1. No category

From www.bloodjournal.org by guest on October 28, 2014. For personal use only.
1987 70: 1284-1290
Effect of flow on polymorphonuclear leukocyte/endothelial cell adhesion
MB Lawrence, LV McIntire and SG Eskin
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Copyright 2011 by The American Society of Hematology; all rights reserved.
From www.bloodjournal.org by guest on October 28, 2014. For personal use only.
Effect
The
of Flow
effect
of flow
leukocytes
gated
using
flow
on
(PMNL)
on Polymorphonuclear
the
a parallel
plate
field.
Washed
PMNL
of primary
human
umbilical
pretreated
with
(FMLP.
HUVEC
U/mL)
for
control
HUVEC
wall
shear
face
was
to
NE
the
also
L.V.
minutes.
or
vessel
wall.
studied
While
the
of
sur-
stress
of
± SEM)
20.3
±
ofthe
system
conditions,
leukocytes
(PMNL)
can
independent
role
for
be quantitated
mediators.
investigations
the
with
have
endothelium
From
Houston;
and
Baylor
Engineering
College
by Grune
& Stratton,
0006-4971/87/7005-0140$3.OO/O
1284
Inc.
1987
by Grune
to
at
the
interaction
under
1.96
28
± 2.9
adhesion
10.2
to
3.8
±
3.01
adhesion
& Stratton,
adhesion
appear
and
dynes/cm2.
appears
outcome
to
of the
these
be
an
PMNL/
experimental
to
EC
Inc.
and,
in the
to act selectively
cases
on the
under
varying
FMLPand
One
of
the
more
interesting
able
distances
before
detaching,
through
the blood vessel wall.9’2
surements
have
of
and
in
IL I to stimuadhesion
to
Our
data
characteristics
to
B4,
I and
show
a
of
IL 1.
pointed
FMNL
IL
In a related
strong
flow dependence
of PMNL
adhesion
to endothelium
that modulates
the previously
reported
chemical
effects
to the
an
of
EC.”
flow conditions.
tissue preparations
of postcapillary
of PMNL
rolling
of
is their
venules
stopping,
This report
velocities
PMNL
ability
to roll
for consideror emigrating
presents
mea-
on control
and
FMLP-
treated
endothelium
at varying
wall shear stresses.
It has been postulated
that granulocytes
play a significant
role in clinical
complications
resulting
from extracorporeal
blood flow, such as occurs
during
hemodialysis
and cardiopulmonary
bypass
procedures.’3’5
and pulmonary
sequestration
The transient
neutropenia
of leukocytes
associated
with
these procedures
of complement
are thought
to be caused
by the activation
by blood-contacting
surfaces.’3”5’7
We report
data on PMNL
human
plasma
adhesion
to nylon surfaces
under conditions
in which
of
the
adhesive
fragment
C3b.
interaction
University,
after exposure
to
the likely mediator
is surface-bound
MATERIALS
of Surgery.
Houston.
Submitted
February
13, 1987; accepted June 1 7, 1987.
Supported
by the National
Institutes
of Health
Grants
No.
ilL-I 7432 and HL-l8636
and the Robert
A. Welch
Foundation
Grant No. C-938.
Address
reprint requests
to Larry V. Mclntire,
Phd, Biomedical
Engineering
Laboratory,
Department
of Chemical
Engineering,
Rice University,
P0 Box 1892. Houston.
TX 77251.
The publication
costs ofthis article were defrayed
in part by page
charge payment.
This article must therefore
be hereby marked
“advertisement”
in accordance
with
18 U.S.C.
§1734
solely
to
indicate this fact.
C /987
PMNL
respect
Rice
PMNL
dropped
in determining
adhesive
thrombin,
on the endothewe used
the
Department
on
control
at
while
endothelium
adhesion
Laboratory.
factor
HUVEC.
HUVEC
observed
in various
along the endothelium
to increase
of Medicine,
control
± 6.0
dynes/cm2.
HUVEC
adhered
respectively,
flow
leukocyte
and
1), tumor
necrosis
factor,
thrombin,
These
agents
induce
large
increases
the Biomedical
to
control
to
bacterial
endotoxin
analog,
FMLP,
to stimulate
adhesion
of
PMNL
to endothelium.
