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Short Communication |
Dynamic Investigation of Leukocyte-Endothelial
Cell Adhesion Interaction under Fluid Shear Stress in Vitro
LING Xu, YE Jian-Feng, ZHENG
Xiao-Xiang*
( Department
of Biomedical Engineering, Zhejiang University, Hangzhou 310027, China )
Abstract To
establish a method to investigate the dynamic adhesion between leukocytes and
human umbilical vein endothelial cells (HUVECs) under definite shear stress. A
parallel plate flow chamber system was developed to produce the definite shear
stress in vitro. After the cultured HUVECs were loaded in the flow chamber, the
circulation solution containing acridine orange (AO)-labeled leukocytes was
perfused to flow through chamber at 0.71 dynes/cm2. In this case,
leukocyte-endothelial cell adhesion process was induced.
Lipopolysaccharide(LPS) was used as the chemical stimulus and
dexamethasone(DXM) was used as the anti-inflammatory reagent. The adhesion
process was recorded in videotape by Olympus IX70 fluorescence microscope and
CCD-camera. Then the number of adhesion leukocyte, slow and fast rolling
velocities of leukocytes on the surface of HUVECs were measured based on the
captured images. The number of static adhering and slow rolling leukocytes on
the HUVECs treated with LPS was significantly increased by 23.7-fold and
4.1-fold compared with that of the control group. Meanwhile, both the slow and
fast rolling velocities of the leukocytes on HUVECs treated with LPS were
significantly decreased by 25.6% and 26.1%. When HUVECs were treated with both
LPS and DXM, the effect of LPS was inhibited obviously. This developed method
can be used in studying ECs adhesion function affected by different chemical
and physical stimulus and evaluating the various compounds interfering with
cell adhesion.
Key words endothelial cells;
shear stress; parallel plate flow chamber; leukocytes; adhesion
Endothelium
forms a smooth layer between blood and vessel wall to prevent blood cells from interacting
with vessel wall when they move in the blood vessels. Therefore, it played a
critical role in the mechanics of blood flow, vascular smooth muscle cell
growth and leukocyte circulation, etc.[1]. Detailed knowledge on the mechanism
of blood cells-endothelial cells interaction is fundamental to the
understanding of many pathological processes. Adhesion of activated leukocytes
to vascular endothelium, an essential process for leukocyte emigration, was one
of the most important responses to tissue injury and infection[2]. This
interaction might be modulated by the expression of diverse adhesion molecules
and their receptors[3].
Generally, the
emigration of circulating leukocytes from the blood into the inflamed tissues
included “three steps”[4,5]: (1) rolling along the vessel
wall mediated by the selectin family[6－8]; (2) firm adhesion to endothelial cells facilitated by activated
integrins; (3) migration into tissue. Much attention had been paid to the
mechanism of the rolling process and many glycoproteins had been found as the
carriers of the carbohydrate ligands of selectins[9－11].
In response to
variations of hemodynamic forces such as fluid shear stress, vascular
endothelial cells modulate their structures and functions in many ways.
Moreover, studies indicated that the torque induced by the fluid shear was very
important to sustain leukocyte’s rolling process, and leukocyte adhesion was
significantly enhanced by fluid shear stress above the threshold level. For
example, the fluid shear above the threshold of 0.5 dynes/cm2 shear stress
significantly enhanced HL-60 myelocyte rolling on P- and E-selectin at site
densities of ≤200/μm2[12]. And the adhesion of
leukocyte through L-selectin to peripheral node addressin (PNAd) required a
minimum level of fluid shear stress to maintain rolling interactions[13].
Therefore, it is very important to establish a method to study the
leukocyte-endothelial cell adhesion under the flow condition. A variety of
parallel plate flow chambers were designed and widely used for this purpose[14－16]. Thus in vitro flow chamber
assays can be designed to construct adhesion systems under a defined shear
stress. In addition, with the help of diverse flow chamber systems, some
studies in vitro also investigated the endothelial cell adhesion molecules
characters of being targets for drug delivery system in response to
inflammatory signals, thus opened new perspectives of anti-inflammatory
therapies[17].
To investigate
the adhesive properties of cells, adhesion assays have appeared as powerful
tools, especially in screening reagents that interfere with or promote cell
adhesion to various substrates. The quantification of cell adhesion in the
assays is essential to determine the capacity of cells to adhere to a substrate
and evaluate various compounds interfering with cell adhesion. Thus, simple and
reliable methods were required in quantification of cell adhesion for both
research and application purposes[18]. For instance, rolling is an important
dynamic process in cell adhesion and needs to be measured in quantification.
Therefore, to some extent, the measuring technique of the leukocytes rolling
velocities would be critical to the study on cell adhesion process.
