|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 95 No. 3 (February 1), 2000:
pp. 911-920
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Sequential binding of CD11a/CD18 and CD11b/CD18 defines neutrophil
capture and stable adhesion to intercellular adhesion
molecule-1
Eric R. Hentzen,
Sriram Neelamegham,
Geoffrey S. Kansas,
Jennifer A. Benanti,
Larry V. McIntire,
C. Wayne Smith, and
Scott I. Simon
From the Speros Martel Section of Leukocyte Biology, Department of
Pediatrics, Baylor College of Medicine, Houston, TX; The Cox Laboratory
for Biomedical Engineering, Institute of Biosciences and
Bioengineering, Rice University, Houston, TX; Department of Chemical
Engineering, State University of New York, Buffalo, NY; and the
Department of Microbiology and Immunology, Northwestern University
Medical School, Chicago, IL.
 |
Abstract |
The relative contributions of CD11a/CD18 and CD11b/CD18 to the
dynamics and strength of neutrophil adhesion to intercellular adhesion
molecule (ICAM)-1-transfected cells were examined over the time course
of chemotactic stimulation. Suspensions of neutrophils and
transfectants were sheared in a cone-plate viscometer, and formation of
heterotypic aggregates was measured by 2-color flow cytometry. The
2-body collision theory was used to compute adhesion efficiency,
defined as the proportion of collisions between neutrophils and target
cells that resulted in capture. ICAM-1 surface density and shear rate
both regulated adhesion efficiency. Target cells expressing
approximately 1000 ICAM-1 sites/µm2 (Ilow)
were captured with an efficiency of 0.15 at 100 s 1,
which decreased to zero at 300 s1. At 8-fold higher
ICAM-1 expression (Ihigh) corresponding to levels measured
on interleukin-1-stimulated endothelium, efficiency was 0.3 at 100 s1 and remained above background to 900 s1. Shear alone was sufficient for CD11a/CD18-mediated
adhesion to ICAM-1, and stimulation with
formyl-methionyl-leucyl-phenylalanine boosted capture efficiency
through CD11a/CD18 by 4-fold. In comparison, CD11b/CD18 supported one
third of this efficiency, but was necessary for aggregate stability
over several minutes of shear and at shear stresses exceeding 5 dyne/cm2. Hydrodynamics influenced capture efficiency
predominantly through the collisional contact duration, predicted
to be approximately 9 milliseconds for successful capture of
Ilow and 4 milliseconds for Ihigh. The
implication is that an increase in ICAM-1 from resting levels to those
on inflamed endothelium effectively increases the permissible shear in
which capture through  2-integrins may occur. Neutrophil
adhesion to ICAM-1 appears to be a cooperative and sequential process
of CD11a-dependent capture followed by CD11b-mediated stabilization.
(Blood. 2000;95:911-920)
© 2000 by The American Society of Hematology.
| |
Introduction |
The multistep model of leukocyte emigration provides a
conceptual framework describing the molecular recognition events that lead from cell capture to adhesion strengthening and transmigration. Neutrophils use selectins to tether to inflamed endothelium and subsequently arrest at specific sites in the vasculature through binding of the 2-integrins.1,2 The
2-integrin family members CD11a/CD18 (LFA-1) and
CD11b/CD18 (Mac-1) are involved in firm adhesion of
neutrophils to endothelial cells.3 Intravital microscopy in
mice4 and rats5,6 has shown that CD11a and
CD11b each contribute significantly to firm adhesion, and concurrent
blocking of both 2-integrins is required for complete
inhibition of human and canine neutrophil
transmigration.3,7,8 ICAM-1 (CD54) is a member of the
immunoglobulin superfamily that is widely expressed on vascular
endothelium and certain parenchymal cells in response to activation
with cytokines or endotoxin.9-11 ICAM-1 has been shown to be the primary endothelial adhesive ligand for
CD11a/CD18,12 whereas CD11b/CD18 binds ICAM-1 and
at least 1 other undefined ligand in supporting arrest and
transmigration.13
The efficiency and location of cell recruitment are apparently
regulated through the interplay of hemodynamics and the binding and
mechanical properties intrinsic to adhesion receptors.2 Integrin activation is required to augment cellular adhesion in numerous experimental systems.14 At shear rates
characteristic of normal blood flow, selectin-mediated leukocyte
rolling can be converted rapidly to shear-resistant stable adhesion
through integrin binding.1,15 This pattern of adhesive
interactions is observed for neutrophil emigration in response to acute
inflammatory stimuli in the microcirculation16-18 and in
flow chambers.19,20 Direct adhesion through
