Effects of recirculating flow on U-937 cell adhesion
to human umbilical vein endothelial cells
KEVIN M. BARBER, AARON PINERO, AND GEORGE A. TRUSKEY
Department of Biomedical Engineering, Duke University, Durham, North Carolina 27708-0281
monocyte; atherosclerosis; hemodynamics
to arterial endothelium at
lesion-prone sites and subsequent transmigration and
formation of lipid-filled macrophages are early events
in atherosclerosis (8, 15, 23, 36). The localization of
monocyte binding may, in part, depend on the local fluid
dynamics. Hemodynamics may affect the transport of
monocytes directly to the endothelium and the subsequent adherence of monocytes to the endothelium.
Hemodynamic behavior at arterial bifurcations may
result in monocytes impinging against the endothelium, leading to increased monocyte adhesion at lesionprone sites. Furthermore, the local fluid dynamics may
produce focal upregulation of adhesion proteins (37).
In vitro investigations of the behavior of blood cells
and microspheres in recirculating flow have demonstrated that fluid dynamics can affect cell adhesion to
biologically inert surfaces and collagen-coated surfaces
(16, 17, 29). For example, adhesion of platelets to
collagen-coated glass in an annular vortex was highest
within the vortex and downstream of the point of flow
reattachment and minimal at the reattachment site
(16, 17). U-937 cell rolling velocities on a silicone
surface in recirculating flow in a sudden expansion
varied linearly with wall shear stress, whereas particle
residence times and cell adhesion varied inversely with
wall shear stress (29).
Because monocytes play a major role in atherogenesis and the localization of atherosclerosis is linked to
THE ADHESION OF MONOCYTES
hemodynamic effects, investigations of monocytes interacting with endothelium in recirculating flow may
provide insight into the factors that influence monocyte
adhesion in vivo. The purpose of this investigation was
to examine the effects of steady recirculating flow on
the frequency of U-937 cell arrests to human umbilical
vein endothelial cells (HUVEC) in a sudden-expansion
flow chamber. Experimental variables included flow
rate, concentration of U-937 cells, and HUVEC activation by tumor necrosis factor (TNF)-a. Predictions of
trajectories of spherical particles in recirculating flow
under the experimental conditions were obtained using
a computational model, and the simulations were compared with the experimental results.
MATERIALS AND METHODS
Endothelial cell culture. HUVEC were isolated by collagenase treatment of human umbilical cord veins (9, 14) and
characterized as endothelial cells by acetylated low-density
lipoprotein uptake, factor VIII expression, and cobblestone
morphology. Cell cultures were maintained in medium 199
(M199) with Earle’s salts (Sigma Chemical, St. Louis, MO)
supplemented with 10% heat-inactivated fetal bovine serum
(FBS) (Sigma), 1% antibiotic-antimycotic solution (1003 stock)
(Sigma), 2 mM L-glutamine, 120 µg/ml heparin (Sigma), and
100 µg/ml endothelial cell growth supplement (Collaborative
Biomedical, Bedford, MA). Cells were grown in tissue culture
flasks (Corning, Corning, NY) coated with 0.1% porcine
gelatin (Sigma) in M199, and confluent monolayers were split
1:4 using 0.05% trypsin-EDTA (Sigma). For adhesion assays,
HUVEC (passage 2–5) were plated on gelatin-coated glass
microscope slides (Fisher Scientific, Medford, MA) and grown
to confluency. After splitting, HUVEC reached confluency in
2–4 days.
U-937 cell culture. U-937 cells were obtained from American Type Culture Collection (Rockville, MD) and fed RPMI
1640 medium (Sigma) supplemented with 10% heat-inactivated FBS, 1% antibiotic-antimycotic solution (1003 stock),
and 2 mM L-glutamine. The cells were grown in either tissue
culture flasks or spinner flasks (Corning) and split every 3–5
days to maintain a cell concentration of 1.0–2.0 3 106
cells/ml. For adhesion assays, U-937 cells were centrifuged
and resuspended at concentrations of either 105 or 106
cells/ml in M199 containing 15 mM HEPES to maintain pH.
From experimental measurements, the mean diameter of
U-937 cells is 13.5 6 2.1 (SD) µm (n 5 50), whereas the mean
cell diameter reported in the literature is 12.5 6 2.2 µm (32).
The mass density of monocytes is 1.07 g/cm3 (6, 30).
Sudden-expansion flow chamber. A sudden-expansion flow
chamber (Fig. 1) was used in this investigation to study the
effects of recirculating flow on interactions between U-937
cells and unactivated or TNF-a-activated HUVEC (34). The
sudden expansion creates a region of flow recirculation with
flow reattachment occurring downstream from the expansion
(Fig. 1). Downstream of the reattachment point, flow becomes
fully developed. Flow through the chamber can be characterized in terms of the Reynolds number (Re) and the expansion
ratio H/h, where h is the chamber height upstream of the
0363-6135/98 $5.00 Copyright r 1998 the American Physiological Society
H591
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Barber, Kevin M., Aaron Pinero, and George A. Truskey. Effects of recirculating flow on U-937 cell adhesion to
human umbilical vein endothelial cells. Am. J. Physiol. 275
(Heart Circ. Physiol. 44): H591–H599, 1998.—We used a
sudden-expansion flow chamber to examine U-937 cell adhesion to unactivated and tumor necrosis factor (TNF)-aactivated human umbilical vein endothelial cells (HUVEC) in
recirculating flow. For both unactivated and TNF-a-activated
HUVEC, U-937 cells exhibited transient arrests within ,150
µm of flow reattachment. Few arrests occurred directly at the
reattachment site. U-937 cell rolling was not observed. At all
other locations within the recirculation zone, U-937 cells did
not exhibit transient arrests or rolling. TNF-a activation
increased the frequency of U-937 cell arrests near reattachment but did not change the median arrest duration. Numerically simulated cell trajectories failed to predict attachment
near the reattachment point. Deviations between experiment
and theory may result from the nonspherical shape and
deformability of U-937 cells. These results demonstrate that
U-937 cell transient arrests occur preferentially in the vicinity of the reattachment point in recirculating flow. Possible
mechanisms for adhesion include low shear stress, curved
streamlines, fluid velocity components normal to the endothelium, and formation of larger contact areas.
