© 2000 American Heart Association, Inc.
Cellular Biology |
Direct Observations In Vivo on the Role of Endothelial Selectins and 
4 Integrin in Cytokine-Induced Leukocyte-Endothelium Interactions in the Mouse Aorta
From the Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden.
Correspondence to Einar Eriksson, Department of Physiology and Pharmacology, Karolinska Institutet, S-171 77 Stockholm, Sweden. E-mail einar.eriksson{at}fyfa.ki.se
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Abstract |
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Abstract—The molecular mechanisms underlying leukocyte recruitment in large arteries have been extensively studied using histological techniques on fixed tissues. However, there are no reports that address the dynamics of leukocyte recruitment in large arteries in vivo. We developed an intravital microscopy technique for direct observation of leukocyte-endothelium interactions in the mouse aorta. Circulating leukocytes were labeled intravasally with rhodamine 6G and microscopically visualized within the aorta, allowing direct analysis of leukocyte rolling and adhesion. In untreated vessels, leukocyte-endothelium interactions were virtually absent. However, local pretreatment with cytokines interleukin-1ß and tumor necrosis factor-
induced clear-cut
leukocyte rolling and adhesion, compatible with normal blood flow and
wall shear rate. High shear decreased rolling leukocyte flux and
increased leukocyte rolling velocity, thus decreasing the tendency for
firm adhesion. Leukocyte rolling was almost abolished by an antibody
blocking the function of P-selectin, whereas function-blocking
antibodies against E-selectin and the 4-integrin subunit
decreased rolling leukocyte flux to 51±34% (mean±SD) and 59±11% of
the value before antibody treatment, respectively. In addition,
inhibition of E-selectin function, but not of 4
integrin, resulted in increased leukocyte rolling velocity (from 48±32
to 71±32 µm per second). Taken together, we introduce the first
model for direct studies of leukocyte-endothelium
interactions in a large artery in vivo and demonstrate
cytokine-induced shear-sensitive leukocyte rolling that is
critically dependent on P-selectin and modulated by E-selectin and
4 integrin.
Key Words: leukocyte • rolling • intravital • atherosclerosis • cytokine
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Adhesive interactions between leukocytes and arterial endothelium precede leukocyte infiltration to the subendothelial space in pathological events such as atherosclerosis and vasculitis.1 2 Characteristics of such interactions have been extensively studied in postcapillary venules in relation to the inflammatory process. The recruitment of leukocytes has in these vessels been shown to require a multistep process including leukocyte rolling on and firm adhesion to the endothelium and subsequent transmigration through the endothelial barrier.3 Similar mechanisms have been postulated to recruit leukocytes to the subendothelial space in atherosclerosis. However, the detailed characteristics of leukocyte-endothelium interactions in large arteries susceptible to development of atherosclerotic lesions have not been subjected to in vivo investigation.
The molecular mechanisms involved in leukocyte rolling along the
endothelium in venules involve several cell adhesion
molecules (CAMs) on leukocytes and endothelial cells.
Rolling interactions are mediated by endothelial P- and
E-selectin interacting with their ligands on leukocytes4 5
and also through L-selectin on leukocytes interacting with its
endothelial ligand(s).4 6 In addition,
endothelial vascular CAM (VCAM)–1, as well as the
intestinal homing receptor for lymphocytes, mucosal addressin CAM-1
(MAdCAM-1), has been shown to mediate rolling through interaction with
4 integrins on leukocytes,7 8
mechanisms bypassing the need for selectins in the adhesion
cascade.
Because arterial endothelium has been shown
to express the same CAMs as those expressed in
venules,9 10 11 the molecular basis for adhesive
interactions is supposedly similar in both vessel types. However,
whereas venular endothelium is sensitive to several
stimuli for CAMs to be expressed and for subsequent leukocyte rolling
and adhesion to be induced, this is not the case for the
arterial vessels so far studied with intravital microscopy,
ie, the arterioles.12 Nevertheless, arteriolar rolling can
be induced by certain stimuli such as perivascular laser
injury13 or treatment with cytokines such as
interleukin (IL)–1ß and tumor necrosis factor
(TNF)–.14 15 Interestingly, leukocyte rolling in
arterioles is also induced by stimuli such as oxidized LDL and
cigarette smoke,16 17 known risk factors for development
of atherosclerosis in humans. However, several factors
are different in large arteries compared with arterioles or venules,
some of which are likely to influence
leukocyte-endothelium interactions. This calls for
studies on leukocyte-endothelium interactions in large
arteries in vivo. However, no such studies have been performed, as a
result of the lack of an adequate experimental model.