2-integrins without selectin tethering may significantly
contribute to emigration under low shear conditions that predominate in
microvascular beds of highly perfused organs such as
liver21,22 and lung23 and more generally in the
peripheral microcirculation during inflammation.24
CD11b/CD18 can mediate firm attachment of neutrophils under flow
conditions within seconds of chemotactic
stimulation.3,25-27 CD11a/CD18 on lymphocytes can likewise
effect attachment within seconds.28
Our objective in this study was to determine the hydrodynamic
influences of shear rate (which determines the duration of
intercellular contact) and shear stress (which translates to the
compressive and tensile forces at the adhesive contact region) on
adhesion mediated by CD11a/CD18 and CD11b/CD18 binding to ICAM-1 on
target cells. ICAM-1 was expressed in a murine pre-B cell
line29 lacking other neutrophil adhesion molecules such as
selectins or their carbohydrate ligands.30 Two clones were
selected that spanned an 8-fold range in ICAM-1 level, reflecting
expression on resting and cytokine-stimulated endothelia. This report
focuses on the sequence of molecular events that supports
2-integrin-mediated neutrophil adhesion to ICAM-1 under
defined hydrodynamic shear.
We used a rotational viscometric technique to expose cell suspensions
to defined shears simulating those experienced by leukocytes traversing
the microcirculation. Suspensions of neutrophils and ICAM-1 target
cells were exposed to a linear gradient of velocity streamlines in a
cone-plate viscometer. Intercellular collisions occurred as cells
traveling near the rotating cone overtook those in slower streamlines
adjacent to the stationary plate. The viscometer's defined shear field
allowed theoretical predictions of the frequency of cell-cell
collisions, the average duration of collisions, and stresses exerted on
aggregates. Stable adhesion occurred when the strength of bonds formed
during collisional contact was greater than tensile forces generated as
aggregates tumbled in the shear field. The numerical efficiency of this
process was derived experimentally from the rate of aggregation as
measured by 2-color flow cytometry.
| |
Materials and methods |
Materials
Formyl-methionyl-leucyl-phenylalanine (fMLP) and Ficoll were
purchased from Sigma (St. Louis, MO); 20% formaldehyde solution was
from Tousimis Research (Rockville, MD). ZK36 374, a stable prostacyclin (PGI2) derivative that inhibits platelet
activation and aggregation, was a gift from Schering Company (Berlin,
Germany). Fluorescein isothiocyanate (FITC)-labeled antibodies to CD45
and CD54 were from Caltag Laboratories (Burlingame, CA). Anti-CD11a monoclonal antibody (mAb) R3.1 (IgG1) and anti-ICAM-1 domain 2 mAb
R6.5 (IgG2a) were generously provided by Dr Kei Kishimoto (Boehringer-Ingelheim, Ridgefield, CT). Lora Whitehouse (Repligen, Cambridge, MA) generously provided humanized anti-CD11b mAb 60.1 (denoted h60.1, IgG1). This isotype does not bind significantly to
human neutrophils through FcR. Fab fragments of R6.5 and R3.1 were
produced using an ImmunoPure Fab kit from Pierce (Rockford, IL). Of
note, use of R6.5Fab was required to block ICAM-1 because whole IgG
failed to inhibit adhesion completely. Anti-CD11c mAb Bly6 (IgG1) was
from Pharmingen (San Diego, CA). Anti-CD50 mAb ICR1.1 was obtained from
Dr Donald Staunton (ICOS, Bothell, WA). W6/32, an IgG2a that binds to
human HLA class I, was from American Type Culture
Collection (Manassas, VA). In all experiments, blocking antibodies were used at saturating concentrations: R3.1 at 10 µg/mL;
h60.1, R6.5Fab, Bly6, and W6/32 at 20 µg/mL; and ICR1.1 at
30 µg/mL. The blocking ability of these mAbs at saturation concentrations has been documented previously.31,32
Cell preparation
Following the protocol approved by the Baylor Institutional Review
Board, human blood was collected by venipuncture into a sterile syringe
containing 10 U/mL heparin (Elkins-Sinn, Cherry Hill, NJ). Neutrophils
were isolated using Mono-Poly resolving medium (ICN Biomedicals,
Aurora, OH), as described previously.33 They were kept at
4°C in Ca++-free HEPES buffer (110 mmol/L NaCl, 10 mmol/L KCl, 10 mmol/L glucose, 1 mmol/L MgCl2, and 30 mmol/L HEPES, pH 7.35) containing 0.1% human serum albumin (Armour
Pharmaceuticals, Kankanee, IL) and were used within 4 hours. Indicators
of activation including cell shape change, L-selectin shedding, or
CD11b/CD18 up-regulation were observed in less than 5% of the isolated
neutrophil population after incubation of cell suspensions at 37°C
for 20 minutes, indicating that they remained in a resting state.