H592
U-937 CELL ADHESION TO ENDOTHELIUM IN RECIRCULATING FLOW
expansion and H is the chamber height downstream. The
Reynolds number is defined as
Re 5
2rf Q
µ(w 1 H)
(1)
where rf is the fluid density, µ is the fluid viscosity, Q is the
volumetric flow rate, and w is the chamber width. This
sudden-expansion flow chamber design was previously characterized by two- and three-dimensional numerical flow
simulations and experimental measurements of reattachment distances across the width of the chamber by flow
visualization using light-reflecting particles (34).
For the current study the flow chamber dimensions were
modified using computational fluid dynamics to maximize the
size of the recirculation region while the volumetric flow rate
was minimized. The resulting flow chamber dimensions,
obtained using a 0.1016-cm-thick Silastic gasket, were h 5
0.0123 cm, H 5 0.1399 cm, w/H 5 13.6, and H/h 5 11.4.
Flow of M199 media through the chamber was generated
using either a 60-ml syringe mounted on a syringe pump
(Orion Research M362, Boston, MA) or a gravity feed system.
A 500-ml cell suspension flowed by gravity through an
adjustable valve into a constant-height pressure head and
then into the flow chamber. The height of the pressure head
was adjusted to obtain the desired steady volumetric flow
rate. The maximum flow rate obtained using this gravity feed
system was ,20 ml/min. Higher flow rates were obtained
using the syringe pump.
Flow assay. Confluent HUVEC monolayers on glass slides
either remained unactivated or were activated by incubation
at 37°C for 4 h with M199 containing 10% FBS and 100 U/ml
TNF-a. After the incubation, the HUVEC monolayers were
rinsed with M199 containing 10% FBS and mounted in the
sudden-expansion flow chamber. The flow chamber was assembled and mounted on an inverted microscope (Nikon
Diaphot-TMD) with a 320 phase-contrast objective. A heat
lamp maintained the temperature at 37°C. The monolayer
was perfused with M199 containing 10% FBS and 15 mM
HEPES for 10–15 min at a flow rate of 10 ml/min to rinse the
cells and verify that the monolayer was intact.
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Fig. 1. Schematic of sudden-expansion flow chamber (not drawn to
scale). H, chamber height downstream of expansion; h, chamber
height upstream of expansion; S, gasket thickness; x, coordinate
parallel to glass slide; y, coordinate normal to glass slide.
HUVEC were then perfused with 105 or 106 U-937 cells/ml
at flow rates of 12, 20, or 30 ml/min (Re 5 24, 40, and 60,
respectively). A typical physiological concentration of monocytes in the blood is ,4.6 3 105 monocytes/ml (6). The region
of flow reattachment was identified within 30 s to 1 min after
initiation of perfusion. Immediately after this, the motions of
U-937 cells within the recirculation region and in the vicinity
of the reattachment location were recorded on videotape for
times ranging from 2 to 5 min, using a video camera (MTI
PA-70, Michigan City, IN) and video recorder (RCA TC3930,
Lancaster, PA) equipped with a time-date generator (VTG-33,
FOR.A, Cypress, CA). The duration of the experiment depended on the flow rate. Using the 320 objective, the field of
view was 640 µm in the direction of flow by 480 µm wide.
Within the recirculation region and downstream of reattachment, cells were tracked to determine if any exhibited rolling
or transient arrests. At the reattachment location, within a
field of view encompassing an area 440 µm wide and ,300 µm
upstream of the reattachment to 300 µm downstream of the
reattachment, U-937 cell motions were analyzed to quantify
the number of transient arrests exhibited. For each condition,
an arrest frequency (arrests · mm22 · min21 ) was calculated.
The duration of each arrest was also measured, and the
median arrest duration was determined for each condition.
The reattachment distance from the expansion site was
measured for each experiment.
Shear flow assays were conducted in a variable-height flow
chamber that exposes U-937 cells to laminar shear flow over a
HUVEC monolayer (40). The durations of transient arrests of
U-937 cells to TNF-a-activated HUVEC were measured at
shear stresses ranging from 0.043 to 0.774 dyn/cm2 (3). Flow
of U-937 cell suspensions through the flow chamber was
generated using a 60-ml syringe mounted on a syringe pump
(Orion Research).
Statistics. Recirculating flow experiments at each flow rate
and U-937 cell concentration were conducted in triplicate for
TNF-a-activated HUVEC and twice for unactivated HUVEC.
To calculate statistical significance between different experimental conditions, we analyzed data by Student’s t-test and
ANOVA with Tukey-Kramer multiple comparisons posttest.
Statistical calculations were performed using INSTAT (Version 2.00, GraphPad Software), and P values #0.05 were
considered significant.
Numerical simulations of flow and particle trajectories. As
previously described (34), flow through the sudden-expansion
flow chamber was numerically simulated using a finite element model. The Fluid Dynamics Analysis Package (FIDAP
version 7.6, Fluid Dynamics International, Evanston, IL) was
used to numerically solve the Navier-Stokes equations for
two-dimensional steady flow of an incompressible Newtonian
fluid in conjunction with the continuity equation. These
simulations provided estimates for the wall shear stress (tw )
along the lower wall of the flow chamber, as well as estimates
for the size of the recirculation zone from the expansion to the
reattachment site (where tw 5 0 at the lower wall). This
approach was previously validated (34) using numerical
solutions of the two- and three-dimensional forms of the
Navier-Stokes equations to determine the wall shear stress
distribution and predict the location of reattachment.