Perhaps
the most dramatic
modulators of the adhesive
interaction
now being
investigated
are
interleukin
I (IL
lipopolysaccharide.
important
HUVEC
PMNL/mm2
PMNL/mm2,
of
IL 1 -treated
14.1
±
42
on either
work
in mediating
by action
experiments
3.5
± 25.8
On
and
At 3.92
noted
little
laboratories
apparently
one set of
and
effect
HUVEC.
was
monocyte-derived
measured
PMNL
been
PMNL
to endothelium,
hal cell (EC).’3
In
to control
68
HUVEC
set of experiments
we used
late the endothelium
and
compounds
such as leukotriene
C5a,
and
formyl-methionyl-leucylin nanomolar
concentrations
in several
adhered
IL 1-treated
dynes/cm2,
been
adhesion.
Chemotactic
complement
fragment
phenylalanine
(FMLP)
shown
PMNL/mm2
±
1 .96
adherence
HUVEC.
to
has been done in characterizing
the magnitude
and significance
of physical
forces
on the process
of leukocyte
attachment to the vessel wall. With the well-defined
flow field that
exists
in a parallel-plate
flow chamber,
the importance
of
wall shear
stress
in the margination
of polymorphonuclear
effects
of inflammatory
A number
of recent
adhered
no PMNL
371
The
At
to FMLP-treated
have
leukocytes
mediators
HUVEC.
adhered
dynes/cm2.
S
leukocytes
Eskin
FMLP-treated
6.8
Adhesion
conditions.
PMNL/mm2
of
on
0.98
adhered
immune
effects
quiescent
and
range
nylon-coated
shear
2
.
stimulated
margination
inflammatory
under
to
195
responses
is the
of various
to
(mean
while
experi-
Cell
control
PMNL/mm2
1
a physiologic
a wall
PMNL/mm2
FIRST
infection
over
adhesion
HUVEC
In other
to
virtually
(HUVEC)
interleukin
and S.G.
PMNL/mm2
a monolayer
cells
Mclntire,
adhered
investi-
a well-defined
over
adhesion
At
± 37.3
or
extensively
was
with
with
PMNL
PMNL
OF THE
blood
Lawrence,
endothelial
five
measured
studied.
283
endothelium
polymorphonuclear
perfused
vein
for
hours.
stresses.
injury
of
chamber
pretreated
was
FMLP-treated
O
M.B.
endothelium
were
mol/L)
were
four
dynes/cm2,
By
formyl-methionyl-leucyl-phenylalanine
1 x i07
ments
adhesion
to vascular
Leukocyte/Endothelial
AND
complement
METHODS
Endothelial
cell cultures.
Human
umbilical
vein endothelial
cells (HUVEC)
were harvested
using
collagenase
digestion,’8
pooled,
seeded onto glass slides (34 x 74 mm, Fisher Scientific
Co.
Springfield,
NJ), and cultured
in Ml99 (GIBCO
Laboratories,
New
York) supplemented
with 20% fetal calf serum (FCS,
HyClone
Laboratories
cm
Inc.
(PSN,
Logan,
UT),
1% by volume,
penicillin,
GIBCO).
ments two to four days after reaching
Preparation
of human
PMNL.
healthy
male
donors
was
drawn
streptomycin,
Cultures
and
were
used
confluence.
Venous
blood
into
heparin
(10
neomy-
in experi-
from
U/mL,
normal,
final
PMNL
were isolated from heparinized
whole blood
by dextran sedimentation
followed by hypotonic
lysis of erythrocytes
and centrifugation
over Ficoll-Hypaque
(FH).’9’#{176}
The final pellet
was resuspended
in M199 at pH 7.4 to which 3.75 mg/mL
of bovine
concentration).
Blood,
Vol
70, No 5 (November),
1987: pp 1284-1290
From www.bloodjournal.org by guest on October 28, 2014. For personal use only.
FLOW
EFFECTS
ON PMNL-EC
ADHESION
1285
serum albumin
(BSA, Sigma Chemical
Co, St. Louis) was added
(M I 99 + BSA). PMNL
were counted
with a model ZBI Coulter
Counter
(Coulter
Electronics
Inc. Hialeah,
FL) connected
to a
Coulter Channelyzer
(Coulter
Electronics)
to determine
the appropriate dilution ratio. Trypan blue exclusion showed greater than 96%
viability.