Additionally, aggregation in response to shear alone of
neutrophils kept at room temperature was within 5% of that observed
for those kept on ice before the experiment.
Murine pre-B lymphocytes, 300.19 cells, were stably transfected with
human ICAM-1 cDNA using a modified SRa vector containing a neomycin
resistance marker. Positive clones were identified by flow cytometry
using R6.5 mAb. The 300.19 cells were cultured in RPMI medium (Gibco,
Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine
serum (Hyclone, Logan, UT), 1% penicillin-streptomycin (Gibco), and
100 µmol/L 2-mercaptoethanol (Fischer, Fair Lawn, NJ). Before the
experiment, the cells were pelleted and washed twice in HEPES buffer
plus 0.1% human serum albumin and 1.5 mmol/L CaCl2, and
were kept at room temperature for up to 4 hours.
Determination of ICAM-1 binding sites
R6.5 mAb was conjugated with FITC using the FluoReporter kit from
Molecular Probes (Eugene, OR). A binding curve was generated to
ascertain the concentration of R6.5-FITC sufficient for saturation of
binding sites. Labeled cells were washed and analyzed on the FL1
fluorescence channel on a FACScan flow cytometer with Cell Quest
analysis software (Becton Dickinson, San Jose, CA). The absolute number
of cell surface binding sites was quantified using Quantum Simply
Cellular microbeads (Flow Cytometry Standards, San Juan, PR), which
have calibrated numbers of goat anti-mouse IgG sites, as described
previously.34 ICAM-1 site densities were also confirmed
using commercially available anti-CD54-FITC (Caltag, Burlingame, CA).
ICAM-1 levels on 300.19 transfectants were compared with those on
first-passage human umbilical vein endothelial cells (HUVEC) grown to
confluence in culture dishes, as described previously.33
Confluent HUVEC were brought into suspension with 0.05% trypsin and
0.02% EDTA in phosphate-buffered saline and washed twice in HEPES
buffer. Unstimulated HUVEC labeled with anti-CD54-FITC expressed
2.3 × 105 sites per cell, a level comparable to the
Ilow 300.19 clone. Stimulation of the HUVEC for 4 hours
with interleukin-1 resulted in an increase in ICAM-1 expression to
8.5 × 105 sites per cell, a copy number
approximately half that detected on the Ihigh 300.19 clone.
In similar labeling experiments, expression of ICAM-2, ICAM-3, or
L-selectin by 300.19 cells was not detectable (data not shown).
Cone-plate viscometry
Aggregation assays were performed at 37°C in a Haake VT550
cone-plate viscometer (Haake, Paramus, NJ), consisting of a stationary plate beneath a rotating truncated cone with an angle of 2°. This apparatus applies a uniform and linear shear field to the entire fluid
sample in the gap between the cone and plate.35 Shear rate
and shear stress are related through the fluid viscosity as  = µ
G, where is the shear stress in dyne/cm2, G is the
shear rate in s1, and µ is the viscosity in
poise.36 In some experiments, buffer viscosity was
increased from approximately 0.7 to 1.7 cp by the addition of 6%
(wt/vol) Ficoll, a high-molecular-weight hydrophilic polymer
of sucrose.