After the finite element flow simulations were performed
and the flow field was obtained, U-937 cell trajectories were
calculated by treating the U-937 cell as a spherical particle
with a mean particle diameter (Dp ) of 13.5 µm based on
experimental measurements and a particle density (rp ) of
1.07 g/cm3 (6, 30). We used a Lagrangian approach to calculate dispersed two-phase flow, in which the dispersed phase
consisted of an infinitely dilute stream of particles moving
U-937 CELL ADHESION TO ENDOTHELIUM IN RECIRCULATING FLOW
H593
through the carrier fluid (7). This approach neglected particleparticle interactions, but it was valid for these simulations
because dilute solutions of U-937 cells at either 105 or 106
cells/ml yield volume fractions of 0.00013 and 0.0013, respectively, assuming a U-937 cell diameter of 13.5 µm.
The following force balance governs the trajectory of a
particle (7)
dup
dt
5
(uf 2 up)
TR
1
(rp 2 rf)
rp
g1f
(2)
TR 5
4rp D2p
(3)
3µCDRep
where Rep is the particle Reynolds number given by (7)
Rep 5
Dp 0 uf 2 up 0 rf
(4)
µ
and CD is the particle drag coefficient given by the power-law
model (7)
CD 5
24
Rep
3
4
1 1 0.15(Rep)0.687
(5)
For a flow rate of 30 ml/min, the maximum value of Rep was
5.0, resulting in a minimum value of CD equal to ,7.0 and a
minimum value of TR equal to 8.6 3 1026 s.
For a rigid sphere translating far from any planar boundaries, f in Eq. 2 equals zero. If the sphere is in the vicinity of a
plane wall, f corrects for drag effects due to sphere-wall
interactions that occur (5, 10, 11). These force corrections
were calculated using the methods of Goldman et al. (10, 11)
and Brenner (5) and were validated by comparison with
published results for the trajectory of a particle in channel
flow (38).
To determine if any particles became entrained in the
recirculating flow region, particles were introduced a distance
of 0.1 cm upstream of the sudden expansion. These particles
were placed at heights of 7.75–16.75 µm, measured from the
center of the particle, above the lower surface of the inlet.
Also, particles were introduced within the recirculating flow
region at various locations along adjacent streamlines that
diverged at the reattachment point, to determine how closely
the particles approached the lower surface upstream and
downstream of the reattachment location. An implicit solver
numerically integrated Eq. 2 to determine the trajectory of
each particle.
RESULTS
Characterization of flow through sudden expansion.
Numerical simulations of two-dimensional steady flow
through the sudden expansion with H/h 5 11.4 were
performed for flow rates of 12, 20, and 30 ml/min (Re 5
24, 40, and 60, respectively). The sudden expansion
produced recirculation regions with reattachment distances that varied, as a function of flow rate, between
Fig. 2. A: wall shear stress vs. distance from sudden expansion. B:
wall shear stress vs. distance from reattachment. Re, Reynolds
number.
approximately 1,000 and 2,500 µm. Figure 2A shows
predictions for the distributions of tw along the lower
wall. Wall shear stresses ranged from 23 to 6 dyn/cm2
within the recirculation region, with downstream wall
shear stresses from 20.28 to 20.69 dyn/cm2. In the
aorta and large arteries, physiological mean wall shear
stress magnitudes range from approximately 0.5 to 10
dyn/cm2, and instantaneous values can vary from zero
to as high as 200 dyn/cm2 (27, 35). Mean physiological
values of Re range from approximately 200 to 6,000 (27,
35). For the in vitro assays, we could not match exactly
the physiological range of the wall shear stress and Re
due to the excessive flow rates required. Figure 2B
shows an expanded view of the distributions of wall
shear stresses along the lower wall for the region in the
vicinity of reattachment that was analyzed to quantify
transient arrests exhibited by U-937 cells.
U-937 cell behavior in recirculating flow. Upstream of
the reattachment, U-937 cells became entrained in the
recirculating flow. At the reattachment location, the
paths of U-937 cells in the fluid flow diverged, with
some U-937 cells traveling downstream toward the
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where up is the particle velocity, uf is the fluid velocity, g is the
acceleration due to gravity, f represents forces acting on the
particle, and TR is the particle relaxation time. The first term
on the righthand side in Eq. 2 is the generalized drag on the
particle, the second term is the buoyancy force, and the third
term corrects for drag effects due to particle-wall interactions
that occur when a sphere is in the vicinity of a plane surface
(5, 10, 11). The particle relaxation time is (7)
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U-937 CELL ADHESION TO ENDOTHELIUM IN RECIRCULATING FLOW
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fully developed flow region and others traveling upstream into the recirculating flow region. U-937 cells
exhibited transient arrests to the HUVEC monolayer
immediately upstream and downstream of the reattachment. These transient arrests occurred as individual
events, with no leukocyte-leukocyte interactions. U-937
cell rolling was not observed.
To demonstrate the observed transient arrest behavior, Fig. 3 shows three images, 0.4 s apart, in the
reattachment region for TNF-a-activated HUVEC at a
flow rate of 20 ml/min (Re 5 40). The vertical dashed
line in each image indicates the line of reattachment.
The black arrow in each image indicates a U-937 cell
that translated downstream from the reattachment
line (Fig. 3A), transiently arrested ,48 µm downstream from the reattachment for 0.63 s (Fig. 3B), and
then detached and accelerated downstream (Fig. 3C).
The white arrow in each image indicates a U-937 cell
that accelerated downstream away from the reattachment region without transiently arresting.
From a sample of 145 transient arrests at all of the
experimental conditions studied, 126 (87%) occurred
within 150 µm of the reattachment line. Of these 145
arrests, 120 (83%) occurred at least 30 µm (approximately 1 or 2 endothelial cell widths) away from the
measured reattachment line. Thus few arrests occurred directly on the line of reattachment, where the
wall shear stress is zero. Of this sample of 145 arrests,
74 occurred upstream of the reattachment line and 71
occurred downstream of the reattachment line. Therefore, the cells did not exhibit a preference for arresting
upstream or downstream of the reattachment line.