Preparation
into
ofplatelet-poor
siliconized
glass
Venous
plasma.
tubes
containing
blood
heparin
(10
was drawn
U/mL,
final
concentration).
After centrifugation
at 400 x g for ten minutes, the
supernatant
was drawn off with a Pasteur
pipet and centrifuged
again at I ,000 x g for ten minutes.
The resultant
platelet-poor
plasma
(PPP)
was
transferred
to a clean
plastic
tube.2#{176}
Experimental
apparatus.
To produce a well-defined
wall shear
stress, we used a parallel
plate geometry
for our flow chamber
(Fig
I A).224 One side of the chamber
was a slide on which HUVEC
were
cultured.
The other side was machined
from polycarbonate.
Once
assembled
these two flat’surfaces
were held approximately
250 im
apart
by a Silastic
rubber
gasket
(Dow
Corning,
Midland,
MI).
Because
of the small gap-to-width
ratio, the channel
could be
approximated
over most of its surface as two infinite parallel plates.
Under
laminar
flow conditions
such as used in our experiments
(Reynolds
number < 2), the velocity
profile
is parabolic
and the wall
shear
stress
can
be calculated
Newtonian
fluid.24
shear
is equal
stress
utilizing
As described
the
in the
momentum
following
balance
equation,
the
=
2a
-
b
shear
stress
Coefficient
of viscosity
Volumetric
Channel
height
Channel
width
syringe
control.
Experiments
rates:
140 sec’,
pump
(South
were
280
Natick,
and
3.92
those
seen
in venules
these
flow conditions
dynes/cm2.
videomicroscopy
then
PMNL
range
veins.
were
sec’,
430
sec’,
digital
rinsed
with
concentration
at the
and
560
processing
using
to
under
phase.contrast
(Fig
I).
at
with
and
37#{176}Cfor
30
Ml99+BSA
30 minutes
minutes.
The
at 37#{176}C
to a cell
was
allowed
for the
PMNL
to stabilize.
The PMNL suspension,
the connecting
tubing,
and the microscope
with flow chamber
(Fig 2 B) were all maintained
at 37#{176}C
by an air-curtain
incubator
(Laboratory
Products,
Boston;
Model 279).
Approximately
30 minutes
after
the PMNL
were
diluted,
the
minutes
PMNL
Using a
mounted
and then rinsed for five minutes
with MI99+BSA.
The
suspension
was then perfused
through
the flow chamber.
Hamamatsu
video camera
(Waltham,
MA; Model C 1012)
on a Nikon Diaphot-TMD
inverted phase-contrast
micro-
scope
(Garden
HUVEC
City,
were
NY),
exposed
the
to I x 10’
experiments
mol/L
were
FMLP
videotaped.
number of PMNL attached
to the HUVEC
monolayer
after
ten minutes
of flow. The control
consisted
experimental
procedure
without the incubation
of the
FMLP. For experiments
involving IL I (Genzyme
Inc.,
tL of IL I (200 U/mL)
in 0.5% BSA was introduced
dish
containing
the
Microscope
HUVEC
monolayer
and
diluted
for five
The
were counted
of the same
HUVEC
with
Boston),
100
into the petri
in the
culture medium to 2 U/mL.
The HUVEC
were incubated
with the
IL I for four hours preceding
the experiment.5
The control consisted
of untreated
HUVEC.
The PMNL
for each experiment
were
isolated
tissue
from
a single
donor,
the same pooled seeding.
PMNL rolling velocities
ing
covered slide was fitted onto the
to hold it in place. The chamber
Ml99+BSA
cells/izL
is similar
interactions
directly
image
was diluted
of 1,000
stresses
PMNL/HUVEC
The HUVEC
and vacuum applied
suspension
ofshear
visualized
assay.
flow chamber
was
This
and
and
Adhesion
Inverted
Video
Camera
MA)
conducted
sec’.
These shear rates, for a flowing tissue culture
medium
at
37#{176}C,
correspond
respectively
to wall shear stresses of 0.98, 1.96,
3.01,
Pump
(A) Parallel
plate flow chamber
machined
from transpolycarbonate.
The cover
slip is covered
with confluent
HIJVEC or coated with nylon. The silastic gasket separates
the
cover slip from the deck of the flow chamber
and. when vacuum
is
applied,
results in a channel
gap of 250 zm. (B) The flow chamber
is attached
to the stage of the inverted-stage
microscope.