Determination of aggregation
To resolve the cellular composition of aggregates, we incubated
neutrophils and 300.19 cells with spectrally distinct fluorescent labels. The 300.19 cells were stained with the vital nucleic acid dye
LDS-751 (Molecular Probes) at a concentration of 0.5 µg/mL for
detection in the red (FL3) channel. Neutrophils were labeled with 5 µg/mL anti-CD45-FITC for detection in the green (FL1) fluorescence channel. These labels did not affect 2-integrin
expression on resting neutrophils and did not alter the increase in
2-integrin expression or adhesivity that occurred with
fMLP stimulation of the cells.32,37 Neutrophils or 300.19 cells were preincubated with anti-CD11a (R3.1 Fab or whole), anti-CD11b
(h60.1), anti-CD11c (Bly6), anti-CD54 (R6.5Fab), anti-CD50 (ICR1.1), or
anti-HLA class 1 (W6/32) for 10 minutes at room temperature. Excess
LDS-751 was removed from 300.19 cells by centrifugation. The 2 cell
populations were mixed in HEPES buffer containing 0.1% human serum
albumin and 1.5 mmol/L CaCl2 and were introduced into the
viscometer at final concentrations of 1 × 106
neutrophils/mL and 2 × 106 300.19 cells/mL. The
suspension was equilibrated at 37°C for 2 minutes before shear was
applied at rates from 40 s1 to 1500 s1. In some experiments, 1 µmol/L fMLP was added 1 second before application of shear. At prescribed time points, 40-µL
samples were obtained by pipet and immediately fixed in 150 µL cold
0.5% formaldehyde in HEPES buffer. For whole-blood aggregation assays, 60 nmol/L ZK36 374 was added to the syringe with heparin before venipuncture. Neutrophils in whole blood were labeled with
anti-CD45-FITC before mixing with LDS-751-labeled 300.19 cells. Whole
blood/300.19 cell suspensions were diluted 1:5 with HEPES buffer before
addition to the viscometer to allow adequate flow cytometric detection of binding events.38
Neutrophil and 300.19 cell populations were identified by flow
cytometry based on their characteristic side versus forward scatter.
Quantification of heterotypic aggregation between neutrophils (N) and
300.19 cells (I) was performed by analysis of dot plots of green versus
red fluorescence (Figure 1).
Homotypic doublets (N2) or larger aggregates
(N3+) composed solely of neutrophils, as well as
heterotypic aggregates composed of 1 300.19 cell and either 1 (IN1), 2 (IN2), or 3 or more neutrophils
(IN3+), were resolved. Flow cytometry and light microscopy
documented that 300.19 cells did not aggregate with each other. The
fraction of neutrophils in heterotypic aggregates was quantified as
follows: % neutrophils in heterotypic
aggregates = (IN1 + 2IN2 + 3IN3+)/(N1 + 2N2 + 3N3+ + IN1 + 2IN2 + 3IN3+).
Aggregates containing more than 3 neutrophils typically represented
less than 10% of the total neutrophil population and were grouped into
IN3+, resulting in an estimated error of less than 3% in
the preceding equation.
View larger version (29K):
[in this window]
[in a new window]
| Fig 1.
Flow cytometric detection of neutrophil-ICAM-1 cell
aggregation.
Neutrophils (1 × 106 cells/mL) were fluorescently
labeled green (CD45-FITC) and ICAM-1 300.19 cells
(2 × 106 cells/mL) were labeled red (LDS-751) for
10 minutes at room temperature. The 2 cell populations were combined in
the cone-plate viscometer and equilibrated to 37°C in buffer
containing 1.5 mmol/L Ca++ for 2 minutes before stimulation
with 1 µmol/L fMLP and initiation of fluid shear. Samples were taken
at prescribed time points and immediately fixed in 0.5% cold
formaldehyde. Two-color flow cytometry was used to detect distinct
populations of single neutrophils (N) and 300.19 ICAM-1 cells (I);
homotypic neutrophil doublets (N2) and triplets
(N3); and 2-color heterotypic aggregates containing a
single 300.19 cell bound to 1 (IN), 2 (IN2), or 3 or more
neutrophils (IN3+). Representative dot plot depicts
aggregation of neutrophils with Ihigh at 600 s1 at 1 minute after stimulation and initiation of
shear. Each dot represents a single particle event containing 1 or more
cells.
|
|
Adhesion efficiency
The probability with which colliding cells bind to each other and
form stable aggregates is termed adhesion efficiency.25 Mathematically, this is expressed as follows: Adhesion
efficiency = (number of collisions resulting in adhesion per unit
time)/(total number of collisions per unit time).