Within the recirculation zone upstream of the reattachment region, U-937 cells exhibited very few transient arrests and no rolling behavior. Instead, U-937
cells translated across the endothelial monolayer, accelerating as they moved upstream from the reattachment
toward the recirculation zone where the wall shear
stress was a maximum, until they attained relatively
high translational velocities. As the U-937 cells approached the expansion site, they moved away from the
endothelial monolayer and became entrained in the
recirculating flow.
Frequencies of cell arrests in the vicinity of reattachment. Results from in vitro recirculating flow experiments indicated that U-937 cells exhibited significantly
higher arrest frequencies in the vicinity of reattachment to TNF-a-activated HUVEC compared with unactivated HUVEC (Fig. 4). These higher arrest frequencies occurred at both U-937 cell concentrations and all
three flow rates except for the condition of 105 U-937
cells/ml at a flow rate of 20 ml/min. Increasing the
U-937 cell concentration from 105 to 106 cells/ml resulted in a roughly two- to threefold increase in arrest
frequencies to TNF-a-activated HUVEC, but no increase occurred for unactivated HUVEC. For TNF-aactivated HUVEC at a concentration of 106 U-937
cells/ml, the cell arrest frequency at a flow rate of 20
ml/min was significantly higher than at 12 or 30
ml/min. However, for TNF-a-activated HUVEC at a
concentration of 105 U-937 cells/ml, flow rate did not
Fig. 3. Three images, 0.4 s apart, in reattachment region for tumor
necrosis factor (TNF)-a-activated human umbilical vein endothelial
cells (HUVEC) at flow rate of 20 ml/min (Re 5 40). Vertical dashed
line in each image indicates line of reattachment. Black arrow
indicates a U-937 cell that translated downstream from reattachment line (A), transiently arrested ,48 µm downstream from reattachment for 0.63 s (B), and then detached and accelerated downstream
(C). White arrow in A–C indicates a U-937 cell that accelerated
downstream away from reattachment region without transiently
arresting.
U-937 CELL ADHESION TO ENDOTHELIUM IN RECIRCULATING FLOW
significantly affect arrest frequencies. For unactivated
HUVEC, flow rate did not affect arrest frequencies.
Predicted U-937 cell trajectories in recirculating flow.
Using results from the numerical simulations for the
flow field, U-937 cell trajectories were calculated at
each experimental flow rate by assuming that the
U-937 cell is a spherical particle. Figure 5A shows
predicted trajectories at 30 ml/min for particles seeded
0.1 cm upstream of the sudden expansion at distances
of 7.75–16.75 µm above the lower surface of the inlet.
At each flow rate of 12, 20, and 30 ml/min, the simulations predicted that none of the particles became entrained in the recirculating flow or encountered the
lower wall of the flow chamber. Instead, all of the
particles traveled downstream beyond the reattachment point.
Experimental results clearly showed that U-937 cells
transiently arrested to the endothelial monolayer in
the vicinity of reattachment and also became entrained
in the recirculating flow. Therefore, in the numerical
simulations, particles were also seeded within the
recirculation zone near the reattachment point on
adjacent streamlines that diverged at the reattachment site, to determine how closely the particles approached the lower surface. Figure 5, B and C, shows
examples of the resulting particle trajectories at 30
ml/min. Particles seeded on streamlines that curved
back upstream became entrained in the recirculating
flow. These particles gradually spiraled outward and
left the recirculation zone (Fig. 5B). This outward
migration is similar to experimental observations by
Karino and Goldsmith (17) for the motions of red blood
cells, platelets, and latex spheres in an axisymmetric
annular expansion. For each flow rate, particles approached no closer than ,10 µm to the lower wall (Fig.
5C). This was true for all flow rates studied. Because
typical bond lengths are several orders of magnitude
smaller than this approach distance (1), none of these
particles in the simulations came close enough to the
lower wall to allow bond formation. Thus the numerical
simulations of U-937 cell trajectories in flow through
the sudden expansion predicted that U-937 cells treated
as spherical particles do not become entrained in the
recirculating flow when seeded upstream of the expansion. Spherical particles seeded near the reattachment
on diverging streamlines did not contact the lower wall
in the flow chamber.
Median arrest duration. As expected, increasing the
U-937 cell density from 105 to 106 cells/ml did not cause
a significant change in arrest duration. Despite a
significant increase in U-937 cell attachment (Fig. 4),
TNF-a activation did not cause significant changes in
median arrest duration for U-937 cell transient arrests
in the vicinity of reattachment (Fig. 6A). For TNF-aactivated HUVEC at both 105 and 106 cells/ml, the
median arrest duration at a flow rate of 30 ml/min was
significantly less than the median arrest duration at 12
ml/min (P 5 0.013 for 105 cells/ml and P 5 0.037 for 106
cells/ml). Flow rate did not affect the median arrest
durations for unactivated HUVEC.
For TNF-a-treated HUVEC in shear flow, U-937 cells
exhibited a shear stress-dependent tethering behavior
(3). At shear stresses of 0.043–0.172 dyn/cm2, U-937
cells exhibited transient cell arrests. The fractions of
U-937 cells that arrest and the frequencies of U-937 cell
Fig. 5. Predicted particle trajectories in sudden expansion for a flow
rate of 30 ml/min. A: particles seeded 0.1 cm upstream of sudden
expansion. B: particles seeded within recirculation zone upstream
and downstream of reattachment point. C: particles seeded near
reattachment point on adjacent streamlines.
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Fig. 4. Arrest frequencies vs. flow rate for unactivated and TNF-aactivated HUVEC at U-937 cell concentrations of 105 and 106
cells/ml. * P , 0.05 vs. unactivated HUVEC at same cell density and
flow rate. ** P , 0.05 vs. TNF-a-activated HUVEC at same flow rate
and 105 U-937 cells/ml. *** P , 0.05 vs. TNF-a-activated HUVEC at
flow rate 5 12 and 30 ml/min and 106 U-937 cells/ml.