Rolling
leukocytes were generally defined as those that moved through microvessels at a
lower velocity than that of freely flowing erythrocytes. Some studies pointed
out that leukocyte rolling velocities varied widely in vivo. They
depended on not only the tissue and the inflammatory stimulus, but also the
adhesion molecules expressed, such as the selectins[19,20]. E-selectin
preferentially promoted leukocyte rolling at about 5 μm/s[19]; L-selectin mainly mediated
leukocyte rolling at faster velocities while P-selectin supported leukocyte
rolling at slower velocities (<50 μm/s). Under physiological conditions, P- and L-selectin synergized
to mediate rolling at velocities of 20－70 μm/s[20].
In this paper,
the method of investigating leukocyte-endothelial cell dynamic adhesion in
vitro was established. And a new technique for analyzing the adhesion process
between leukocytes and HUVECs was also developed, especially for measuring the
rolling velocity.
1 Materials
and Methods
1.1 Leukocytes
isolation and labeling
Sprague-Dawley
rat (♀, about
300 g, Zhejiang Center of Laboratory Animals) was anesthetized, and then blood
(about 8 mL) was withdrawn from carotid artery via a polyethylene catheter into
a sterile 10-mL syringe containing 0.8 mL 3.8% sodium citrate. Blood was
immediately transferred into a sterile 50-mL centrifuge tube, then 8 mL
phosphate buffered saline (PBS) and 2.67 mL 3% gelatin solution were added.
This mixed liquid was blended by shaking and blowing, and then left to sediment
at room temperature for 1 h. The leukocyte-rich upper layer was removed into
another tube and centrifuged (Labofuge 400R, Heraeus) at 1500 r/min for 10 min.
The resultant sediment (containing a few erythrocytes) was suspended in 1 mL 9
g/L NaCl. Then 24 mL distilled water was added to destroy erythrocytes. After
45 s, osmotic pressure of the solution was recovered by adding 8 mL 36 g/L NaCl
immediately. Leukocyte sediment was acquired by the second centrifugation at
1500 r/min for 10 min, and then diluted with PBS to get a suspension containing
about 2×107 cells/mL.
The leukocyte
suspension was labeled using acridine orange (AO, 15 mg/L) in dark at room
temperature for 20 min. After being washed with PBS for 3 times, leukocytes
were suspended in PBS to 2×106 cells/mL for the later experiment.
1.2 Endothelial
cells monolayer preparation
Human umbilical
vein endothelial cells (HUVECs) were isolated from human umbilical cord veins
according to Jaffe et al.[21], using the trypsin digestion. The
endothelial cells (5×104 cells/mL, 2.4 mL/plate), were primarily
cultured in RPMI 1640 (Gibco BRL) containing 20% heat-inactivated CBS (calf
bovine serum), 100 ku/L penicillin and 100 ku/L streptomycin with 35 mm culture
dishes (Nunc, Danmark) coated by 0.02% gelatin previously. The cell monolayers
were maintained in a humidified incubator with 95% air and 5% CO2 at
37 ℃ and grew to
confluence in 5－7 d. These
endothelial cells were divided into 3 groups: control, lipopolysaccharide(LPS),
and LPS+dexam-ethasone(DXM) for the perfusion experiments. LPS group was
treated with 4 mg/L LPS (final concentration) for 6 h before perfusion. LPS+DXM
group was treated with 50 mg/L DXM (final concentration) for 20 min, then
stimulated together with 4 mg/L LPS (final concentration) for 6 h before
perfusion.
1.3 Perfusion
system
The 35 mm
culture dish with HUVECs, which were divided to control group, LPS group and
LPS+DXM group, were loaded separately in parallel plate flow chamber
(GlycoTech) (Fig.1). Since the flowing bubbles might disrupt HUVEC monolayer,
care should be taken to eliminate air bubbles in the flow chamber before placing
it on the HUVEC monolayer. Labeled leukocytes were perfused under steady flow
into parallel plate flow chamber by syringe pump (ZCZ-50, Zhejiang Medical
University, China). In this experiment, a gasket with the height of 0.0254 mm
and width of 10.0 mm was used to establish the chamber where the leukocytes
would dynamically interact with HUVECs under a definite shear stress.
Fig.1 chematic of
the parallel plate flow chamber
With the
parallel plate flow chamber system, the definite shear stress could be produced
to investigate the dynamic adhesion process. The wall shear stress (τ)
was calculated according to the Navier Stokes equation[7] as following [Formula
(1)].