The numerator is derived from flow cytometric quantitation of the
kinetics of heterotypic aggregation over the first 60 seconds after
application of shear. The denominator is estimated using 2-body
collision theory to predict the number of collisions per unit time, as
described previously.31,34 Adhesion efficiency computed in
this manner accounts for influences of the magnitude of shear rate and
particle volume fraction on the frequency of collisions. As such, it is
solely a function of biologic and biophysical properties of the cell,
such as extent of activation, adhesion receptor affinity, receptor
topography, and tensile strength of receptor-ligand bonds. For typical
experiments, neutrophils (ri = 3.75 µm) were sheared
with 300.19 cells (rj = 5.5 µm) at a concentration ratio (neutrophils/300.19) of approximately 0.5. Under these
conditions, heterotypic collisions occur approximately 6 times more
frequently than homotypic collisions.
We wish to state an erratum in a previous article.34 In
previous publications using this model, the total number of collisions per unit time was calculated using a prefactor of 2/3 instead of the
correct constant of 4/3 from 2-body collision theory.39 As
a result, the collision frequency estimated was half the correct value,
and the adhesion efficiencies were twice those actually fit to the
kinetic aggregation data. This error was carried over to 2 other
articles and a review published by the authors.25,31,40 Therefore, a correction factor of 0.5 must be applied to
the efficiencies computed in those manuscripts.
Statistics
Data are presented as mean values, and error bars indicate standard
error of the mean. One-way analysis of variance was used for multiple
comparisons and t tests for comparisons between 2 groups.
Posttests were performed using the Newman-Keuls method. P < .05 was considered significant.
| |
Results |
Detection and kinetics of neutrophil adhesion to 300.19 cells
expressing ICAM-1
Application of shear and chemotactic stimulation resulted in the
formation of homotypic neutrophil aggregates containing up to 3 or more
neutrophils (labeled with anti-CD45-FITC) and heterotypic aggregates
consisting of a single 300.19 cell (labeled with LDS-751) bound to 1 or
more neutrophils (Figure 1). The 300.19 cells were not activated after
application of shear or fMLP and did not homotypically aggregate. Two
300.19 clones were selected based on their surface expression of
ICAM-1. These clones were denoted Ilow and
Ihigh and expressed approximately
2.2 × 105 and 1.7 × 106 ICAM-1
sites per cell, respectively, compared with HUVEC, which expressed
2.3 × 105 sites per cell before and
8.5 × 105 sites per cell after 4 hours of
stimulation with interleukin-1. The nontransfected parent cell line did
not express detectable surface ICAM-1.
The kinetics of heterotypic aggregation were measured over a range of
shear rates typical for the microcirculation (40 s1
to 1500 s1). Neutrophil adhesion to parent
cells peaked at approximately 25% singlet recruitment and was
significantly above background only at the low shear rate of 90 s1 (Figure 2A). This
adhesion was inhibited by anti-CD18 but not by anti-ICAM-1 mAbs (data
not shown). Adhesion to ICAM-1-transfected 300.19 cells was more rapid
and extensive. For example, at 90 s1, 50% of
neutrophils adhered to Ilow and 75% to Ihigh
within 1 minute of stimulation (Figures 2B and 2C). Maximum aggregation with Ihigh was maintained at shear rates to 600 s1 and decreased to background levels as shear was
increased to 1500 s1. Neutrophil aggregation with
Ilow was maximal up to 300 s1 and
decreased to baseline at 900 s1. Adhesion
mediated by 2-integrins and ICAM-1 was more stable than neutrophil-neutrophil adhesion. Whereas breakup of homotypic aggregates began within 2 minutes and was complete by 5 minutes at shear rates above 400 s1,25,34 neutrophil-300.19-ICAM-1
aggregates were maintained under these conditions. For example,
aggregation with Ihigh was stable for 15 minutes at
shear rates less than 600 s1 (Figure 2C). At shear
rates greater than 600 s1, the peak extent of
heterotypic aggregation was diminished.
View larger version (20K):
[in this window]
[in a new window]
| Fig 2.
The kinetics of heterotypic aggregation following shear
and fMLP stimulation.
