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Immunology Research Group and Departments of Physiology and Biophysics and Medicine, 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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With the use of a whole blood laminar flow
chamber system, we examined the types of leukocytes, adhesion molecules
and the role of nuclear factor-
B (NF-B) in thrombin-induced
leukocyte recruitment. Primary human umbilical vein endothelial cells
(HUVEC) stimulated with thrombin induced a significant increase in
P-selectin-dependent neutrophil recruitment. Unexpectedly, brief
thrombin stimulation (3 min) of endothelium also induced a significant
lymphocyte recruitment 4 h later in addition to neutrophil
recruitment. E-selectin antibody reduced neutrophil recruitment by
>90%, whereas vascular adhesion molecule-1
(VCAM-1)/
4-integrin were primarily responsible for lymphocyte recruitment. To examine whether NF-B contributed to leukocyte recruitment 4 h post thrombin stimulation, we treated HUVEC with the NF-B inhibitor MG-132 for 1 h before thrombin stimulation. MG-132 significantly reduced the number of rolling (77.1%) and adherent (79.9%) leukocytes compared with thrombin stimulation alone. The inhibitor was more effective at preventing lymphocyte than neutrophil recruitment, consistent with its greater effect on VCAM-1 versus E-selectin expression. Tumor necrosis factor-- and MG-132-treated HUVEC displayed no inhibition of leukocyte recruitment despite a decrease in NF-B activation. In
summary, thrombin causes predominant neutrophil recruitment via rapid
P-selectin expression but also a delayed E-selectin- and
VCAM-1-dependent neutrophil and lymphocyte recruitment via de novo
protein synthesis. Although NF-B mobilization was essential for
thrombin-mediated VCAM-1-dependent recruitment, it only partially contributed to E-selectin-dependent recruitment.
selectins; vascular adhesion molecule-1; integrin; tumor necrosis
factor-; endothelium
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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 blood stream into inflammatory tissue sites involves a
sequence of steps: initial leukocyte tethering and rolling, followed by
firm adhesion to the endothelium, and subsequent transmigration into
the tissue. Briefly, leukocyte tethering and rolling is mediated by
interactions with endothelial selectins (E- and P-selectin). P-selectin
is stored in the Weibel-Palade bodies of endothelial cells and is
rapidly expressed on the endothelial cell surface in response to
thrombin, histamine, and reactive oxygen metabolites (15, 18, 25,
33). E-selectin is synthesized de novo and expressed optimally
on the endothelial cell surface 4-8 h after stimulation with tumor
necrosis factor- (TNF-), interleukin-1 (IL-1),
lipopolysaccharides (LPS), and thrombin (3, 4). Therefore,
thrombin has the potential to induce both early P-selectin- and delayed
E-selectin-dependent leukocyte recruitment. An unrelated adhesion
pathway vascular adhesion molecule-1 (VCAM-1)/4-integrin
has been reported to function in rolling and firm adhesion (2,
17), but whether thrombin can induce leukocyte recruitment via
this pathway or exclusively via selectins, and whether neutrophils are
the only leukocyte type recruited, is not known.
Although thrombin, a multifunctional serine protease, is generated through activation of the coagulation cascade, its effects also clearly contribute to inflammation. Indeed, studies (12, 41) are emerging to suggest inhibition of thrombin reduces the inflammatory response. Studies (30) from our lab have clearly demonstrated that following ischemia-reperfusion, neutrophil rolling and adhesion are blocked by anti-thrombin III, an endogenous inhibitor of thrombin. In addition, there is growing evidence (16, 27, 41) that thrombin may contribute to atherosclerosis, arthritis, and diseases where mononuclear leukocyte recruitment is a prominent feature. Therefore, understanding the mechanisms by which thrombin induces leukocyte recruitment may be very important to defining novel anti-inflammatory therapeutics. The first objective was to fully elucidate the types of leukocytes and adhesion molecules mediating the thrombin-induced inflammatory response.