τ=(6Q・η)/(w・h2) (1)
where Q is the volumetric flow rate
(mL/s) of the perfusion solution, η is viscosity coefficient of the
perfusion solution (assumed to be equal to water as 0.01 poise at room
temperature of 20 ℃),
w is the channel width (cm), and h is the channel height (cm).
The shear stress was proportional to the volumetric flow rate Q of the
perfusion solution and could be calculated as dynes/cm2.
1.4 Leukocytes
perfusion
Leukocytes in suspension (2.5×105
cells/mL in PBS incubation buffer) were pushed from a reservoir through plastic
tubing and chamber with a syringe pump at 20 ℃. The leukocyte perfusion lasted for 15 min at
shear stress of 0.71 dynes/cm2. A 40× objective was used to observe
the adhesion process and the whole process was recorded on videotape in real
time. The microscope was adjusted to focus on the ECs surface. After that,
continuous perfusion of PBS without leukocytes for 5 min at the same shear
stress was applied to differentiate leukocytes adhering to the HUVECs monolayer
from those coincidentally residing on it. Then 10 images of totally about 1 mm2
field were captured for every perfusion experiment to determine the number of
the adhering leukocytes.
1.5 Image
capture and analysis
The whole experiment
system was set up as Fig.2. The assembled chamber system was placed under an
inverted fluorescence microscope (Olympus IX70), and the fluorescence emitted
from the leukocytes was excited by light from a mercury lamp (Olympus). U-MWG
filter cube (BP510－550, DM570, BA590) was used to yield the exciting light of 510－550 nm and the emitted light of
> 590 nm. The whole adhesion process was imaged by a black and white CCD
video camera (MTV-1881Ex, MINTRON) and recorded on videotape in real time by a
video-cassette recorder (NV-HD350, Panasonic). Then, with the video card
(DH-VRT-CG200, Daheng, China), digital images were sampled and saved with
personal computer. Afterwards, the number of adhered cells and the velocity of
the rolling cells were evaluated off-line using the image analysis method
established in this research.
Fig.2 Schematic
of the experiment system
The tethered
cells were defined as cells that maintained an adhesive interaction with the
substrate for at least 1 s, followed by rolling or rapid detaching. While those
maintained adhesion without moving in 10 s during the perfusion were referred
as static adherent cells, distinguished from the tethered and slowly rolling cells.
Slow rolling and fast rolling cells could be clearly distinguished from the
images. A fast moving object appears as a long bar in video image, thus those
fast rolling cells appeared as bright white bars in the captured images
(Fig.3), while those slow rolling cells were bright white dots (Fig.4). The
velocity of the rolling leukocytes, which were in focus, was calculated from
the formula (2).
v=dL/dt (2)
Fig.3 A fast rolling
leukocyte in two sequent images
(A) Begin of rolling. (B) End of
rolling. The bright white dot in the upper part of the images was the static
adhering leukocytes. While the bright white bar in the right part of the images
was the fast rolling leukocyte. The displacement dL of the leukocyte was
measured using the image processing method, and dt is 20 ms. The velocity of
the fast rolling downwards leukocyte here was calculated as about 600 μm/s.
Shear stress, 0.71 dynes/cm2; Scale bars, 50 μm.
Fig.4 A slow
rolling leukocyte in two sequent images
(A) Begin of rolling. (B) End of rolling.
The rolling direction was downwards. The displacement dL was shown in
the figure. The bright white dot representing the slow rolling leukocyte was
moving down. The interval of time of these two sequent images was 7 s. The
rolling velocity was about 4.21 μm/s. Shear stress, 0.71 dynes/cm2;
Scale bars, 50 μm.
In which dL
is the displacement of the leukocyte in μm and dt is the time interval of the
movement. To properly measuring the rolling velocity, different image sampling
intervals were used for the slow and fast rolling leukocytes. For the fast
rolling leukocytes, frame images were captured every 5 s to get totally 120
frames while the perfusion process was replayed off-line for every experiment.
A frame of image was consisted of two images of sequent fields with interval (dt)
of 20 ms, and the distance (dL) between the profiles of the same fast
rolling leukocyte in these two sequent fields could be measured by the
custom-made software developed for this research. Thus, the instantaneous
velocities of the fast rolling leukocytes in the captured images were
efficiently calculated. While for slow rolling leukocyte, a series of images
with the interval of 200 ms were captured to calculate the rolling velocity.
1.6 Statistical
analysis
The experiments
were repeated for 3 times. Data were reported as the x±s.
Student’s t-test was used in statistical analysis. A level of P<0.05
was considered as statistical significance.
2 Results
The leukocytes
in experiments could be separated into three kinds according to their moving
modes: fast rolling, slow rolling, tethered or firm adhering.