H595
H596
U-937 CELL ADHESION TO ENDOTHELIUM IN RECIRCULATING FLOW
DISCUSSION
Fig. 6. A: median arrest duration vs. flow rate for unactivated and
TNF-a-activated HUVEC at U-937 cell concentrations of 105 and 106
cells/ml. B: shear flow median arrest durations for TNF-a-activated
HUVEC plotted vs. wall shear stress (tw ) for shear flow compared
with recirculating flow median arrest durations for TNF-a-activated
HUVEC plotted vs. root mean square value of wall shear stress
(tw-rms ).
arrests decreased with increasing shear stresses. However, at shear stresses . 0.172 dyn/cm2, U-937 cells
exhibited rolling across the endothelium in an erratic
fashion at nonuniform velocities that were much lower
than the hydrodynamic velocity, with very few transient arrests.
Although the wall shear stress does not vary across
the field of view in shear flow, large shear stress
gradients occur in the reattachment region in recirculating flow, with shear stress changing from positive to
negative at the reattachment location. To compare the
median arrest durations in shear flow with the median
arrest durations for the transient arrests in recirculating flow, a root-mean-square (rms) value of the wall
shear stress, tw-rms, was calculated for each recirculating flow condition from the wall shear stress distributions in the vicinity of reattachment (shown in Fig. 2B)
tw-rms 5
Îo
1
n
n i51
2
tw
i
(6)
The value of tw-rms accounts for differences in the sign of
the shear stress and represents the average magnitude
of the shear stress exerted on U-937 cells near flow
This study represents the first report of monocytic
cell adhesion to activated endothelium in recirculating
flow conditions similar to mean flow conditions in
arteries. The factors that govern U-937 cell adhesion to
HUVEC in recirculating flow include transport effects
such as diffusion and convection, the intrinsic forward
and reverse rate coefficients for bond formation and
dissociation, the numbers and types of receptors ex-
Fig. 7. Fraction of U-937 cells remaining bound as a function of time
after initiation of arrest, calculated from measured arrest durations
for unactivated HUVEC in recirculating flow at tw-rms 5 0.162
dyn/cm2, TNF-a-activated HUVEC in recirculating flow at tw-rms 5
0.162 dyn/cm2, and TNF-a-activated HUVEC in shear flow at tw 5
0.172 dyn/cm2. The 2 straight lines represent fits of single exponential model to cell arrest durations for each recirculating flow case, and
curved line represents fit of biexponential model to cell arrest
durations for shear flow case.
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reattachment. As Fig. 6B shows for TNF-a-treated
HUVEC, the recirculating flow median arrest durations at tw-rms 5 0.162 dyn/cm2 were not significantly
different from the shear flow median arrest durations
at shear stresses of 0.129–0.172 dyn/cm2. However, the
recirculating flow median arrest durations decreased
by ,50% as tw-rms increased by over a factor of 10.
Furthermore, under shear flow very few transient
arrests were seen at shear stresses .0.172 dyn/cm2,
indicated by the dashed line in Fig. 6B.
Although the recirculating flow median arrest duration at tw-rms 5 0.162 dyn/cm2 was not significantly
different from the shear flow median arrest duration at
a shear stress of 0.172 dyn/cm2, the fractions of cells
remaining bound vs. time after initiation of arrest,
calculated from measured arrest durations, are very
different (Fig. 7). In shear flow, the cell arrest durations
are best fit by a biexponential model (indicated by the
curved line in Fig. 7), which suggests that cells bind by
a single bond to two classes of receptors with very
different dissociation constants (3). However, in recirculating flow, the cell arrest durations are best fit by a
single exponential model (indicated by the straight
lines in Fig. 7), which suggests that the cells bind to a
single receptor (1). This difference in bond lifetimes for
cells adherent in shear flow and near flow reattachment suggests that receptors that detach slowly cannot
form near flow reattachment.
U-937 CELL ADHESION TO ENDOTHELIUM IN RECIRCULATING FLOW
k5
kf NR
ġ
(7)
where NR is the endothelial surface receptor density
and kf is the overall forward reaction rate coefficient,
assuming that the number of counterreceptors on the
U-937 cell is very large. The coefficient kf depends on
fluid transport effects and the intrinsic rate constants
for bond formation. The parameter k is a measure of the
ratio of the contact time to the time for binding. If k ..
1, then the probability of binding is very high, whereas
if k ,, 1, then the binding probability is very small. In
shear flow, the adhesion of rat basophilic leukemia cells
to antigen-coated substrates increased as the shear
rate decreased, and this increased adhesion scales with
k in the simple diffusion-limited case (33). Expressions
for diffusion-limited values of kf exist for the shear flow
case; however, recirculating flow is more complicated
than shear flow because of the functional dependence of
kf on the flow field (33).
The parameter k is sensitive to adhesion receptor
levels on endothelial cells, flow rate, and location of flow
reattachment. TNF-a treatment induces HUVEC to
upregulate expression of adhesion receptors to which
U-937 cells may bind. This results in an increase in NR,
due to the presence of more receptors on the HUVEC
monolayer, increasing the value of k and causing a
higher frequency of arrests compared with unactivated
HUVEC.
The frequencies of U-937 cell arrests (Fig. 4) show the
effect of increasing flow rate on the magnitude of k. We
speculate that increasing the flow rate causes more
rapid delivery of U-937 cells to the endothelial monolayer. As a result, kf increases as the adhesion shifts
from transport limited to reaction limited, and k increases. At the highest flow rate, kf reaches a maximum
while ġ continues to increase, so that k decreases. The
overall effect is an increase and then a decrease in k as
the flow rate increases, resulting in the variation in the
frequency of U-937 cell arrests to TNF-a-activated
HUVEC at 106 U-937 cells/ml (Fig. 4).
