoagulants, such as citrate, abolished interactions (27). The perfusion rate was set at 10 dyn/cm2. Experiments were recorded via a charge-coupled device camera (Hitachi Denshi; San Jose, CA) and a videocassette recorder (Panasonic; Secaucus, NJ) attached to the microscope. Rolling and adherent cell counts were made through video analysis.
Experimental protocol.
Leukocyte recruitment was examined on endothelial monolayers exposed to
thrombin for 10 min (P-selectin dependent), 4-h postthrombin treatment
or, for comparison, monolayers treated with TNF- (25 ng/ml) for
4 h. The concentration of TNF used is a standard dose based on
optimal concentrations from our laboratory and what others have used.
To determine the effects of thrombin (0.5 U/ml, from human plasma,
Sigma-Aldrich; Ontario, Canada) on endothelium at the 4-h time point,
the medium was removed, and the coverslips were washed once with warm
sterile PBS. The endothelium was stimulated with thrombin for 3 min.
Thrombin was removed and the media were placed back onto the
coverslips. Four hours later, the coverslips were placed in the flow
chamber and perfused briefly with Hanks' buffered salt solution (HBSS;
composed of Ca2+, Mg2+, and sodium
bicarbonate). We (23) previously reported that this
concentration of thrombin induced optimal E-selectin expression, whereas higher concentrations induced less E-selectin expression due to
significant injury to the endothelium. In addition, 3 min were optimal
for E-selectin expression because prolonged exposure to thrombin (4 h)
injured the endothelium (detachment from substratum).
treatment.
Preliminary experiments involving ATF-2 phosphorylation had indicated
the need for the p38 MAPK inhibitors to be present throughout the kinase assay to significantly inhibit the p38 activity. Therefore, the
inhibitors were readded during the kinase reaction for 10 min at 37°C
before the addition of ATP and ATF-2 fusion protein. In some
experiments, 2 mg/ml Refludan, a recombinant hirudin analog and a
specific thrombin inhibitor, was incubated with thrombin (0.1 U/ml) for
15 min before stimulation of endothelium.
Statistics. All data are reported as means ± SE. Student's t-test was used to compare between groups with a Bonferroni correction for multiple comparisons. Significance was set at P < 0.05. All experiments within a series (including controls) were done on the same day with the use of endothelial cells from the same cord to minimize variability.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Thrombin induces phosphorylation of p38 MAPK. Preliminary experiments were performed to determine the time course for thrombin-induced phosphorylation of p38 MAPK. HUVEC were exposed to thrombin (0.5 U/ml) for 3 min and after 5 min, 20 min, or 1 h, cell lysates were prepared and stained with the use of a phospho-p38 MAPK antibody. At 20 min, the phosphorylated p38 MAPK was optimal compared with the other two time points (data not shown). The 20-min time point was subsequently used for all the MAPK studies.
Figure 1 shows thrombin-induced phosphorylation of p38 MAPK. HUVEC were pretreated with the p38 MAPK inhibitors SB-203580 and SKF86002 for 1 h before thrombin treatment. These inhibitors function by competitive binding to the ATP pocket of p38 MAPK (10). HUVEC were treated with thrombin for 3 min washed, and medium was placed back into the dish for 20 min, after which cell lysates were prepared. As Fig. 1A demonstrates, there was an increase in phosphorylated p38 MAPK in response to thrombin stimulation (lane 2) compared with untreated HUVEC (lane 1). As expected, the inhibitors did not affect phosphorylation of p38 MAPK (lanes 3 and 4). A large increase in phosphorylated p38 MAPK was observed with TNF- (lane 5). Neither SB-203580 nor
SKF86002 affected TNF--induced phosphorylation of p38 MAPK
(lanes 6 and 7) consistent with
previous literature (15). The gels were stripped and
reprobed with an antibody against p38 MAPK to determine that equivalent
amounts of protein were placed in each lane (Fig. 1B).
Densitometry was carried out to quantitate the relative amounts of
phosphorylated p38 MAPK. As Fig. 1C demonstrates, there was
an approximately sevenfold increase in phosphorylated p38 MAPK in
thrombin-treated compared with untreated endothelium. The increase in
phosphorylated p38 MAPK was not affected by the inhibitors. A similar
pattern was observed in TNF--treated HUVEC, and this was also
unaffected by the inhibitors.