Neutrophils (1 × 106 cells/mL) and 300.19 cells
(2 × 106 cells/mL) were labeled, stimulated, and
sheared as described in Figure 1. Aggregation was monitored as
described in "Materials and Methods." Heterotypic aggregation is
presented as the percentage of total neutrophils bound to a 300.19 cell. Aggregation kinetics over a range of shear rates as denoted are
plotted for the parent 300.19 cell line (A) and for transfected clones
expressing ICAM-1 at (B) low levels (Ilow) and (C) 8-fold
higher levels (Ihigh). *P < .05 compared with
peak aggregation at the same shear rate. Data are presented as
mean ± SEM from 3 to 7 separate experiments.
|
|
The efficiency of neutrophil capture of ICAM-1 cells provides a measure
of the intrinsic properties of the cells that determine its adhesivity
by accounting for physical parameters that regulate collision
frequency, such as shear rate, aggregate size, and concentrations of
neutrophils and 300.19 cells. Figure 3
shows the dependence of adhesion efficiency on shear rate and ICAM-1
expression. Peak efficiency occurred at the lowest shear rate and
diminished with increased shear. For example, stable adhesion between
neutrophils and Ihigh at 40 s1 occurred
in 27 of 100 collisions, versus 15 of 100 for Ilow. These
efficiencies were significantly higher than the 9 in 100 collisions
between neutrophils and the parent cells that resulted in capture.
Comparison of exponential decay functions used to fit efficiency versus
shear rate indicated that neutrophils captured Ihigh twice
as effectively as Ilow. Furthermore, the efficiency of
adhesion to Ihigh was significantly above baseline up to
900 s1, as compared with 300 s1
for Ilow. The efficiency of adhesion to non-ICAM-1 ligands
expressed on parent 300.19 cells approached zero at 200 s1. Adhesion efficiency decreased directly with
increased shear rate over a log range in ICAM-1 from approximately
2 × 105 to 2 × 106 receptors
per cell. Together these data indicate that the efficiency and extent
of 300.19 cell recruitment into aggregates were dependent upon ICAM-1
expression and the magnitude of applied shear rate.
View larger version (19K):
[in this window]
[in a new window]
| Fig 3.
Adhesion efficiency as a function of shear rate and
ICAM-1 expression level.
The kinetics of heterotypic aggregation over the first 60 seconds of
fMLP stimulation and initiation of shear were fit with a mathematical
model at each shear rate, as described in "Materials and
Methods." Figure shows the efficiency of neutrophil capture of the
parent 300.19 cell line and of transfected clones expressing low
(Ilow) and high (Ihigh) levels of ICAM-1. The
experimental data were fit as smooth curves with a first-order
exponential decay function. Data are presented as mean ± SEM from
3 to 7 separate experiments.
|
|
Shear-induced adhesion of unstimulated neutrophils to ICAM-1
Unstimulated neutrophils have been shown to adhere to
purified ICAM-1 in a parallel plate flow chamber.3 In the
absence of chemotactic stimulation, application of shear resulted in
significant formation of heterotypic aggregates that plateaued within 1 minute and remained stable to 5 minutes (Figure
4A). Most aggregates comprised a single
neutrophil bound to a single 300.19 cell, as compared with the
multicellular aggregates formed after fMLP stimulation (Figure 1).
Subsequent activation by fMLP addition at 2 or 3 minutes after
initiation of shear boosted aggregation to the same peak level
(approximately 75%) observed for concurrent application of shear and
fMLP. Preincubation of neutrophils with anti-CD11a significantly
inhibited shear-induced adhesion at all shear rates tested (Figure 4B).
Significant inhibition of unstimulated adhesion with anti-CD11b was
detected only at 600 s1. However, CD11b/CD18 did
contribute to the stability of adhesion because disaggregation occurred
by 5 minutes with anti-CD11b (Figure 4A). Further, complete inhibition
of aggregation at each shear rate required concomitant blocking of
CD11a and CD11b (Figure 4B).
View larger version (20K):
[in this window]
[in a new window]
| Fig 4.
Shear-induced adhesion of unstimulated neutrophils to
ICAM-1 transfectants.
Neutrophils (1 × 106 cells/mL) were preincubated
for 10 minutes with saturating concentrations of monoclonal antibodies
to CD11a or CD11b. They were then mixed with Ihigh
(2 × 106 cells/mL), added to the cone-plate
viscometer, and allowed to equilibrate for 2 minutes before initiation
of fluid shear. Samples were taken at prescribed time points, fixed,
and detected by flow cytometry as described in "Materials and
Methods." (A) Kinetics of aggregation are plotted for
Ihigh at a shear rate of 466 s1. (B)
Peak extent of shear-induced aggregation occurred within 1 minute of
initiation of shear and is plotted for Ihigh over a range
of shear rates and antibody blocking conditions. *P < .01
compared with control at the same shear rate; **P < .05
compared with control and with concurrent block of CD11a and CD11b at
the same shear rate. (C) Data from Figure 4B were modeled to calculate
adhesion efficiency over the first 60 seconds after initiation of
shear, as described in "Materials and Methods." Smooth lines
represent curves fit to experimental data with a first-order
exponential decay function. *P < .05 compared with control
at the same shear rate. Data are presented as mean ± SEM from 3 to 6 separate experiments.