Nuclear factor-B (NF-B) has been shown to be an important
transcription factor in the development of inflammation in numerous animal models, including LPS (5, 23), organ transplant
(9, 13, 38), carrageenin-induced pleurisy
(10), and streptococcal cell wall-induced polyarthitis
(31). These authors have postulated that one potential
anti-inflammatory mechanism is the reduction in adhesion molecule
expression and leukocyte recruitment. In vitro work has supported this
view; however, most of the studies (6, 7, 24) have been
restricted to the agonist TNF-. NF-B has been shown to regulate a
number of adhesion molecules. Promoters of E-selectin and VCAM-1 are
well characterized and contain NF-B-binding sites necessary for
transcriptional activation (8, 22). Furthermore,
TNF--induced expression of E-selectin and VCAM-1 can be partially
blocked with an NF-B inhibitor, MG-132 (34). In
addition to NF-B, transcription factors, including activator
protein-1 (AP-1), have been evoked in TNF--induced adhesion molecule
expression and appear to compensate for or overlap with NF-B
(1, 8, 11). It is not known whether the transcription factor NF-B mediates thrombin-induced gene transcription,
upregulation of adhesion molecules, and ultimately leukocyte
recruitment. Therefore, a second objective of this study was to examine
if the transcription factor NF-B is mobilized in response to
thrombin treatment, and, if so, what effect does this have on the
recruitment of leukocytes.
The data presented herein suggest that thrombin induces three separate
functional pathways of endothelial activation, which translate into
recruitment of a particular type of leukocyte. Rapid activation of
endothelium resulted in 1) exclusive P-selectin-dependent neutrophil recruitment, 2) de novo synthesis via a partial
NF-B-mediated, E-selectin-dependent neutrophil recruitment, and
3) NF-B-mediated, VCAM-1-dependent lymphocyte 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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Monoclonal antibodies.
E-selectin antibody (ES-1) was kindly donated by Dr. K. D. Patel
of the University of Calgary (Calgary, Alberta, Canada). 4-Integrin antibody (HP1/2) and VCAM-1 antibody (4B9)
were generously supplied by Dr. R. Lobb (Biogen; Cambridge, MA), and
P-selectin antibody was a gift from Dr. R. P. McEver.
Endothelium isolation. Human umbilical vein endothelial cells (HUVEC) were harvested and cultured from fresh human umbilical cords as previously described (29, 30). Briefly, fresh cords were perfused with sterile phosphate-buffered saline (PBS). The cords were filled with collagenase (320 U/ml, Worthington Biochemical; Freehold, NJ) and incubated for 20 min in warm PBS. After being incubated, 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 fetal bovine serum and centrifuged for 8-10 min at 1,100 rpm. The cell pellet was resuspended in medium 199 (GIBCO BRL; Grand Island, NY), supplemented with 20% fetal bovine serum, 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.
Flow chamber assay. To study the leukocyte-endothelial cell interactions under shear conditions in vitro, a flow chamber assay was used as previously described (36, 37). Glass coverslips plated with confluent endothelial cells were mounted onto a polycarbonate chamber with parallel plate geometry. The flow chamber was placed onto a stage of an inverted microscope (Zeiss; Don Mills, Ontario, Canada), which was enclosed in a warm-air cabinet maintained at 37°C. The endothelial monolayers were visualized at ×200 with the use of phase-contrast microscopy. A syringe pump (Harvard Apparatus; S. Natick, MA) was used to draw whole blood over the endothelium monolayer. Whole blood was taken from healthy individuals, and 30 U/ml of heparin sodium (1,000 U/ml) was added to prevent coagulation. The heparin was shown not to affect leukocyte-endothelium interactions, whereas other anticoagulants, such as citrate, abolished interactions (37). The perfusion rate was set at 10 dyn/cm2. Experiments were video recorded via a charge-coupled device (CCD) 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.