In addition to the numbers and types of receptors
expressed by the endothelium, U-937 cell adhesion
depends on the nature of the flow itself. U-937 cells
transiently arrested within 150 µm upstream and
downstream of the reattachment line, but few U-937
cells arrested directly at the reattachment line. This is
similar to previous studies of platelet adhesion to
collagen-coated glass in an annular vortex (16) and
U-937 cell adhesion to a silicone wall in a sudden
expansion (29). At the reattachment location, the shear
rate ġ is zero and the particle residence time is high.
However, few cells may arrest due to a very low flux of
U-937 cells along fluid streamlines with low velocities
that reach the reattachment location (16, 17), resulting
in a low overall forward reaction rate kf and a low value
of k. Immediately upstream and downstream of the
reattachment site, ġ increases but the flux of cells to the
endothelium also increases so that k reaches a maximum, resulting in cell arrests. Farther away from the
reattachment region within the recirculation zone, k is
small because of higher values of ġ; thus very little
binding occurs.
At shear rates .0.172 dyn/cm2, few measurable
transient arrests of U-937 cells occur in shear flow (3).
Nevertheless, U-937 cells adhere near the reattachment point in recirculating flow. Unlike those in shear
flow, U-937 cells in recirculating flow are exposed to
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pressed by the HUVEC and U-937 cells, and the local
wall shear rate ġ.
Unactivated HUVEC constitutively express low levels of intercellular adhesion molecule-1 (ICAM-1) (4,
18, 39). TNF-a treatment induces HUVEC to upregulate expression of vascular cell adhesion molecule-1
(VCAM-1), ICAM-1, E-selectin, and P-selectin (20, 21,
31). U-937 cells express the b1 and b2 integrins very
late antigen-4 (VLA-4) and lymphocyte functionassociated antigen-1 (LFA-1), as well as sialyl Lewisx
counterreceptors to E-selectin and P-selectin (28). However, U-937 cells lack L-selectin (22) and may not
express P-selectin glycoprotein ligand-1 (PSGL-1) as a
counterreceptor to P-selectin. Unfortunately, we could
not use isolated blood monocytes in our cell adhesion
assays. Each experiment required between 12 million
and 120 million suspended cells because of the high
flow rates necessary to create the recirculating flow. We
selected the U-937 cell as the most appropriate monocytic cell line to use in our experiments. Due to differences in types and numbers of receptors expressed
between U-937 cells and monocytes, isolated monocytes
may yield results different from those we obtained
using U-937 cells.
In shear flow, the predominant pattern of monocyte
adhesion is immediate arrest on initial contact with the
endothelium, with a few monocytes rolling before and
after firm arrest (19, 21). Previous studies of monocyte
adhesion to TNF-a or IL-4-activated HUVEC have
implicated L-selectin as the major molecule that mediates initial arrest and rolling under flow (21, 22).
However, a more recent study of monocyte adhesion to
IL-1b-activated HUVEC showed that monocytes may
adhere via three independent pathways: 1) L-selectin,
2) VLA-4/VCAM-1, and 3) a sialyl-Lewisx pathway that
may involve P-selectin, E-selectin, or some unidentified
receptor (19). Thus U-937 cells may bind to HUVEC via
several different adhesion molecules under recirculating flow conditions. In support of this, our recent shear
flow assays indicate that U-937 cell transient arrests to
unactivated HUVEC can be blocked by monoclonal
antibodies (MAb) to ICAM-1, whereas U-937 cell transient arrests to TNF-a-activated HUVEC can be blocked
by MAb to VCAM-1, ICAM-1 and E-selectin (3). Flow
cytometry also revealed the presence of these adhesion
molecules after TNF-a treatment of the endothelium.
The absence of L-selectin may not be critical for U-937
cell arrests to occur, but it may affect the numbers of
cells that arrest.
The complex effects of fluid transport and receptorligand binding on U-937 cell adhesion can be understood in terms of a dimensionless forward reaction rate
(12, 13, 33)
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H598
U-937 CELL ADHESION TO ENDOTHELIUM IN RECIRCULATING FLOW
The mechanisms outlined here that affect the localization of U-937 cell transient arrests in these experiments may also play a role in monocyte adhesion in
arteries. Our results suggest that, in addition to shear
stress-induced alterations in endothelial cell function,
fluid dynamics may also affect atherosclerotic lesion
localization by influencing monocyte transport and
adhesion to the vessel wall. Monocytes that encounter
the vessel wall near a stagnation or reattachment point
where the wall shear stress is low may exhibit transient arrests, which could lead to long-term arrest and
subsequent transmigration to the intima.
We thank Dr. Robert Lindberg for assistance in isolation of human
umbilical vein endothelial cells (HUVEC) and Dr. Tracey duLaney for
assistance in the characterization of HUVEC.
This work was supported, in part, by National Heart, Lung, and
Blood Institute Grants HL-41372 and HL-57446 and a resource
allocation grant from the North Carolina Supercomputing Center.
K. M. Barber was supported by National Institutes of Health
Training Fellowship GM-08555.
Address for reprint requests: G. A. Truskey, Dept. of Biomedical
Engineering, Duke Univ., 136 Hudson Hall, Durham, NC 277080281.
Received 13 June 1997; accepted in final form 30 April 1998.
REFERENCES
1. Alon, R., D. A. Hammer, and T. A. Springer. Lifetime of the
P-selectin-carbohydrate bond and its response to tensile force in
hydrodynamic flow. Nature 374: 539–542, 1995.
2. Barbee, K. A., P. F. Davies, and R. Lal. Shear stress-induced
reorganization of the surface topography of living endothelial
cells imaged by atomic force microscopy. Circ. Res. 74: 163–171,
1994.