|
|
Modeling of adhesion efficiency of unstimulated neutrophils to 300.19 cells revealed that 7 of 100 collisions at the lowest shear rate
resulted in capture of Ihigh (Figure 4C), a significantly greater level than that observed for adhesion to parent cells (< 2%). As indicated earlier, shear-induced capture of
ICAM-1-expressing cells was attributed entirely to CD11a/CD18 because
no significant difference from control efficiency was detected upon
blocking of CD11b.
To determine whether constitutive adhesion attributed to CD11a/CD18 was
due to activation of neutrophils during isolation, we assessed
heterotypic aggregation in whole blood. Venous blood was drawn into a
syringe containing heparin and the prostaglandin analogue ZK36 374,
which inhibits activation and aggregation of platelets.38
Whole blood was diluted 1:5 in HEPES buffer, mixed with
Ihigh cells (2 × 106/mL), and sheared
in a manner identical to that in the isolated cell suspension
experiments. Kinetics of shear-induced heterotypic aggregation were
comparable to those of isolated neutrophils, with a peak heterotypic
aggregation of 50% ± 6% at 600 s1 (n = 3).
Anti-CD11a blocked this aggregation and anti-CD11b had no effect. We
also examined the possibility of activation through release of
chemotactic stimuli by 300.19 cells. Pretreatment of neutrophils with
pertussis toxin to block Gi -mediated signal transduction
did not inhibit shear-induced adhesion to Ihigh, but did
block the boost in avidity after fMLP stimulation (data not shown).
Together these data indicate that neutrophils can constitutively bind
ICAM-1 through CD11a/CD18 without chemotactic activation.
Relative contributions of CD11a/CD18 and CD11b/CD18 in
neutrophil adhesion to ICAM-1 with stimulation
2-Integrins were required for neutrophil adhesion to
Ilow and Ihigh cells over the time course of
fMLP stimulation (Figure 5). For adhesion
to Ilow at a shear rate of 90 s1,
blocking of CD11b did not significantly alter the rate of aggregation, and CD11a/CD18 alone supported adhesion up to the peak level observed for untreated control at 1 minute (Figure 5A). However, after maximum
aggregation at 1 minute, disaggregation proceeded more rapidly in the
presence of anti-CD11b as compared with untreated control. In
comparison, CD11b/CD18-dependent adhesion (in the presence of
anti-CD11a) proceeded more slowly and reached a level only
approximately 20% of control. These aggregates remained stable over 5 minutes (Figure 5A).
View larger version (21K):
[in this window]
[in a new window]
| Fig 5.
Contributions of CD11a and CD11b to neutrophil-ICAM-1
adhesion over the time course of fMLP stimulation.
Neutrophils (1 × 106 cells/mL) were preincubated
with saturating concentrations of anti-CD11a, anti-CD11b, or both
concurrently. They were then mixed with Ihigh
(2 × 106 cells/mL), added to the cone-plate
viscometer, and allowed to equilibrate for 2 minutes before stimulation
with 1 µmol/L fMLP and initiation of fluid shear. Samples were taken
at prescribed time points, fixed, and detected by flow cytometry as
described in "Materials and Methods." The contribution of each
2-integrin subunit to capture of 300.19-ICAM-1 was
assessed under the following conditions: (A) Ilow at 90 s1, (B) Ihigh at 90 s1, and (C) Ihigh at 600 s1. *P < .05 compared with control at
the same time point. Data are presented as mean ± SEM from 3 to 6 separate experiments.
|
|
Adhesion to Ihigh at 90 s1 was also
dependent on 2-integrins and, as indicated earlier,
blocking of CD11b did not significantly alter the initial boost in
aggregate formation (Figure 5B). Anti-CD11a effectively slowed the rate
of aggregation as demonstrated for Ilow. However, the
increased availability of ICAM-1 on Ihigh markedly increased the extent of CD11b/CD18-dependent capture. In contrast to
Ilow, aggregation with Ihigh was sustained for
more than 5 minutes, demonstrating persistence of adhesion through
either CD11a/CD18- or CD11b/CD18-ICAM-1 bonds at this relatively low shear rate.