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SB-203580 and SKF86002 decreased p38 MAPK activity.
The activity of p38 MAPK was then examined with the use of the
downstream effector of p38 MAPK activation, phospho-ATF2. Figure 2A demonstrates an increase in
phospho-ATF2 (lane 2) versus unstimulated HUVEC (lane
1). SB-203580 and SKF86002 reduced the activity of p38 MAPK
(lanes 3 and 4). TNF- also increased activity
of p38 MAPK (lane 5) versus unstimulated HUVEC
(lane 1). SB-203580 and SKF86002 reduced TNF-
induced p38 MAPK activity (lanes 6 and 7). The
membranes were stripped and reprobed with an antibody against ATF-2 to
confirm equal loading (Fig. 2B). Figure 2C shows densitometry of ATF-2 phosphorylation and reveals a significant increase in response to thrombin stimulation, which is blocked by
SB-203580 and SKF86002. The twofold increase in phosphorylated ATF-2
was decreased to control levels for thrombin as well as for TNF-.
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(lane 5). Neither SB-203580
nor SKF86002 affected TNF--induced phosphorylation of JNK MAPK
(lanes 6 and 7). The gels were stripped and
reprobed with an antibody against JNK MAPK to determine that equivalent
amounts of protein were placed in each lane (Fig. 3B).
Figure 3C shows that the ERK MAPK pathway was not
significantly induced in response to thrombin stimulation and that
neither SB-203580 nor SKF86002 affected this MAPK.
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Thrombin-induced mobilization of NF-B is blocked with p38 MAPK
inhibitor SKF86002.
We previously reported that thrombin induced NF-B mobilization. We
next asked whether p38 MAPK contributed to this process. Figure
4 illustrates NF-B mobilization in
response to thrombin stimulation. HUVEC were pretreated for 1 h
with the p38 MAPK inhibitor SKF86002 before thrombin treatment. As
previously reported by us and others, thrombin specifically induced
NF-B DNA binding complexes in HUVEC (lane 3) compared
with the untreated HUVEC (lane 1). Competitive labeling
verified the specificity of the binding (lanes 2,
4, and 6). Most importantly, Fig. 4, lane
5, shows that SKF86002 reduced NF-B activation to levels below
those observed in controls. Quantitation of the EMSA showed a
significant increase in NF-B DNA binding, and this was reduced by
83% in the presence of the inhibitor (data not shown).
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P38 MAPK inhibition reduced E-selectin expression on
thrombin-treated endothelium.
Table 1 shows the data for cell surface
E-selectin expression on endothelium treated for 4 h
with two concentrations of thrombin. There was a significant increase
in E-selectin expression with both concentrations of thrombin compared
with untreated endothelium. Pretreating the endothelium with SB-203580
for 1 h eliminated E-selectin expression at the lower
concentrations of thrombin and partly inhibited E-selectin expression
by 55% at the higher concentrations. The value of 0.188 U of
fluorescence under basal conditions reflects background fluorescence
because no leukocyte recruitment is seen under basal conditions.
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P38 inhibitors blocked leukocyte recruitment on 4 h
postthrombin-stimulated HUVEC.
Figure 5 shows leukocyte rolling and
adhesion on untreated endothelium and endothelium treated with
thrombin. When whole blood was perfused over untreated endothelium,
5 ± 2 rolling leukocytes per field of view were noted, whereas
endothelium treated with thrombin for 3 min and whole blood perfused
4 h later supported an average of 50-60 rolling leukocytes
per field of view (Fig. 5A). Pretreating with SKF86002 for
1 h reduced rolling leukocytes to untreated levels (5 ± 3 rolling leukocytes per field of view). The number of adherent cells
followed a similar pattern. The number of adherent leukocytes after
thrombin stimulation increased more than fivefold. SKF86002 reduced the
number of adherent leukocytes to control levels (Fig. 5B).
At the end of the experiments, the coverslips were stained
(Geimsa-Wright stains), revealing that 70% of the leukocytes were
neutrophils. The other 30% were mononuclear cells. An
insufficient number of cells were seen on SKF86002-treated endothelium
for analysis of leukocyte cell type.