3. Barber, K. M. Effects of Shear and Recirculating Flow on U-937
Cell Adhesion to Activated Endothelium: Implications in Atherosclerosis (PhD Thesis). Durham, NC: Duke University, 1997, p.
322.
4. Biffl, W. L., E. E. Moore, F. A. Moore, and C. Barnett. Nitric
oxide reduces endothelial expression of intercellular adhesion
molecule (ICAM)-1. J. Surg. Res. 63: 328–332, 1996.
5. Brenner, H. The slow motion of a sphere through a viscous fluid
towards a plane surface. Chem. Eng. Sci. 16: 242–251, 1961.
6. Cokelet, G. R. The rheology and tube flow of blood. In: Handbook of Bioengineering, edited by R. Skalak and S. Chien. New
York: McGraw-Hill, 1987, p. 14.1–14.17.
7. Fluid Dynamics International. FIDAP 7.0 User’s Guide.
Evanston, IL: FDI, 1994.
8. Gerrity, R. G., J. A. Goss, and L. Soby. Control of monocyte
recruitment by chemotactic factor(s) in lesion-prone areas of
swine aorta. Arteriosclerosis 5: 55–66, 1985.
9. Gimbrone, M. A., R. S. Cotran, and J. Folkman. Human
vascular endothelial cells in culture. J. Cell Biol. 60: 673–684,
1974.
10. Goldman, A. J., R. G. Cox, and H. Brenner. Slow viscous
motion of a sphere parallel to a plane wall. I. Motion through a
quiescent fluid. Chem. Eng. Sci. 22: 637–651, 1967.
11. Goldman, A. J., R. G. Cox, and H. Brenner. Slow viscous
motion of a sphere parallel to a plane wall. II. Couette flow. Chem.
Eng. Sci. 22: 653–660, 1967.
12. Hammer, D. A., and S. M. Apte. Simulation of cell rolling and
adhesion on surfaces in shear flow: general results and analysis
of selectin-mediated neutrophil adhesion. Biophys. J. 63: 35–37,
1992.
13. Hammer, D. A., and D. A. Lauffenburger. A dynamical model
for receptor-mediated cell adhesion to surfaces. Biophys. J. 52:
475–487, 1987.
14. Jaffe, E. A., R. L. Nachman, C. G. Becker, and C. R. Minick.
Culture of human endothelial cells derived from umbilical veins.
J. Clin. Invest. 52: 2745–2756, 1973.
15. Joris, I., T. Zand, J. Nunnari, F. J. Krolikowski, and G.
Majno. Studies on the pathogenesis of atherosclerosis. I. Adhe-
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.2 on June 18, 2017
fluid velocity components normal to the endothelium.
As a result, a U-937 cell may impact the endothelium
with a sufficiently greater force that results in compression of microvilli with a larger contact area between the
U-937 cell and the endothelium. This larger contact
area may increase the chance of forming a bond before
the cell moves away. Such interactions may be brief,
further influencing the types of adhesion molecules
that bind, as well as the kinetics of binding (Fig. 7).
Although multiple receptors are present on HUVEC
after TNF-a activation, the forces and contact duration
affect which actually form bonds near flow reattachment.
Computer simulations of spherical particle trajectories within the recirculating flow through the sudden
expansion predicted that rigid spheres do not enter the
recirculation region or contact the lower wall of the
sudden expansion. However, experimental results demonstrated that U-937 cells transiently adhere to the
endothelium in the vicinity of the reattachment site.
Thus the numerical model cannot accurately simulate
this result from the experiments. However, the numerical model did simulate trajectories of particles that
gradually spiraled outward and left the recirculation
zone (Fig. 5B), which agrees with experimental observations for the motions of particles in an axisymmetric
annular expansion (17).
Major assumptions of the computational model include treating the U-937 cells as rigid spherical particles with no microvilli, neglecting cell-cell collisions,
and neglecting any disturbances in the local flow field
that the cells may cause. These assumptions may not
be valid. Cell deformability, microvilli, and aspherical
cell shape may cause the observed deviations between
the computational predictions of cell trajectories and
the experimental results. Inclusion of these effects will
require solving the coupled motion of the particle and
fluid, a more complex problem than the one addressed
in this study. An additional experimental condition not
included in the model is the wavy surface of the
endothelial monolayer, where variations in height as
much as 4–5 µm exist (2). Variations in the local flow
over this wavy surface, compared with a flat surface
modeled in the simulations, may also account for the
experimental deviations from the model predictions.
In vivo, convective transport of monocytes to the
vessel walls along curved streamlines that have large
radial velocity components may be partly responsible
for the flux of monocytes to the walls. In addition,
monocyte heterogeneities and variations in cell size
and shape, cell-cell collisions, and pulsatile flow can
affect transport of monocytes to the wall. Collisions
between monocytes and red blood cells are an important aspect due to the high concentrations of red blood
cells that are present at physiological hematocrit levels
in whole blood. As demonstrated in vitro, leukocyte
collisions with red blood cells may enhance transport of
leukocytes to the endothelium, due to radial dispersion
of leukocytes (24–26, 35). This effect can increase the
frequency of encounters between monocytes and vessel
walls.
U-937 CELL ADHESION TO ENDOTHELIUM IN RECIRCULATING FLOW
16.
17.
18.
19.
20.
22.
23.
24.
25.
26.
27.
28. Prieto, J., A. Eklund, and M. Patarroyo. Regulated expression of integrins and other adhesion molecules during differentiation of monocytes into macrophages. Cell. Immunol. 156: 191–
211, 1994.
29. Pritchard, W. F., P. F. Davies, Z. Derafshi, D. C. Polacek, R.
Tsao, R. O. Dull, S. A. Jones, and D. P. Giddens. Influence of
hemodynamic factors on the adhesion pattern of U-937 cells in a
flow model: implications in atherosclerosis. J. Biomech. 28: 1459,
1995.