Enzyme-linked immunosorbent assay for cell surface adhesion molecule expression. Briefly, confluent HUVEC were seeded onto fibronectin-coated 48-well plates, and they were treated with various stimuli, fixed with 1% formalin, and then blocked with 1% bovine serum albumin. The endothelial cells were either labeled with 2 µg/ml ES-1 (E-selectin Ab) or with 2 µg/ml of 4B9 (VCAM-1 Ab). They were then washed and labeled with a peroxidase-labeled goat anti-mouse immunoglobulin G (IgG) (1 µg/ml, Dako). After undergoing a final wash, the 3,3',5, 5'-tetramethylbenzidine one-step substrate system (Dako) was added for color development, and the color reaction was stopped with 0.18 M of H2SO4. The plates were read at 450 nm.
Electrophoretic mobility shift assay.
After HUVEC treatment, cells were lysed and nuclear extracts were
prepared as described elsewhere with modifications (22, 34). Briefly, monolayers were incubated on ice for 15 min in buffer [10 mM HEPES, 0.1 mM EDTA, 10 mM KCl, 1 M dithiothreitol (DTT),
0.33 M phenylmethylsulfonyl fluoride (PMSF), 5 mg/ml of leupeptin, 2.1 mg/ml of aprotinin, and 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 rpm) 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). 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 the Bio-Rad Protein Assay
(Bradford assay).
B were synthesized on a
Beckman Oligo 1000 Synthesizer, 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 then were slowly cooled to room temperature.
Unincorporated label was removed by gel filtration (Microspin G-25
columns, Pharmacia). Binding reaction mixtures contained 5 µl of
binding buffer [50 mM Tris · HCl, 500 mM NaCl, 5 mM EDTA, 1 M
MgCl2 (5 mM), 20% glycerol, and 1 M DTT (5 mM)], 2-5
µg nuclear extract protein, and 3.3 µg/µl poly dI:dC (2.95 mg/ml), and 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-labeled 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. The 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.
Experimental protocol. Leukocyte recruitment was examined on endothelial monolayers exposed to thrombin for 10 min or 4 h post thrombin treatment. To determine the effects of thrombin (0.5 U/ml) on endothelium 4 h after stimulation, the coverslips were kept in petri dishes in medium 199. The media were 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 were perfused briefly with Hanks' balanced salt solution (HBSS, with Ca2+, Mg2+, and sodium bicarbonate). Ostrovsky et al. (29) 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. Additionally, 3 min was optimal for E-selectin expression because prolonged periods (4 h) also injured the endothelium (detachment from substratum).
To determine the effect of thrombin on endothelium at 10 min, confluent monolayers of endothelium were perfused with HBSS buffer containing thrombin (0.5 U/ml) for 10 min and were used immediately in experiments. All of the experiments were done with 0.5 U/ml of thrombin. Whole blood was perfused at 10 dyn/cm2 over thrombin-stimulated endothelium for 5 min, followed by perfusion with HBSS to clear nonattached red blood cells and leukocytes. Five fields of view were recorded for 20 s each and rolling and adhesion was determined by using playback analysis as previously described (37). If a leukocyte remained stationary for at least 10 s, it was defined as adherent. In control experiments, the endothelium was perfused with HBSS without thrombin for 10 min and then whole blood was perfused as described above. The use of whole blood permits identification of types of leukocytes recruited onto the endothelium. Briefly, the coverslips were removed from the chambers, allowed to dry, and then stained with Geimsa-Wright stains. Differentials were then performed on the coverslips to determine the subpopulations of leukocytes recruited onto the endothelium (200 cells were counted for differentials). Careful removal of the coverslips was demonstrated to avoid the loss of leukocytes from the endothelial surface (37). It has been shown (37) that the cell recruitment profiles do not change between shear forces of 5 and 40 dyn/cm2. For the antibody studies, monoclonal antibodies were added directly to whole blood for 10 min (incubated in 37°C water bath) before perfusion. For the NF-B studies, the coverslips were pretreated with
the NF-B inhibitor MG-132 (10 µM; Calbiochem; San Diego, CA) for
1 h before thrombin treatment.
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 were done on the same day by using endothelial cells from the same cord to minimize variability in the response between cords.
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RESULTS |
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Subsets of leukocytes recruited onto thrombin-treated endothelium.
