ISSN 0582-9879 ACTA BIOCHIMICA et BIOPHYSICA SINICA 2003, 35(6): 567-572 CN 31-1300/Q
Dynamic Investigation of Leukocyte-Endothelial Cell Adhesion Interaction under Fluid Shear Stress in Vitro
( 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.. 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. This interaction might be modulated by the expression of diverse adhesion molecules and their receptors.
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. 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. 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.
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. 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; 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.
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.
1.1 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., 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.
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 as following [Formula (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.
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).
(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.
(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.
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.
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
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