The
syringe pump controls
the flow and the water
bath containing
the
PMNL cell suspension.
An air-curtain
incubator
maintains
the flow
chamber,
inlet tubing. and microscope
stage at 37#{176}C.
The output of
the video camera
is recorded
for later
image
processing
and
analysis.
flow rate
Apparatus
for flow
wall
Wall shear
=
IL
following
Syringe
Fig 1 .
Q
935 Harvard
Both
parent
2a2b
employed
Woter
wall
to:
T
was
Recorder
for a
3tiQ
A model
B
Video
(Perceptive
Systems
and
the
HUVEC
were measured
Inc.,
Houston;
monolayers
by digital
Model
327)
were
image
from
process-
of videotapes.
The time interval
used for these velocity
measurements
was 30
seconds to resolve PMNL velocities
below I sm/sec.
Accordingly,
a
stable
adhesion
was defined as one that lasted 30 seconds or longer.
Normalized
histograms
of velocity distributions
and the averages
were determined
from analyzing
two to four frames of videotape
separated
by at least one minute in each experiment.
The same procedure
for PMNL preparation
was used with studies
of PMNL adhesion to nylon. Glass slides were coated with Elvamide
nylon (DuPont,
Wilmington,
DE) by solvent casting
(MeOH,
1%
solution).
In some experiments
the nylon surface was incubated
with
PPP for 20 minutes
followed by a two-minute
washout
before the
PMNL suspension
was perfused
through the chamber.
Statistical
analysis.
The variation
in adhesion
ratios was analyzed using a one-tailed
t test
for paired data since the control,
FMLP-treated
and IL 1-treated
HUVEC,
were from the same
population
of pooled cells, and the washed
PMNL
used in the
experiment
were from the same donor. Ratios
of the means of
PMNL rolling velocities on FMLP-treated
HUVEC
and controls as
well as ratios of the means at two shear stresses were compared
using
a two-tailed
t test.
A two-tailed
t test
was also used to compare
PMNL
rolling velocity distributions.
Error bars on all histograms
represent
the SEM.
From www.bloodjournal.org by guest on October 28, 2014. For personal use only.
LAWRENCE,
1286
McINTIRE,
AND
ESKIN
0
0
(‘8
0
In
(
0
E0
‘0
08
C
C0
a) In
V
(0
-I
z
Xe
0.
0
In
0
A
Fig 2.
PMNL
adherent
to FMLP-treated
HUVEC
monolayer
under flow taken from digitized
frame of videotape
(magnification
20 x objective).
The large white
dots are PMNL adhering
to the
HUVEC
monolayer
(dark background
with barely
visible
HUVEC
outlines).
PMNL
counts
in the field of view are normalized
to
PMNL/mm2.
Wall shear stress = 0.98 dynes/cm2.
(Figure reduced
to 65% of original
size.)
B
C
Fig 3.
Effect of flow and FMLP-treatment
of HUVEC
on PMNL
adhesion.
Experiments
performed
at wall shear stresses
of 0.98
dynes/cm2
(A. 140 sec’).
1 .96 dynes/cm2
(B. 280 sec’),
and 3.92
dynes/cm2
(C. 560 sec1).
PMNL were suspended
in Ml99+
BSA
(1.000
celIs/sL)
at 37#{176}C.
FMLP-treated
HUVEC
(1 x iO
mol/L,
five minutes),
cross-hatched
bars; control
HUVEC,
open bars.
Error bars represent
SEM.
RESULTS
In paired
experiments
at 0.98 dynes/cm2
wall shear stress,
the ratio of PMNL
adhesion
to FMLP-treated
HUVEC
v
control
HUVEC
was 1.45 (P < .01, n = 9, Fig 3). At this
wall shear
stress
283 ± 37.3
PMNL/mm2
(mean
±
SEM)
a
a
adhered
to FMLP-treated
HUVEC
and 195 ± 20.3 PMNL/
mm2 adhered
to control
HUVEC
after ten minutes
of flow.