Figure 1 shows the rolling and adherent
leukocytes on untreated endothelium and endothelium treated with
thrombin for 10 min and 4 h. When whole blood was perfused over
untreated endothelium, <2 rolling leukocytes per field of view were
noted, whereas endothelium stimulated with thrombin for 10 min
supported an average of 20 rolling leukocytes per field of view (Fig.
1A). Perfusing whole blood over thrombin-stimulated
endothelium at 4 h showed ~40 rolling leukocytes per field of
view (Fig. 1A). The number of adherent cells followed a
similar pattern. Endothelium stimulated with thrombin for 10 min
supported 20-fold more adherent leukocytes than the untreated
endothelium (Fig. 1B). This value was further increased at
4 h. It is interesting that only 3 min of thrombin was required to
induce profound endothelial changes at 4 h.
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Adhesion molecules mediating leukocyte interactions with 10 min and
4 h of thrombin treatments.
Figure 3 shows that the thrombin-treated
endothelium, when exposed to P-selectin antibody (G1), completely
blocked rolling leukocytes at this early time point. As a result of the
block in leukocyte rolling, leukocyte adhesion was also prevented. At 4 h this antibody had no effect on leukocyte recruitment (data not
shown). As expected, an E-selectin antibody (ES-1) did not have an
effect on leukocyte rolling or adhesion at the 10-min time point (Fig.
3, C and D).
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4-integrin antibodies (HP1/2) on leukocyte rolling
on endothelium 4-h post thrombin treatment. When ES-1 (5 µg/ml) was
added to whole blood perfused over 4 h thrombin-treated
endothelium, there was a significant reduction in the number of rolling
and adherent leukocytes compared with 4 h of thrombin treatment
alone. Rolling and adherent leukocytes decreased by 75% and 84% per
field of view, respectively. When HP1/2 (2 µg/ml) was added to the
treated endothelium, there was no reduction in rolling leukocytes but a
significant reduction in adherent leukocytes. When
4-integrin antibody was added to the whole blood in
combination with E-selectin antibody, the number of rolling and
adherent leukocytes further decreased to those observed in the
untreated conditions, suggesting that E-selectin and
4-integrin mediated all thrombin rolling at 4 h.
There was no effect with isotype-specific control antibody on rolling
and adhesion 4 h after thrombin treatment (data not shown).
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4-integrin antibodies on the types of
leukocytes (neutrophils and lymphocytes) recruited onto
thrombin-treated endothelium. E-selectin antibody reduced neutrophil
recruitment by 90% (Fig. 5A), whereas lymphocyte recruitment was only reduced by 40% (Fig. 5B). In contrast,
4-integrin antibody inhibited more than 80% of
lymphocytes (Fig. 5B). The antibody directed against
4-integrin did inhibit 40-50% of neutrophil recruitment (Fig. 5A). This observation is inconsistent with
the view that neutrophil recruitment is not
4-integrin-dependent; however, Patel (32)
and Reinhardt and Kubes (37) consistently saw
4-integrin-dependent neutrophil recruitment from whole
blood. Nevertheless, thrombin-induced lymphocyte recruitment is
primarily 4-integrin dependent, whereas the E-selectin
pathway appears to predominantly recruit neutrophils. This pattern and
magnitude of cell recruitment was not affected by IgG-1 nonbinding
antibody (data not shown).
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Role of NF-B mobilization on 4 h of thrombin-induced
leukocyte recruitment.
Figure 6A illustrates
NF-B mobilization in response to thrombin stimulation.