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B inhibitor reduced leukocyte recruitment by 60%, we did
not expect to see 100% inhibition with SKF86002. To ensure that this
was not specific to SKF86002, we repeated the experiments with
SB-203580. Figure 6 demonstrates a
similar pattern of leukocyte recruitment with this second p38 MAPK
inhibitor.
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-treated HUVEC to determine whether the p38 MAPK
inhibitor SKF86002 would inhibit leukocyte recruitment. Figure
7 shows a significant increase in rolling
and adherent leukocytes with TNF- compared with unstimulated
HUVEC. The average number of rolling leukocytes on
TNF--treated HUVEC were observed to be 80 ± 16 cells per field
of view, whereas under untreated conditions there were on average two
cells per field of view (Fig. 7A). Adherent leukocytes with
TNF- stimulation were observed to increase >20-fold (Fig.
7B). The majority of leukocytes were neutrophils. In
contrast to the thrombin results, SKF86002 did not block
TNF--induced leukocyte recruitment (Fig. 7).
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P38 inhibitors did not block leukocyte recruitment on 10-min
thrombin-stimulated HUVEC.
To determine whether p38 MAPK mediated all adhesive responses induced
by thrombin, we examined leukocyte recruitment at 10 min. It has been
well established that 10-min thrombin stimulation induces a
P-selectin-dependent (protein-synthesis independent) pathway of
leukocyte recruitment. Figure 8
demonstrates the rolling (Fig. 8A) and adherent (Fig.
8B) leukocytes on untreated endothelium and endothelium
treated with thrombin for 10 min. Few leukocytes were observed to be
rolling or adherent on untreated endothelium, whereas 10 min of
thrombin treatment supported an average of 28 ± 10 rolling and
101 ± 39 adherent leukocytes per field of view (Fig. 8,
A and B). Pretreatment of the endothelium with
SKF86002 did not affect leukocyte recruitment at this time point.
SB-203580 showed a similar pattern of recruitment (data not shown).
Although the thrombin was kept on the endothelium for the entire time
of this acute experiment, rolling began as early as 3-5 min
postthrombin administration, and the p38 MAPK inhibitors neither
delayed nor reduced leukocyte rolling and adhesion.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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In this study, we demonstrate that thrombin causes the
phosphorylation and activation of p38 MAPK, which leads to
NF-B-dependent and -independent synthesis of E-selectin. Our data
also show that this pathway is functional inasmuch as all
thrombin-induced leukocyte recruitment at 4 h was inhibited by two
well-characterized p38 MAPK inhibitors. By direct contrast, the very
rapid (within 5-10 min) leukocyte recruitment associated with
thrombin, involving the mobilization of presynthesized P-selectin from
Weibel-Palade bodies is not dependent on p38 MAPK. Clearly, thrombin
can activate at least two distinct pathways to induce surface
expression of two distinct adhesion molecules, ultimately resulting in
the recruitment of neutrophils at 5-10 min and neutrophils and
mononuclear cells at 4 h. Finally, our data reveal that inhibition
of p38 MAPK alone is insufficient to inhibit TNF--induced leukocyte
recruitment, suggesting activation of p38-independent pathways for
leukocyte recruitment. Therefore, whereas both thrombin and TNF-
induced phosphorylation of p38 MAPK, this event was absolutely
necessary for thrombin but not TNF--induced leukocyte recruitment.
Activation of p38 MAPK appears to be important in regulating cell
responses to stress. Heat, oxidative stress, cytokines, and even
thrombin have been shown to activate this MAPK member in various cell
types (16, 17, 20, 28). Recently, the focus on p38 MAPK
has revealed an important role for this MAPK in numerous important
endothelial responses. Endothelial morphological changes to shear
stress, endothelial adhesion molecule expression in response to
TNF-, endothelial motility, and growth in response to VEGF or
IGF-, and even endothelial chemokine production have all been shown
to involve p38 MAPK (1, 20, 26, 29). Our study adds to the
growing list of stimuli and growing list of functions played by
endothelial p38 MAPK. These data also suggest that different pathways
must be used to induce morphological changes versus inflammatory
changes versus motility and even growth. Clearly, on activation of p38
MAPK with shear stress or VEGF, downstream phosphorylation of two
kinases, MAPK-activated protein kinase-2 and -3, leads to
phosphorylation of a small heat shock protein-27 that enhances F-actin
polymerization and cytoskeletal changes (9). In this
study, we show that activation of endothelium with thrombin also leads
to activation of p38 MAPK; however, this results in phosphorylation of
ATF-2 and c-Jun (26), which then presumably act as
transcriptional activators, leading to adhesion molecule synthesis. The
mechanisms that regulate which p38-dependent pathway is invoked are
likely related to phosphorylation of different sites on p38 or
phosphorylation of different p38 subunits (, 
, , and 
),
which dictate differential downstream events.