At 1.96 dynes/cm2
wall shear
stress,
the ratio
of PMNL
adhesion
to FMLP-treated
I .58 (P < .025,
mm2 adhered
n
=
to
HUVEC
PMNL/mm2
adhered
to control
of flow. At 3.92 dynes/cm2
wall
adherence
was
HUVEC
transient
v control
8). At this wall stress 68
FMLF-treated
HUVEC
noted
on
=
control
was
I 4. 1 PMNL/
and 42 ± 6.0
±
HUVEC
after ten
shear stress, virtually
either
(<I cell/mm2,
n
white cell/endothelial
HUVEC
a
U,
(‘8
or
minutes
no cell
FMLP-treated
3), although
there
cell interactions.
were
many
E0
La
a) a
.c
Co
_J
a
z
X
In paired
experiments
25.8
PMNL/mm2
371
±
and 28
resulting
Fig
4).
±
At
adhered
of 13.8
wall
wall
IL
±
no
to the
77
±
wall
nylon
21.7
stress,
surface
while
PMNL/mm2
ratio of 7.9:1 (P < .01, n
adhesion
(<I
cell/mm2,
nylon
at I .96 dynes/cm2
(data
0
a
a
HUVEC,
.01, n = 5,
<
stress,
10.2
and
6.8
giving
±
3.8
a
U,
± 3.5
an adhe-
a
A
9.7
± 3.8
on nylon
4, Fig 5).
n = 3) of
Fig 4.
giving
an
There
PMNL
was
to
not shown).
B
PMNL/
pretreated
adhered,
=
stress,
HUVEC
to control
1.2 (P
HUVEC
shear
shear
1-treated
shear
to IL 1-treated
dynes/cm2
adhered
PPP,
untreated
dynes/cm2
to
adhered
to control
HUVEC,
of I .65 ± 0.25 (P < .05, n = 5).
0.98
adhesion
almost
3.01
adhered
FMNL/mm2
sion ratio
mm2
with
adhered
2.9 PMNL/mm2
in an adhesion
ratio
PMNL/mm2
At
at I .96 dynes/cm2
Effect
of flow
and
IL 1 treatment
of HUVEC
on PMNL
adhesion.
Experiments
performed
at wall shear stresses
of 1.96
dynes/cm2
(A. 280
and 3.01
dynes/cm2
(B. 430 sec’).
PMNL were suspended
in Ml 99 + BSA (1 .000 cells/pL)
at 37#{176}C.
IL
1 -treated
HUVEC
(2 U/mL,
four hours),
cross-hatched
bars; control HUVEC.
open bars. Error bars represent
SEM.
From www.bloodjournal.org by guest on October 28, 2014. For personal use only.
FLOW
EFFECTS
ON
PMNL-EC
ADHESION
1287
At a wall shear stress of0.98
dynes/cm2,
PMNL
that were
attached
to control
HUVEC
had an average
rolling
velocity
0
0
CU
of 0.166
0
sm/sec
means
was
0
,,
0.046
due
not
0
C
C-0
(00
had
(n
to FMLP
rolling
FMNL
rolling
attached
velocity
.4).
>
(0
0
_.J (0
The
z
X
ratio
dynes/cm2
0.0
ID
Between
the
of
of
PMNL
shear
20%
and
0
to
0
CU
dynes/cm2,
less than
tm/sec
(Fig 7), while
A
B
between
of the
HUVEC
a wall
shear
control
0.3 1 3
0.047
±
of 0.1 34
monolayer
stress
of I .96
HUVEC
0.054
HUVEC
zm/sec
rolling
stress
90%
velocities
was
had
tm/sec,
of the PMNL
monolayer
rolled
with
1.96
and
stable
0.98
adhesions
rolled
faster
approximately
1 tm/sec
to FMLP
of
.4).
>
.02).
downstream.
1% of the PMNL
at I .96 dynes/cm2,
than
due
<
an
and
had an average
(n = 5).
The
at
I .94 (P
±
these
means
due to FMLP
treatment
was also not significant
(P
of the FMNL
rolled
faster
ences in velocity
distributions
0
to FMLP-
velocity
difference
to
to FMLF-treated
of 0.269
±
wall
HUVEC
At
attached
velocity
attached
rolling
6). The
difference
between
these
the HUVEC
monolayer
V
PMNL
treatment
(P
FMNL
average
and
an average
3, Fig
=
significant
dynes/cm2,
E
#{163}
tm/sec;
HUVEC
0.026
0
(0
-..,.
±
treated
(0
(Fig 8).
treatment
At
0.98
than
I
5%
Differof the
C
Fig 5.