HUVEC were pretreated with the NF-B inhibitor MG-132 for 1 h
before thrombin treatment. Thrombin specifically induced NF-B
DNA-binding complexes in HUVEC (lane 3) compared with the
untreated HUVEC (lane 1). Competition by unlabeled
oligonucleotides verified the specificity of binding to the sites
(lanes 2, 4, and 6). As shown in Fig. 6,
lane 5, MG-132 reduced NF-B activation below control
levels. Lane 7 shows the NF-B mobilization induced by
TNF-. MG-132 also inhibited TNF--induced NF-B mobilization to
the nucleus (data not shown). In an attempt to quantitate these results, densitometry was performed. Figure 6B shows that a
significant increase in NF-B was consistently observed with thrombin
that was very significantly inhibited by MG-132. This graph is intended to show reproducibility and is unlikely to represent quantitative increases due to nonlinear properties. We have tried other inhibitors including pyrrolidine dithiocarbamate (PDTC, 50 µM) and lactacystin (20 µM). PDTC inhibited NF-B mobilization to the nucleus but also
inappropriately induced profound P-selectin expression, whereas lactacystin was unable to fully inhibit NF-B mobilization. For this
reason, only MG-132 was used. Nevertheless, PDTC was able to inhibit a
significant portion of the E-selectin- and VCAM-1-dependent leukocyte
recruitment (data not shown), confirming the MG-132 data shown in
Fig. 7.
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B
inhibition of leukocyte recruitment on thrombin-treated endothelium.
With thrombin treatment alone there were ~20 ± 8 rolling and
200 ± 62 adherent leukocytes per field of view; however,
pretreatment with MG-132 reduced this to 7 ± 3 rolling and
50 ± 9 adherent leukocytes per field of view. MG-132 in the
absence of thrombin had no effect on leukocyte recruitment. Figure 7,
C and D, shows that pretreating HUVEC with MG-132
primarily reduced lymphocyte recruitment (90%) with a smaller but
significant effect on neutrophil recruitment (60%). Recruitment of
monocytes and eosinophils was not affected by the inhibitor. The lack
of effect on monocytes or eosinophils is not due to the sensitivity of
the system because IL-4-stimulated HUVEC have been shown to preferentially recruit eosinophils from whole blood (32).
Furthermore, there was a significant increase in E-selectin and VCAM-1
expression with thrombin treatment compared with untreated endothelium
(data not shown). Due to variability of magnitude of thrombin-induced
adhesion molecule expression from different cords, the MG-132 data are
presented as percent inhibition. VCAM-1 surface expression was reduced
by 88 ± 6% with the NF-B inhibitor. E-selectin expression was
also reduced by MG-132, but a residual amount of this adhesion molecule
(~40 ± 21%) remained. The remaining E-selectin expression
(independent of NF-B) was functional because rolling occurred on
thrombin and MG-132-treated endothelium and this was reduced further to
baseline levels by E-selectin antibody (data not shown).
In a final series of experiments, we examined how the thrombin-induced
leukocyte recruitment compared with TNF-. As Fig. 8 demonstrates, at 4 h, optimal
concentrations of TNF--induced leukocyte-endothelial cell
interactions were comparable with those of thrombin. Although MG-132
inhibited TNF--induced NFB translocation, neither leukocyte
rolling nor leukocyte adhesion were altered. This recruitment was
dependent on E-selectin (Fig. 8, A and B). These
data suggest a much greater role for NF-B in thrombin- versus
TNF--induced cell recruitment.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The leukocyte recruitment paradigm has been presented as a
coordinated cascade of events, including initial tethering and rolling
of leukocytes by selectins, followed by firm adhesion due to integrins
(3, 28, 40). This is almost certainly true for
neutrophils, which can be recruited to roll via P-selectin within
minutes or via E-selectin within hours of de novo synthesis of this
molecule (15, 21, 42). Neutrophils can then firmly adhere
via the 
2-integrins (42). However, the same
paradigm may not apply to other leukocyte populations. For example, it is well appreciated that monocytes, eosinophils, and lymphocytes all
constitutively express the 4-integrin, a molecule that
has been shown to mediate both leukocyte rolling and adhesion through its ligand VCAM-1 (2, 17). Although some lymphocyte
subsets use 4-integrin almost exclusively for
recruitment, monocytes have been shown to use L-selectin, E-selectin,
and 4-integrin, whereas eosinophils rely primarily on
P-selectin and 4-integrin (32). Much of the
aforementioned work has been derived from isolated populations of cells
(e.g., neutrophils) perfused over immobilized adhesion molecules (e.g.,
E-selectin). However, complexity is increased substantially in vivo
because different proinflammatory mediators may express different
adhesion molecules on endothelium and may thereby recruit different
cell populations which themselves may impact on other leukocyte populations.