In this study, we used a newly developed intravital microscopic
technique for direct observation of
leukocyte-endothelium interactions in the mouse aorta
in vivo. By stimulating the aorta with IL-1ß and TNF-, leukocyte
rolling and adhesion were induced. In this setting, the influence of
arterial hemodynamics on leukocyte rolling,
and major molecular mechanisms involved in this event, were
investigated.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Animals
Sixty-nine male C57BL/6 mice weighing 20 to 30 g were obtained from B&K (Stockholm, Sweden). The animals were fed a standard diet and water ad libitum. The experiments were approved by the regional ethical committee for animal experimentation.
Cytokine Stimulation
Four hours before microscopic observation, mice were
anesthetized by inhalation of 1% to 2% isoflurane (Forene,
Abbott) in 35% O2. The abdomen was opened
through a midline incision and the intestines were retracted, giving
access to the dorsal peritoneum. Injections of cytokines (0.5
µg TNF- and 0.125 µg IL-1ß in 0.3 mL in PBS) were given in the
retroperitoneal space around the aorta inferior to the
renal arteries. The abdomen was then closed with a 5-0 suture.
Experimental Procedure
Under isoflurane anesthesia, catheters were placed
in the left carotid artery and in the left jugular vein. Through the
venular catheter a continuous infusion of bicarbonate-buffered glucose
was allowed. The arterial catheter was connected to a
pressure transducer, a Grass amplifier, and an infusion of saline. The
total rate of fluid infusion was 0.8 mL/hour. Blood pressure and heart
rate were continuously monitored. The rectal temperature was kept at
37°C with a heating pad and an infrared heat lamp. The exposed tissue
was superfused with a thermostated (37°C) bicarbonate-buffered saline
solution equilibrated with 5% CO2 in nitrogen to
maintain physiological pH, or covered with a
physiologically buffered hyaluronic acid
solution (Healon, 14 mg/mL, Pharmacia-Upjohn). All
parameters were recorded on computer using Grass
Polyview software and stored for later analysis. Serial blood
samples (10 µL) were taken through the carotid catheter. Samples were
later analyzed for white blood count (WBC) in a Bürker
chamber.
Surgical Procedure
The abdomen was reopened in the midline, and the intestines were
retracted and kept moist during the experiment. The aorta was carefully
exposed and separated from the vena cava for a distance of 2 to 3
mm immediately inferior to the renal arteries. The mouse
was placed under the microscope, and an ultrasonic flowprobe connected
to a flowmeter (Transonic T-106 flowmeter, 0.7v flowprobe;
sample rate, 200 per second) was placed around the artery. Flow could
be manipulated by elevating the flowprobe using a micromanipulator
causing partial vessel obstruction. Increased flow as compared with
baseline level was seen after obstruction was released. Direct
intravital microscopic observations were performed on the abdominal
aorta 4 to 5 mm downstream of the flowprobe where laminar flow had
been reestablished.
Intravital Microscopy
Microscopic observations were made using an intravital
microscope (Leitz Biomed) with a water-immersion objective (Leitz U-O,
23x). Epi-illumination fluorescence microscopy (Leitz
Ploem-o-pac, filter block M2 illuminated by a cooled
infrared-filtered lamp [Osram HBO 200W/4]) was started 2
minutes after labeling of circulating leukocytes with an
intravenous injection of rhodamine 6G (0.3 mg/mL, 0.67
mg/kg). Images were televised and recorded on videotape using a
Panasonic WV-1900 video camera.
Analysis of In Vivo Experiments
In the aorta, rolling and adhering leukocytes were visualized on
the anterior half of the vessel facing the objective. Rolling leukocyte
flux was determined as the number of leukocytes passing a 150-µm-long
reference line perpendicular to blood flow. Leukocyte rolling velocity
was measured as the mean velocity of individual rolling leukocytes
during a minimum time of 0.5 seconds. Leukocyte rolling distance was
defined as the distance a rolling cell covered either before leaving
the field of vision or before detaching from the
endothelium. Wall shear rate (WSR) in the aorta was
calculated from the online flow measurement (q) and the
vessel radius (r) measured from the microscopic image
according to the formula
w=4q/
r3 . Mean WSR
(MWSR) was defined as the average WSR during a specified time of
microscopic observation, and maximum WSR (MaxWSR) was calculated as the
highest WSR that occurred during the same time period. Peak
systolic WSR for every heartbeat was stable during each period
of observation, rendering MaxWSR relevant for systolic
shear.