30. Schmid-Schönbein, G. W. Rheology of leukocytes. In: Handbook of Bioengineering, edited by R. Skalak and S. Chien. New
York: McGraw-Hill, 1987, p. 13.1–13.25.
31. Spertini, O., F. W. Luscinskas, M. A. Gimbrone, and T. F.
Tedder. Monocyte attachment to activated human vascular
endothelium in vitro is mediated by leukocyte adhesion molecule-1 (L-selectin) under nonstatic conditions. J. Exp. Med. 175:
1789–1792, 1992.
32. Sundström, C., and K. Nilsson. Establishment and characterization of a human histiocytic lymphoma cell line (U-937). Int. J.
Cancer 17: 565–577, 1976.
33. Tempelman, L. A., and D. A. Hammer. Receptor-mediated
binding of IgE-sensitized rat basophilic leukemia cells to antigencoated substrates under hydrodynamic flow. Biophys. J. 66:
1231–1243, 1994.
34. Truskey, G. A., K. M. Barber, T. C. Robey, L. A. Olivier, and
M. P. Combs. Characterization of a sudden expansion flow
chamber to study the response of endothelium to flow recirculation. J. Biomech. Eng. 117: 203–210, 1995.
35. Turrito, V. T., and H. L. Goldsmith. Rheology, transport, and
thrombosis in the circulation. In: Vascular Medicine, edited by J.
Loscalzo, M. Creager and V. Dzau. Boston, MA: Little, Brown,
1992, p. 157–204.
36. Walker, L. N., M. A. Reidy, and D. E. Bowyer. Morphology and
cell kinetics of fatty streak lesion formation in the hypercholesterolemic rabbit. Am. J. Pathol. 125: 450–459, 1986.
37. Walpoa, P. L., A. I. Gotlieb, M. I. Cybulsky, and B. L.
Langille. Expression of ICAM-1 and VCAM-1 and monocyte
adherence in arteries exposed to altered shear stress. Arterioscler. Thromb. Vasc. Biol. 15: 2–10, 1995.
38. Wattenbarger, M. R., D. J. Graves, and D. A. Lauffenburger.
Specific adhesion of glycophorin liposomes to a lectin surface in
shear flow. Biophys. J. 57: 765–777, 1990.
39. Wildner, O., T. Lipkow, and J. Knop. Increased expression of
ICAM-1, E-selectin, and VCAM-1 by cultured human endothelial
cells upon exposure to haptens. Exp. Dermatol. 1: 191–198, 1992.
40. Xiao, Y., and G. A. Truskey. Effect of receptor-ligand affinity on
the strength of endothelial cell adhesion. Biophys. J. 71: 2869–
2884, 1996.
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.2 on June 18, 2017
21.
sion and emigration of mononuclear cells in the aorta of hypercholesterolemic rats. Am. J. Pathol. 113: 341–358, 1983.
Karino, T., and H. L. Goldsmith. Adhesion of human platelets
to collagen on the walls distal to a tubular expansion. Microvasc.
Res. 17: 238–262, 1979.
Karino, T., and H. L. Goldsmith. Flow behaviour of blood cells
and rigid spheres in an annular vortex. Philos. Trans. R. Soc.
Lond. 279: 413–445, 1977.
Klein, C. L., H. Kohler, F. Bittinger, M. Wagner, I. Hermanns, K. Grant, J. C. Lewis, and C. J. Kirkpatrick.
Comparative studies on vascular endothelium in vitro. I. Cytokine effects on the expression of adhesion molecules by human
umbilical vein, saphenous vein and femoral artery endothelial
cells. Pathobiology 62: 199–208, 1994.
Kukreti, S., K. Konstantopoulos, C. W. Smith, and L. V.
McIntire. Molecular mechanisms of monocyte adhesion to interleukin-1b-stimulated endothelial cells under physiologic flow
conditions. Blood 89: 4104–4111, 1997.
Luscinskas, F. W., H. Ding, and A. H. Lichtman. P-selectin
and vascular cell adhesion molecule 1 mediate rolling and arrest,
respectively, of CD41 T lymphocytes on tumor necrosis factor-aactivated vascular endothelium under flow. J. Exp. Med. 181:
1179–1186, 1995.
Luscinskas, F. W., H. Ding, P. Tan, D. Cumming, T. F.
Tedder, and M. E. Gerritsen. L- and P-selectins, but not
CD49d (VLA-4) integrins, mediate monocyte initial attachment
to TNF-a-activated vascular endothelium under flow in vitro. J.
Immunol. 156: 326–335, 1996.
Luscinskas, F. W., G. S. Kansas, H. Ding, P. Pizcueta, B. E.
Schleiffenbaum, T. F. Tedder, and M. A. J. Gimbrone.
Monocyte rolling, arrest and spreading on IL-4-activated vascular endothelium under flow is mediated via sequential action of
L-selectin, b1-integrins, and b2-integrins. J. Cell Biol. 125:
1417–1427, 1994.
Malinauskas, R. A., R. A. Herrmann, and G. A. Truskey. The
distribution of intimal white blood cells in the normal rabbit
aorta. Atherosclerosis 115: 147–163, 1995.
Melder, R. J., L. L. Munn, S. Yamada, C. Ohkubo, and R. K.
Jain. Selectin- and integrin-mediated T-lymphocyte rolling and
arrest on TNF-a-activated endothelium: augmentation by erythrocytes. Biophys. J. 69: 2131–2138, 1995.
Munn, L. L., R. J. Melder, and R. K. Jain. Analysis of cell flux
in the parallel plate flow chamber: implications for cell capture
studies. Biophys. J. 67: 889–895, 1994.
Munn, L. L., R. J. Melder, and R. K. Jain. Role of erythrocytes
in leukocyte-endothelial interactions: mathematical model and
experimental validation. Biophys. J. 71: 466–478, 1996.
Nichols, W. W., and M. F. O’Rourke. McDonald’s Blood Flow in
Arteries. Philadelphia, PA: Lea and Febiger, 1990.
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