In this study, we used a whole blood perfusion system and documented
the types of cells recruited by thrombin under flow conditions and
established the importance of each of the endothelial adhesion molecules. Indeed, early thrombin activation of endothelium
preferentially induced rapid neutrophil rolling and adhesion, which was
entirely P-selectin dependent. Indiscriminate recruitment of all cell
types would have resulted in ~60% neutrophils and 30% lymphocytes,
with the remainder composed of other cell types. Clearly, the
80-90% neutrophils on the surface of endothelium, which was
treated briefly with thrombin, suggests that this adhesion molecule
profile favored neutrophils. In contrast, the adhesive profile at
4 h was clearly different (E-selectin and VCAM-1 but not
P-selectin) and was associated with a very significant increase in
lymphocyte and neutrophil recruitment. Although some overlap between
the adhesion molecules existed, it became clear that neutrophils were
recruited predominantly via E-selectin and lymphocytes via
4-integrin.
These data on leukocyte recruitment by thrombin are in line with a growing body of evidence demonstrating that this serine protease can contribute to inappropriate leukocyte recruitment in numerous inflammatory diseases. For instance, in an in vivo model of ischemia-reperfusion, the endogenous inhibitor of thrombin, anti-thrombin III, prevented P-selectin-dependent rolling and CD18-dependent adhesion after ischemia-reperfusion (30). Unfortunately, in that study, the ischemia-reperfusion-induced leukocyte recruitment was examined for only the first 2 h, when primarily neutrophils are recruited. Because both an early neutrophil and delayed neutrophil and lymphocyte recruitment were induced by thrombin, it requires some reassessment of leukocyte recruitment at later times in ischemia-reperfusion. Indeed, a recent report by Kokura et al. (20) suggests a potential role for lymphocytes as regulators of neutrophil recruitment at 4 h of ischemia-reperfusion: a time point consistent with thrombin-induced lymphocyte recruitment in our study. In addition, a few studies (14) reported increased tissue levels of thrombin in certain chronic diseases such as rheumatoid arthritis and osteoarthritis. Taken together, these observations suggest a multifaceted, proinflammatory role for thrombin.
Although there is growing evidence that thrombin may also contribute to
monocyte recruitment in vitro (19, 39), our data reveal
that thrombin-induced leukocyte recruitment is not selective for
monocytes. Although Kaplanski and colleagues (19)
described that thrombin can express sufficient VCAM-1 to allow purified monocytes (no other cells added) to settle onto and adhere to thrombin-activated endothelium under static conditions, our data reveal
that under flow conditions in whole blood, very few monocytes are
recruited relative to other cells. Under flow conditions, Luscinskas
and colleagues (26) reported that TNF--induced monocyte recruitment was dependent on L-selectin-dependent rolling and VCAM-1/4-integrin-dependent adhesion. Our data suggest
that thrombin can induce VCAM-1, but this was not sufficient to recruit
monocytes in a preferential manner. A likely explanation is that the
ligand for L-selectin is expressed on TNF--stimulated endothelium
but perhaps not in sufficient quantities after thrombin stimulation.
Much interest has recently been devoted to elucidating the signaling
pathways that contribute to adhesion molecule expression, although the
majority of investigations have been restricted to TNF--stimulated
endothelium. In this study, we clearly demonstrate for the first time
that thrombin induces NF-B translocation to the nucleus and results
in the synthesis of sufficient E-selectin and VCAM-1 expression to
recruit both neutrophils and lymphocytes under flow conditions.
Moreover, our data also suggest that this transcription factor is
essential for VCAM-1-dependent lymphocyte recruitment with a smaller
but significant effect on E-selectin-dependent neutrophil recruitment.