Scanning Electron Microscopy
Animals were perfusion-fixed through the left carotid artery
with 2.5% glutaraldehyde in 0.1 mol/L cacodylate
buffer (20 minutes, 100 mm Hg) with outflow through severed
jugular veins. The aorta was excised and stored in fixative overnight.
The vessel was then dissected free from perivascular tissue, opened
longitudinally, attached en face on glass slides (Superfrost Plus Gold)
using superglue, and stored in buffer. The specimens were then
dehydrated in increasing concentrations of ethanol and Freon 113 and,
finally, through critical point dehydration with
CO2. After gold sputter coating, the aortas were
examined in a scanning electron microscope (Philips SEM 515).
Antibodies and Reagents
The antibodies used in vivo in this study were monoclonal
antibody (mAb) RB40.34 against mouse P-selectin (30 µg per mouse;
Pharmingen), mAb 9A9 against mouse E-selectin (30 µg per mouse; a
kind gift from B.A. Wolitzky, Hoffman-La Roche Inc), mAb R1-2 against
the mouse 4-integrin subunit (150 µg per
mouse; Pharmingen), and control mAb R3-34 (60 µg per mouse;
Pharmingen). Recombinant human IL-1ß and TNF- were obtained from
R&D systems Inc, and rhodamine 6G was from Sigma.
Statistical Analysis
The data represent the mean±SD of measurements obtained
in the indicated number of experiments. Statistical analysis
was performed using paired t test and Wilcoxon
signed rank test for paired samples. Analysis of the influence
of hemodynamic parameters was performed
using linear regression. Statistical significance was set at
P<0.05. In figures, *, **, and *** denote difference from
control value by significance of P<0.05,
P<0.01, and P<0.001, respectively.
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Results |
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All mice appeared normal and healthy before and during cytokine stimulation. Systemic leukocyte counts were decreased by pretreatment possibly as a result of an inflammatory response in the abdomen after surgery. In addition, stimulation with TNF-
has in
itself been shown to decrease systemic leukocyte count.18
Cytokine stimulation may also favor adhesion of mononuclear
leukocytes in abdominal microvessels of cytokine-treated
animals,8 thereby increasing the percentage of
polymorphonuclear cells in peripheral blood as compared
with animals treated with PBS. The systemic leukocyte counts are
summarized in the Table
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Hemodynamic Parameters in Mice In
Vivo
Blood pressure and heart rate were continuously measured in most
experiments. Mean arterial blood pressure ranged between 60
and 100 mm Hg. Heart rate ranged between 400 and 750 bpm. Blood
flow in the abdominal aorta was monitored online. A typical
recording of aortic blood flow with calculated WSR is shown in
Figure 1. Normal blood flow in the aorta
was 1.86±0.44 mL/min (n=9) with diameters of 510±22 µm.
Baseline values of MWSR and MaxWSR were 2710±2047 and 7646±4676 per
second, respectively.
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Treatment With IL-1ß and TNF- Induces
Leukocyte-Endothelium Interactions in the Mouse Aorta
In Vivo
Leukocyte rolling and adhesion were virtually absent in untreated
arteries, whereas occasional interactions were seen in some aortas
after treatment with PBS. On the other hand, clear-cut
leukocyte-endothelium interactions were induced by
cytokine treatment (Figure 2).
This induction was not totally consistent, and only weak
responses were seen in the aorta using IL-1ß or TNF- separately
(data not shown). A more distinct response was achieved when using both
cytokines in combination, suggesting a synergistic effect of
these substances as previously shown for leukocyte rolling in
arterioles.15 Video images of rolling and adherent
leukocytes in the aorta are shown in Figure 3A through 3F.