PMNL
adhesion
to nylon-coated
surface
under
flow
(0.98
dynes/cm2,
140
sec’).
PMNL
were
suspended
in
M199+BSA
(1.000
cells/zL)
at 37’C.
PMNL/mm2
adhering
to
untreated
nylon surface
(A). PMNL/mm2
adhering
to nylon preincubated
with PPP for 20 minutes
(B), PMNL/mm2
adherent
to
control
HUVEC
(C). Error bars represent
SEM.
(0
0
0)
C
..-I
-
ID
0
0
C.-J
z
0
X
0.’
.8.)
C
C-)
a,
a)
C-
C’)
-.--
a,
‘C
(-3
a
>
a
1
0)
0c’J
L
a
--3
z
0
0
a
A
I
Fig 6.
Effect of flow and FMLP treatment
of HUVEC
on the
mean rolling velocities
of PMNL.
Experiments
performed
at wall
shear stresses
of 0.98 dynes/cm2
(A. 140 sec’)
and 1 .96 dynes/
cm2 (B. 280 sec’).
FMLP-treated
HUVEC.
cross-hatched
bars;
control
HUVEC,
open bars. Error bars represent
SEM.
Fig 7.
Distribution
of PMNL
rolling velocities
at 0.98 dynes/
cm2. FMLP-treated
HUVEC,
cross-hatched
bars; control
HUVEC,
open bars. Each pair of bars represents
the fraction
of adherent
PMNL with rolling velocities
ranging from 0.0 to 0.13 sm/sec
(A).
0.13 to 0.26 tm/sec
(B). 0.26 to 0.39 sm/sec
(C). 0.39 to 0.52
sm/sec
(D). 0.52 to 0.65 sm/sec
(E), 0.65 to 0.78 sm/sec
(F). 0.78
to 0.91 pm/sec
(G). and 0.91 to 1.04 Mm/sec
(H). To generate
a
representative
velocity
distribution
for each experiment.
the normalized
velocity
distributions
from two to four image frames
of
videotapes
(separated
in each case by at least one minute)
were
averaged.
Histograms
from
experiments
under
the same conditions were
again averaged
to create
a final normalized
velocity
distribution.
Error bars represent
SEM.
From www.bloodjournal.org by guest on October 28, 2014. For personal use only.
ID
level
0
of PMNL
hum,
over
with
Lackie’s
r
ID
-.
0
adhesion,
a small
even
range
proposal
delicately
0)
C
r8
McINTIRE,
LAWRENCE.
1288
that
balanced
on highly
of wall
stimulated
shear
the
adhesive
shear
force
The wall shear
rates
examined
in these
close
to those
estimated
for the venules
vessels
but are lower than those estimated
C-
-J
z
X
.8.10
C
bed
where
there
is normally
the
abrupt
drop
in shear
a,
capillaries
to venules
C-
of PMNL
adhesion
a,
‘C
similar
pattern
dilute
suspensions
little
can
B
C
0
E
F
I’);
8
G
H
Fig 8.
Distribution
of PMNL rolling velocities
at 1 .96 dynes/
cm2. FMLP-treated
HUVEC.
cross-hatched
bars; control
HUVEC,
open bars. Each pair of bars represents
the fraction
of adherent
PMNL with rolling velocities
ranging
from 0.0 to 0.13 Mm/sec
(A),
0.13 to 0.26 am/sec
(B). 0.26 to 0.39 Mm/sec
(C). 0.39 to 0.52
two
flow
conditions
examined,
although
a broader
distribution
rolling
velocities
was observed
at I .96 dynes/cm2.
In experiments
with
nylon
surfaces,
we observed
of
monolayer
were
downstream
next
to the
from
300
to 600
to the
not
rolling,
focus
nylon
significant
although
surface
Mm/sec.
PMNL
had
the
spreading
nylon
surface
From
our
impact
Doubling
experiments
fluid
that
ranged
of
PMNL
of
be seen
forces
was observed.
drag
Doubling
the wall
2 to 4 dynes/cm2
tually
zero. On IL 1-treated
sion was I 3-fold
more than
was expected
PMNL/EC
approximately
dropped
both
forces
PMNL
dramatically.
with
the
in our
why
from
of the
bulk
A qualitatively
data
vessel
suggest
data
that
using
PMNL
endothelium)
can
below a threshold
PMNL
geometry
In vivo,
transition
location
model
Our
.