Indeed, whereas MG-132 blocked 90% of VCAM-1 expression and lymphocyte
recruitment, a significant amount of E-selectin expression and
neutrophil recruitment persisted suggesting an NF-B-independent
pathway of E-selectin-associated neutrophil recruitment. In fact, our
data demonstrate that TNF- also induced very profound NF-B
translocation, which was completely inhibited by MG-132, as reported by
Read et al. (34). However, unlike thrombin, TNF- must
have activated other sufficiently important pathways such that no
notable inhibition of cell recruitment was observed under flow
conditions. Indeed, Collins and colleagues (8) have
demonstrated that along with NF-B induction, TNF- also activates
c-Jun NH2-terminal kinase (JNK) and p38 mitogen-activated protein kinase pathways leading to AP-1 activation. The presence of
both NF-B and JNK/p38 was required for maximal expression of
E-selectin (35). Whether AP-1 may also be the
transcription factor contributing to thrombin-induced E-selectin
expression and neutrophil recruitment remains unclear because no
specific inhibitor of AP-1 is presently available.
Although the idea of targeting NF-B as an approach to reduce the
inflammatory response is intriguing, our data raise the following
issues. First, the importance of NF-B may differ for individual
adhesion molecules. For example, complete inhibition of NF-B was
sufficient to inhibit VCAM-1 but not E-selectin expression. Second, our
data suggest that inhibition of NF-B may provide selectivity with
respect to the type of leukocyte that is recruited. Third, our data
suggest that inhibition of NF-B may provide greater anti-inflammatory effects when thrombin is the underlying
proinflammatory molecule than when TNF- is the inflammatory
molecule. Indeed, despite complete inhibition of TNF--induced
NF-B via MG-132, no reduction in leukocyte recruitment was noted.
Some discussion of the approach of using whole blood in a flow chamber is warranted. Reinhardt and Kubes (37) demonstrated that perfusion of whole blood over immobilized adhesion molecules provides data on the types of leukocytes that can be recruited. In that study, both P-selectin and E-selectin preferentially recruited neutrophils, whereas VCAM-1 preferred lymphocytes. In this study, we demonstrate that this technique can also be used with thrombin-activated endothelium to elucidate the type of leukocytes recruited after multiple types of adhesion molecule expression. The more complex adhesion molecule profiles in this model system reveal that a single mediator can be responsible for the recruitment of multiple leukocyte types at different time points. This model also has the advantage that the shear forces used with whole blood more closely approximated the in vivo situation. Generally, 2 dyn/cm2 or less are used when isolated leukocytes are perfused through flow chambers, yet 2 dyn/cm2 is thought to be at the lower end of the shear forces in blood vessels. In contrast, the whole blood technique permits the use of shear forces approaching 40 dyn/cm2, and data by Reinhardt and Kubes (37) suggest similar percentages of leukocytes are recruited on immobilized adhesion molecules across a wide range of shear forces. Finally, although it is essential to heparinize blood in this system, we have systematically determined that the type of heparin used in this study does not affect leukocyte recruitment (37).
In conclusion, thrombin a potentially important mediator of
cardiovascular diseases can activate at least three pathways leading to
the synthesis of multiple adhesion molecules and the subsequent recruitment of multiple cell types. The first pathway leads to rapid
neutrophil recruitment due to P-selectin expression, whereas the other
two pathways require NF-B translocation and VCAM-1 and E-selectin
synthesis with both lymphocyte and neutrophil recruitment, respectively. The thrombin-induced, E-selectin-dependent neutrophil recruitment is also mediated by a second, unknown, NF-B-independent pathway of cell recruitment.
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ACKNOWLEDGEMENTS |
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The work was supported by the Heart and Stroke Foundation of Canada, Bayer Inc. of Canada, and the Canadian Red Cross Society Research and Development Fund. P. Kubes is an Alberta Heritage Foundation for Medical Research (AHFMR) scientist and R. C. Woodman is an AHFMR senior scholar. J. Kaur was supported by AHFMR studentship.
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FOOTNOTES |
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Address for reprint requests and other correspondence: P. Kubes, Immunology Research Group, Univ. of Calgary, 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.
Received 4 November 2000; accepted in final form 20 April 2001.
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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 compared with 10 min of
thrombin treatment (n = 3).