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The characteristics of cytokine-induced leukocyte rolling changed during the time course of the experiments. Rolling leukocyte flux decreased slowly from the start of intravital microscopy (data not shown). The same was found in control experiments using fluorescence microscopy on cytokine-treated cremaster muscle arterioles prepared as described previously,15 despite stable rolling flux for at least 40 minutes when using transillumination microscopy. Thus, fluorescence microscopy using rhodamine 6G decreases rolling leukocyte flux. In contrast, the distance and velocity of rolling leukocytes in the aorta at constant MWSR were stable for at least 20 minutes of fluorescence microscopy. However, to avoid problems in interpreting the data obtained by intravital microscopy, the time of observation in each experiment was kept below 10 minutes.
Rolling Leukocyte Flux in the Mouse Aorta Is Sensitive to
WSR
The influence of WSR on rolling leukocyte flux was measured
in the aorta of 5 cytokine-treated mice in 15-second periods at
different flow rates. Vessel diameter and blood flow were manipulated
by partial vessel obstruction, yielding values of 380 to 540 µm
(472±62 µm) and 0.14 to 5.55 mL/min (1.64±1.11), respectively.
MWSR ranged between 270 and 8911 per second (2533±1833) and MaxWSR
between 1315 and 21 416 per second (7584±4642). The minor stretch
caused when obstructing the vessel by elevating the flowprobe did not
influence the adhesive properties of the endothelium in
control experiments. To adjust for differences in WBC between animals,
rolling flux was normalized by dividing flux values with a factor given
by WBC/1x106.
The influences of MWSR and MaxWSR on rolling leukocyte flux are
shown in Figure 4A and 4B, respectively.
Rolling leukocyte flux decreased with increasing shear at MWSR above
2500 per second. Similarly, rolling flux decreased at MaxWSR
>12 000 per second. WSR exceeding these values may stress the
molecular bonds between leukocytes and endothelium and
destabilize interactions. At MWSR and MaxWSR above 6000 and 18 000 per
second, respectively, hardly any interactions occurred. This
demonstrates a hydrodynamic limit for leukocyte rolling in this in vivo
situation.
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At MWSR below 2000 per second, rolling leukocyte flux showed a
tendency to decrease. However, when we examine the rolling leukocyte
flux fraction in Figure 4C
, it seems that the fraction of
leukocytes rolling along the endothelium continues to
increase with decreasing shear even at MWSR below 2000 per second.
This indicates that the molecular bonds between leukocytes and
endothelium are functional at low shear, in accordance
with leukocyte rolling seen at the low shear levels present in
venules,19 and that the decreased flux was caused by
decreased delivery of leukocytes to the endothelium at
low flow rates. It seems that in the mouse aorta, a peak in rolling
leukocyte flux appears at MWSR 2000 per second where the
relationship between the adhesive capacity of
leukocyte-endothelium interactions and the delivery of
leukocytes to the endothelium is optimal.
WSR Influences Leukocyte Rolling Velocity
The qualitative appearance of rolling interactions was altered by
WSR. At low shear, most rolling leukocytes rolled in a slow and steady
fashion, whereas leukocyte rolling at high shear was unstable and
characterized by tethering interactions. Transit time of leukocytes was
inversely correlated to MWSR resembling a correlation between shear and
leukocyte rolling velocity (r=0.626, P<0.001,
Figure 4D).
Effect of Antibodies Against Endothelial Selectins
and 4 Integrin on Leukocyte Rolling
The molecular events involved in leukocyte rolling were
investigated in separate experiments. Leukocyte rolling was observed
for 3 minutes before intra-arterial injections of mAbs
against specific adhesion molecules followed by a subsequent 3 minutes
of observation. WBC and MWSR were stable during experiments. The effect
of antibodies on rolling leukocyte flux is shown in Figure 5A. The antibody RB40.34 against mouse
P-selectin almost abolished leukocyte rolling (n=7,
P<0.001). In animals treated with the E-selectin mAb 9A9,
the flux of rolling leukocytes decreased to 51±34% of the value
before treatment (n=10, P<0.001), whereas treatment with
the antibody R1-2 against the 4-integrin
subunit decreased rolling leukocyte flux to 59±11% (n=4,
P<0.01). Treatment with control antibody did not alter
rolling leukocyte flux (102±70% of value before treatment, n=5;
P=0.952).