We previously demonstrated that NF-B inhibition significantly
reduces thrombin-induced leukocyte recruitment. In that study, we
proposed that the residual recruitment was NF-B-independent and
perhaps p38 MAPK dependent. To our surprise, the p38 inhibitors SB-203580 and SKF86002 blocked all of the thrombin-induced leukocyte recruitment, suggesting that p38 MAPK mediated both NF-B-dependent and independent cellular recruitment. Although, in some systems p38
MAPK appears to be distinct from NF-B, p38 MAPK has been reported to
be involved in some NF-B-dependent biology (8, 12, 22,
30). For example, the specific involvement of p38 MAPK in
lipopolysaccharide-induced NF-B activation and inducible nitric
oxide synthase expression in macrophages has been demonstrated. In the
presence of SB-203580, NF-B DNA binding and inducible nitric oxide
synthase mRNA production were dramatically reduced (12).
Clearly, in these and our own study, p38 MAPK is an important activator
of NF-B. Our own data suggest that the activation of p38 MAPK to
induce NF-B translocation to the nucleus to induce E-selectin
expression is the major pathway for leukocyte recruitment by thrombin.
Although the p38 MAPK inhibitors eliminated E-selectin-dependent rolling, they reduced E-selectin expression by only 55%. Clearly, inhibition in this E-selectin expression is sufficient to completely inhibit E-selectin function, in the flow chamber assays.
The pyridinyl imidazole compound SB-203580 has been shown to be active
in a variety of animal models of acute and chronic inflammation
(10), making p38 MAPK inhibition a potential
anti-inflammatory strategy. Despite its pharmacological efficacy for
p38 MAPK inhibition, it has been suggested that in certain cell types
this compound nonspecifically activates and/or inhibits signaling
molecules other than p38 MAPK. Birkenkamp et al. (4) have
demonstrated that 10 µM concentrations of SB-203580 activated the ERK
MAPK pathway in an erythroleukemic cell line, which subsequently
enhanced NF-B transcriptional activity. Lali et al.
(18) showed that the same concentration of inhibitor
blocked phosphorylation and activation of PKB kinase through inhibition
of PKB kinase phosphoinositide-dependent protein kinase-1 in an
interleukin-2-stimulated T cell line CT6. There are numerous studies
that have not seen these nonspecific effects of p38 MAPK inhibitors
(1, 20). In our system, using primary HUVEC we did not
observe the activation of ERK MAPK in the presence of SB-203580 nor did
we observe any effect on PKB phosphorylation.
These data may have direct implications with respect to therapeutic
approaches to disease. Whereas TNF- is a ubiquitous cytokine that is
increased in many disease states to induce immune cell recruitment, the
inflammatory spectrum of thrombin is somewhat more restricted to
vascular diseases, including ischemia-reperfusion, atherosclerosis, and vasculitis. Because inhibition of p38 MAPK has
such dramatically different effects with respect to efficacy for
thrombin and TNF-, investigation of the new classes of p38 inhibitors in thrombin-induced inflammatory processes is warranted.
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ACKNOWLEDGEMENTS |
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The work was supported by Canadian Institutes of Health Research and Bayer. P. Kubes is an Alberta Heritage Foundation for Medical Research (AHFMR) Scientist and a Canadian Research Chair recipient. R. C. Woodman is an AHFMR senior scholar. J. Kaur is an AHFMR student.
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FOOTNOTES |
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Address for reprint requests and other correspondence: P. Kubes, Dept. of Physiology and Biophysics, Univ. of Calgary, Health Science Centre, 3330 Hospital Dr. NW, Calgary, Alberta, Canada, T2N 4N1 (E-mail: pkubes{at}ucalgary.ca).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published December 27, 2002;10.1152/ajpheart.00016.2002
Received 16 January 2002; accepted in final form 17 December 2002.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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P < 0.05, relative to thrombin treatment alone
(n = 3).