In our experiments
was used
so that
would
normally
and
the
adhere
resultant
evidence
that
Pretreatment
done,
purpose
as the
the endothelial
a dilute,
washed
any
influence
of
be eliminated.
some recent
is reduced.28
in
com-
However,
FMNL
platelets
there
has
in plasma
PMNL
adhesion
ofthe
PMNL
with FMLP
of this
work
cell substrate
was
to evaluate
on PMNL
adhesion.
susor
been
to EC
was not
the
role
of
Preactiva-
tion of PMNL
would probably
lead to very different
levels of
leukocyte
adhesion
to HUVEC.
It has been
shown
that
FMLP
causes
a 20-minute
aggregation
response
in FMNL,
so the incubation
times
for endothelial
cells were selected
accordingly.2#{176} Tonneson
et a129 have reported
that exposure
of endothelial
cell monolayers
to FMLP
at a comparable
concentration
for periods
of time ranging
from 1 5 minutes
to
4 hours did not increase
PMNL
adhesion
to the monolayer.
the small
increases
in adhesion
3) and that of others
may be due
by low levels of residual
FMLP.”3
endothelium
appear
to have
a
by
endothelium,
on controls
the level of adheat 2 dynes/cm2,
as
of static
the wall
on the
ratio and
This
experiments
shear
stress
IL 1-treated
the absolute
large
decrease
coated
were
seen
to some
in our
work
PMNL
(Fig
activation
of PMNL
sticking
to nylon
was very
with the number
of FMNL
that adhered
at a wall
shear
stress
of 0.98
dynes/cm2.
low
to
When
on
to
HUVEC
number
of
in the
flow chamber
wall with PPP, as would occur
in contact
with
a similar
type
of foreign
dramatically
PMNL
have
and
shear
stress
again
from
reduced
adhesion
to vir-
from earlier
results
adhesion.5
Increasing
3 dynes/cm2
the adhesion
adhesion.27
the wall stress was doubled,
we observed
no PMNL
adhering
to the untreated
nylon
surface.
The contact
of the nylon-
2 dynes/cm2
reduced
the number
of adherent
PMNL
more than a factor of four on both control
and FMLP-treated
adherent
is
fluid
experiments
are
and large
blood
for the capillary
in the
(even activated
have dropped
complex
The number
when compared
on the number
of PMNL
adhering
to endothethe wall shear stress from approximately
1 to
endothelium.
approximately
.
adhesion.
pension
Thus,
no
in the plane
velocities
Extensive
PPP-incubated
at
DISCUSSION
major
hum.
to
plex fluid mechanics,
the flow system
described
simplifies
the type of forces a FMNL
experiences
and permits
a direct
correlation
of wall shear stress with the level of PMNL/EC
erythrocytes
Mm/sec
(D). 0.52 to 0.65 tm/sec
(E). 0.65 to 0.78 him/sec
(F). 0.78
to 0.91
zm/sec
(G). and 0.91 to 1.04 aim/sec
(H). To generate
a
representative
velocity
distribution
for each experiment.
the normalized
velocity
distribution
from two to four image
frames
of
videotape
(separated
in each case by at least one minute)
were
averaged.
Histograms
from experiments
under the same conditions were
again averaged
to create
a final normalized
velocity
distribution.
Error bars represent
SEM.
found
of PMNL.
By eliminating
attached
force
due
in the microcirculation.26
level and may explain
in part
venules
and not in capillaries.
A
PMNL
forces
coincides
adhesion
to endothelium
occur only after shear
PMNL
is in accord
drag.2526
0
HUVEC
endothe-
stresses
the PMNL/EC
with
AND ESKIN
C3bi
facilitating
increased
receptors
for effectively
the
the adhesion
of
for the complement
attaching
phagocytosis
if blood
surface,
PMNL3#{176} (Fig
5).
fragments
C3b
to opsonized
of bacteria.31’32
surfaces
However,
and
when
nylon activates
the C3 convertase
enzyme,
the C3b and C3bi
fragments
formed
probably
bind to nylon’s
polysaccharidelike surface,
possibly
forming
an “opsonized”
surface
for the
PMNL
to attach
An unattached
would
be moving
there
layer,
to.