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The effect of antibody treatment on leukocyte rolling was further
characterized by analyzing the leukocyte rolling distance of individual
rolling leukocytes (Figure 5B). The mean leukocyte rolling
distance after treatment with antibodies blocking function of
E-selectin and 4 integrin decreased to
80±23% (P<0.05) and 74±10% (P<0.05) of the
values before treatment, respectively. Control antibody had no effect
(100.8±17%, P=0.917). Furthermore, leukocyte rolling
velocity increased from 48±32 to 71±32 µm per second after
treatment with the antibody against E-selectin (155±39% of
pretreatment value, P<0.001, Figure 5C) but was not
altered either by control (97±22%, P=0.798) or by
4-integrin blocking antibody (117±27%,
P=0.308). Slow rolling of leukocytes occurred mostly in
certain areas of the observed arteries. This slow rolling was
attenuated by function inhibition of E-selectin, indicating that the
localized slow rolling was E-selectin dependent. In areas of slow
rolling, adherent leukocytes were regularly found.
To further clarify the potential of other mechanisms mediating rolling without involvement of P-selectin, observation time after treatment with P-selectin antibody was prolonged up to 10 minutes after antibody was given. In 7 experiments, with a total observation time after antibody treatment of 60 minutes, only 2 leukocytes passed the reference line in the aorta (0.035 cells/min) as compared with the rolling leukocyte flux of 10.9±7.0 cells/min before antibody treatment. Within the total field of vision of 330x430 µm, 3 leukocytes were observed to attach to and roll on the endothelium, whereas 19 adherent leukocytes released from firm adhesion started to roll and subsequently detached. Thus, rolling was dramatically decreased, but not abolished, by treatment with a function-blocking antibody against P-selectin.
Cytokine Stimulation Induces Platelet-Independent
Adhesion of Leukocytes in the Aorta
Adherent leukocytes were visualized by intravital microscopy.
However, because the distinction between adherent leukocytes and other
cells in the arterial wall could sometimes be difficult,
and to investigate whether platelets were involved in
leukocyte-endothelium interactions, scanning electron
microscopy on en face–mounted aortas was performed. In all specimens,
the endothelial lining was intact without adherent
platelets. In cytokine-treated aortas, leukocytes were seen
adhering to the endothelium both in vessels that had
been exposed to intravital microscopy and in those that had not. Some
leukocytes were deformed in drop shape with the head in the direction
of flow giving the impression that they were rolling on
endothelium (Reference 2020 , Figure 6). Untreated aortas had no adherent
leukocytes. No leukocytes were covered by platelets.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Leukocyte recruitment to the arterial wall is of importance in different physiological and pathophysiological situations. Numerous studies have addressed the molecular events involved in this process using histological techniques. However, no studies have investigated the dynamic events of arterial leukocyte-endothelium interactions in vivo.
In this study, by using direct intravital microscopy on the mouse
aorta, we present data on leukocyte-endothelium
interactions in a large artery in vivo. The described technique will be
an important new tool when studying the rapid dynamics of leukocyte
recruitment in arterial pathophysiology such as in
atherosclerosis and in certain types of vasculitis. In
the present work, we studied IL-1ß– and TNF-–induced
leukocyte-endothelium interactions, because both
cytokines are expressed in
atherosclerosis21 22 23 and also because
they have been shown in various models to induce expression of
endothelial CAMs that can be found both in early and in
late stages of atherogenesis.9 10 11 24 25 Although
cytokine stimulation does not resemble any specific pathology,
the adhesive mechanisms investigated in this study and their potential
in influencing leukocyte rolling in the mouse aorta are likely to be
relevant. Future studies in pathological models will also be possible
by using the technique described. Using cytokines, combined
treatment with both IL-1ß and TNF- proved to be more efficient in
inducing leukocyte-endothelium interactions than use of
either substance alone, as previously shown in
arterioles.15 The mechanism underlying this synergistic
effect may involve a multiplicative effect of these cytokines
on intracellular signaling systems and could be of importance in
upregulation of CAMs on arterial
endothelium. Unfortunately, because the aorta was
perfused by whole blood in vivo and the leukocytes were
intravenously labeled with rhodamine, we could not identify
the subtype(s) of the leukocytes rolling along the
endothelium. However, techniques that allow this
discrimination are currently under development.