Some leukocytes
were observed rolling fast on the HUVECs monolayer (Fig.3). Clearly, these fast
rolling leukocytes had weak interaction with the adhesion molecules on HUVECs.
If no further interaction occurred, these cells would return to free flowing
class. Otherwise, it would become a slow rolling one (Fig.4), being obviously
tethered or moving rather slowly. The “tethering” leukocytes might ultimately
lead to adhering or slow rolling process. For all frames, those fast rolling
leukocytes were represented by the bright white bars. While the “tethering” or
slow rolling process was observed when the video was replayed.
In this
research, LPS was used to activate the adhesion interaction and DXM was used as
an anti-inflammatory reagent. (1) After PBS perfusion, the number of the static
adhering leukocytes was counted (Fig.5). It was clearly indicated that there
were 23.7-fold more leukocytes firmly adhered on the HUVECs treated with LPS (4
mg/L) than the control group (P<0.01). This effect of LPS was
significantly inhibited by decrease to 39.9% when DXM (50 mg/L) was
administrated 20 min before the stimulation of LPS (P<0.05). (2) The
number of the slow rolling leukocytes in one field of about 0.1 mm2 of every
experiment was also counted. And it was significantly (P<0.05) increased by
4.1-fold on the LPS-treated HUVEC monolayer compared with that of the control
group (Fig.5). When HUVECs were treated with LPS+DXM, the number of slow
rolling leukocytes was decreased to 70.6% compared with the LPS group, although
this decrease was not significant (P>0.05).
Fig.5 Number of
the static adhering and slow rolling leukocytes on the surface of 0.1 mm2
HUVECs monolayer (n=3)
Comparisons
were made in staticadhering or slow rolling respectively. **P<0.01
vs. control; #P<0.05 vs. LPS; *P<0.05 vs.
control.
As to the rolling leukocytes, their
velocities were also affected by LPS (Fig.6), while DXM effectively inhibited
LPS-induced adhesion interactions. For the slow rolling leukocytes [Fig.6(A)],
the velocity on the LPS-treated HUVECs was significantly reduced by 25.6%
compared with that of the control group (P<0.05). While in the group
of HUVECs treated with LPS+DXM, the velocity of the slow rolling leukocytes was
115.3% faster than that of the LPS group (P<0.01). Similarly, LPS
also had such effect on the velocity of fast rolling leukocytes [Fig.6(B)]. The
fast rolling velocity was significantly reduced by 26.1% on the HUVECs treated
with LPS compared to that of the control group (P<0.05). However, the
LPS-induced decrease of the fast rolling velocity was not significantly
inhibited by DXM (P>0.05).
Fig.6 Velocity of
the rolling leukocytes (n=3)
*P<0.05 vs. control; ##P<0.01 vs. LPS.
3 Discussion
Previously
studies showed that leukocytes rolling velocities in vivo varied widely, and
different adhesion molecules mediated rolling at different velocities[19,20].
It implied that the change of the adhering leukocytes induced by various
stimulus might attribute to variation of rolling velocity, which was modulated
by adhesion molecules.
In this study
the leukocytes in fast or slow rolling could be distinguished by the characters
of the acquired images, so that to obtain the detailed information of the
rolling leukocytes and had a deep view into the mechanisms of the cell
adhesion.
It was shown
that LPS effectively activated the adhesion function of the endothelial cells
by increasing the adhesion number and reducing the rolling velocity of
leukocytes. This result was consistent with the previous researches indicating
that LPS could induce the high expression of some adhesion molecules on
endothelial cells[3,22]. It was also indicated that DXM effectively inhibited
the LPS-induced activation of the adhesion function of endothelial cells, by
reducing the number of adhering leukocytes and increasing the slow rolling
velocity. This result was also in agreement with the fact that DXM had been
used as an anti-inflammatory drug and its in vivo effects and mechanisms
were also greatly studied on rats[23,24].
Furthermore, the
adhesion quantification method in this research was simple and useful to study
leukocytes adhesion to HUVECs and the mechanisms involved. Based on the results
obtained in the present study, future experiments could be conducted to study
the mechanisms of different shear stress and chemical stimulus on the adhesion
properties of endothelial cells, which were very important factors affecting
some physiological and pathological process, such as inflammation, ischemia
reperfusion injury and arteriosclerosis, etc.
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Received: January 30, 2003 Accepted: March 18, 2003
This work was supported by Zhejiang Provincial
Key Laboratory Chinese Medicine Screening, Exploitation & Medicinal
Effectiveness Appraise For Cardio-Cerebral Vascular & Nervous System and
the Key Laboratory for Biomedical Engineering of Ministry of Education of China
*Corresponding author: Tel,
86-571-87951091; Fax, 86-571-87951676; e-mail, [email protected]