FMNL
near
at a velocity
were no interactions
with
as was the case of PMNL
the wall
of several
of our flow chamber
hundred
jsm/sec
the endothelial
near the nylon
if
cell monosurface.
In
From www.bloodjournal.org by guest on October 28, 2014. For personal use only.
FLOW
EFFECTS
contrast,
ON
the
endothelium
rolling
are
considerably
there
In addition,
ring.
rolling
ity
dynes/cm2
ity
on
PMNL
stress.
to
subjected
FMNL
velocities
at either
experiments,
was to broaden
experiments
that
20%
occur-
monolayer
after
coincubation
of mean
a133 have
reported
mean
rolling
veloc-
of adhesion
marginate
is a property
of leukocytes
from
the bulk flow in the
some
ments
with
mobility
along
PMNL
to
the
adhering
be cell
suggests
the
PMNL/HUVEC
ofrolling
and show
PMNL
Since
to flow,
retain
of the
both FMLF
and
wall shear stress
ofvelocities
a large
fraction
it is possible
that
PMNL
interaction
adhesive
that allows
circulation
vessel
but
In our
we
observed
no rolling
experiwhat
FMNL,
than
treated
and
be more
readily
were
were
not
et
the strength
FMNL
flowing
arrested.
near
However,
very
threshold
shear
FMLP-treated,
close
to each
to HUVEC
suggests
that, while there
IL 1 and FMLP-treated
formed
cell
Charo
observed
between
PMNL
and control
endothelium.
indicated
the
to untreated,
adhesion
types,
increase
be lower,
would
HUVEC
of PMNL
cell
C5a
determined
to endothelium.
A stronger
adhesive
stimulated
endothelium
implies
that
would
our data
adhesion
were many
endothelium,
necessarily
rolling
Addi-
stresses
for
and IL I-
other.
The
at different
pattern
shear
stresses
more adhesive
the adhesive
stronger
sites on
bonds
than
on
the
though
untreated
conditions
in the
blood
rarely
occur,
conducted
in
studies
have
involved
in
leukocyte
adhesion
to endothelium
but say little about
relative
importance
of different
forms of activation
under
wide range
of physiologic
conditions
FMNL
encounter
Our
stresses
data,
can
stimulated
the type and strength
of the bonds
and EC or the actual
forces on the
whether
or not the FMNL
will
static
most studies
of leukocyte
adhesion
have been
systems
without
controlled
flow effects.
These
revealed
much about the biochemical
interactions
vivo.
which
nature
tionally,
PMNL
Even
downlevel
them
to
yet still
wall.
is of a different
interaction.
is known
about
between
PMNL
that
determine
velocities
two
and
that
an endothelial
HUVEC.
consistently
were
rolling
of PMNL
roll
this intermediate
to nylon,
spreading
rolling
from
of the
FMLF
adhesion
with
the
assay
FMNL
only slight differences
were
velocities
on FMLP-treated
rate as well as to raise the mean
rolling
cells. The velocity
distributions
in all the
similar
attached
of PMNL
interaction
that
the EC monolayer
downstream.
stream
due
Little
formed
interface
interaction
to FMLF-pretreatment
the distribution
analyzed
were
to 90%
of the
appeared
to separate
flow rate examined.
With
the effect of increased
higher
flow
of attached
a centrifugation
needed
dynes/cm2.
due
Employing
force
and Born’0 have reported
that in vivo granulovelocities
exhibit
a strong
flow dependence.
there was no significant
difference
in average
rolling
HUVEC
control
to 0.98
adhere.9”
the
dependence
The
on
which
a wall
shear
stress
of 1.96
double
the mean rolling
veloc-
was approximately
Atherton
cyte
rolling
Unexpectedly,
at the
velocity
shear
a strong
we report
1 tm/sec,
than
adhesive
show
subjected
of PMNL
of FMNL
less
is a strong
wall
1289
velocities
our data
velocity
of
ADHESION
mean
that
indicates
PMNL-EC
control
showing
cause
and
determining
where
changes
in wall
in PMNL
HUVEC,
flow-perhaps
the presence
as
slight
changes
unstimulated
of blood
significant
that
large
the
the
in
shear
adhesion
suggest
to both
that
the local
by vessel dilation-may
of inflammatory
mediators
the arrest
of leukocytes
be as
in
occurs.
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