By studying aortas in isoflurane-anesthetized mice, we found that virtually no interactions occurred spontaneously, whereas cytokine treatment induced leukocyte rolling and adhesion that were compatible with normal WSR. However, blood flow and shear may be different, and possibly higher, in other situations, such as in the awake and freely moving animal. Nonetheless, WSR influences rolling leukocyte flux and leukocyte rolling velocity, possibly predisposing leukocyte adhesion to sites of low or fluctuating shear such as the coronary arteries, in curvatures or at branch points, areas that are all prone to development of atherosclerotic lesions.26
In the cytokine-treated mouse aorta, P-selectin was found
to be critical for leukocyte rolling. E-selectin and
4 integrin appeared to stabilize rolling
interactions inasmuch as blocking function of either one of these CAMs
decreased rolling leukocyte flux. This indicates that capture of
leukocytes to the endothelium is dependent on
P-selectin and that E-selectin and 4 integrin
play minor roles in this event. However, limited capture and rolling
can still occur after function inhibition of P-selectin. Importantly,
despite the dominant role for P-selectin in leukocyte rolling, we could
not find any evidence for involvement of activated
platelets, as suggested by the lack of platelets adherent to
any leukocyte in SEM studies on the aorta, including those leukocytes
having a shape typically seen in rolling cells.20 In
addition, in previous studies in which platelet-leukocyte
interactions have been of importance in leukocyte recruitment, clusters
of platelets and leukocytes have been seen tumbling and rolling
down the vessels of the microcirculation.17 27 In this
study, no such clusters were seen.
The finding that an antibody against E-selectin decreases the number of
rolling leukocytes in the aorta contrasts with studies in the
microcirculation, in which inhibition of E-selectin function increases
rolling flux.14 18 The discrepancy between these
observations and the ones observed in the aorta could be due to the
high shear levels in large arteries stressing the remaining adhesive
bonds after antibody treatment, thereby causing leukocytes to detach,
whereas shear may not reach these critical levels in microvessels. This
is supported by the fact that function inhibition of E-selectin
decreased leukocyte rolling distance. A similar function of
4 integrin as of E-selectin in stabilizing
leukocyte rolling is plausible. Thus, these data illustrate that
rolling leukocyte flux is dependent not only on capture but also on
stability of rolling interactions.
Similarly to previous findings in the microcirculation, blocking function of E-selectin in the aorta increased leukocyte rolling velocity, indicating a role for E-selectin in slow leukocyte rolling.18 In addition, E-selectin–mediated slow rolling in areas of the aorta in which adherent cells often were found supports, and extends to large arteries, previous findings of a role for E-selectin and/or slow rolling in leukocyte adhesion.28 29
The importance of endothelial selectins in
cytokine-induced arterial
leukocyte-endothelium interactions could explain the
delayed formation of atherosclerotic lesions in LDL receptor–deficient
mice carrying additional mutations in the genes for these adhesion
molecules.30 Furthermore, our findings indicate that
although the 4 integrin/VCAM-1 pathway has
been implicated in leukocyte recruitment in
atherogenesis,9 10 11 25 31 the selectins appear to
strongly enhance leukocyte recruitment in arteries in vivo, despite
expression of VCAM-1 in cytokine-stimulated mouse aortas as
detected by immunohistochemistry (E.E. Eriksson et al, unpublished
data, 1999). Thus, although VCAM-1 is expressed on arterial
endothelium, it may not be sufficient to efficiently
recruit leukocytes in large arteries in vivo. Instead, additional
contributions of other CAMs are likely to be required.
In summary, we have studied, through direct intravital microscopy for
the first time, leukocyte-endothelium interactions in a
large artery in vivo. We have demonstrated that arterial
shear forces influence leukocyte-endothelium
interactions and could predispose leukocyte adhesion to sites of low
shear. In addition, we have shown that cytokine-induced
leukocyte rolling in the mouse aorta is highly dependent on initial
tethering and rolling on P-selectin, whereas E-selectin and
4 integrin stabilize rolling interactions. The
findings give novel insights into dynamic
leukocyte-endothelium interactions in large arteries
and indicate that leukocyte recruitment in these vessels in vivo is
strongly dependent on expression and function of the
endothelial selectins.
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Acknowledgments |
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This work was supported by the Wallenberg Foundation, the Swedish Medical Research Council (Grants 4764, 14X-4342, and 04P-10738), the Swedish Heart and Lung Foundation, the Swedish Foundation for Health Care Sciences and Allergy Research, IngaBritt and Arne Lundbergs Foundation, and Karolinska Institutet. We thank Ingeborg May for help with the scanning electron microscopy and the Haephtes Society for continuous support. We also thank B.A. Wolitzky for supplying the E-selectin mAb 9A9.
Received July 21, 1999; accepted December 9, 1999.
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