An increase in shear rate up to 600 s1 resulted in a
pattern of adhesion to Ihigh resembling that to
Ilow at 90 s1 (Figure 5C). A significant
contribution to the initial boost in aggregation was again evident only
for CD11a/CD18-dependent adhesion. Aggregation was reversible in the
presence of anti-CD11b, as observed for the low shear and low ICAM-1
condition. Together these data suggest that CD11a/CD18 alone is
sufficient for the initial boost in neutrophil adhesion at low and high
ICAM-1 surface density over a wide range of shear rates. The
contribution of CD11b/CD18 was most apparent in maintaining the
stability of formed aggregates after initial tethering through
CD11a/CD18. A distinct contribution from CD11b/CD18 in capture of
ICAM-1 cells was detected only at high ICAM-1 surface density or at low
shear rates corresponding to relatively long intercellular contact durations.
Specific molecular interactions support neutrophil adhesion to
ICAM-1
We next determined which neutrophil adhesion molecules bound ICAM-1
expressed on 300.19 cells. The peak extent of aggregation after fMLP
stimulation at an optimal shear rate of 600 s1 is
plotted in Figure 6A for samples pretreated
with a panel of antibodies. Under these conditions, approximately 65%
of the neutrophil population formed aggregates with Ihigh,
as compared with 7% for adhesion to parent cells. Addition of
anti-ICAM-1decreased aggregation to background, as did simultaneously
blocking CD11a and CD11b; these results indicate that adhesion through
non-ICAM-1 ligands was minimal. Addition of anti-CD11a alone also
decreased heterotypic aggregation to background. In contrast, blocking
CD11b decreased aggregation by a lesser but significant amount
(approximately 40%). In control experiments, we blocked CD11c, a third
2-integrin subunit that supports leukocyte adhesion to
stimulated endothelium,32 ICAM-3 (CD50), which serves as a
major ligand for LFA-1 in homotypic neutrophil
aggregation,40 and the neutrophil HLA class 1 receptor. None of these molecules contributed to heterotypic aggregation. Under
these conditions, CD11a/CD18 is necessary and sufficient for
collisional adhesion to ICAM-1; however, both CD11a/CD18 and CD11b/CD18
are required for optimal adhesion.
View larger version (25K):
[in this window]
[in a new window]
| Fig 6.
Receptors that support neutrophil adhesion to ICAM-1.
Neutrophils (1 × 106 cells/mL) were preincubated
with saturation concentrations of blocking antibodies, mixed with
either Ihigh or parent 300.19 cells
(2 × 106 cells/mL), stimulated with 1 µmol/L
fMLP, and sheared in the cone-plate viscometer. (A) Peak heterotypic
aggregation at 1 minute after stimulation with fMLP and initiation of
shear at 600 s1 is compared for saturating
concentrations of blocking antibodies as denoted. The open bar depicts
binding to the nontransfected parent 300.19 cell.
*P > .05 compared with other bars with the
same symbol. (B) Adhesion efficiency for aggregation mediated through
CD11a/CD18 and CD11b/CD18. The kinetics of heterotypic aggregation over
the first 60 seconds after stimulation with fMLP and initiation of
shear were fit with a mathematical model at each shear rate. Smooth
lines represent curves fit to experimental data with a first-order
exponential decay function. *P < .05 compared with control
at the same shear rate. Data are presented as mean ± SEM from 3 to 6 separate experiments.
|
|
Activation boosted the efficiency of neutrophil adhesion to 300.19 ICAM-1 cells approximately 4-fold above unstimulated cell suspensions
(Figures 4 and 6B). As observed for unstimulated neutrophils, adhesion
efficiency over the first minute of stimulation was supported almost
entirely by CD11a/CD18. Only at the lowest shear did CD11b/CD18 significantly bind 300.19-Ihigh cells, at approximately one
third the efficiency of CD11a/CD18. Concurrent blocking of CD11a and ICAM-1 indicated that adhesion was largely attributable to
binding through CD11b/CD18-ICAM-1.
Effects of shear stress on neutrophil adhesion to ICAM-1
The efficiency of neutrophil adhesion to ICAM-1 300.19 cells was
found to decrease with increasing shear rate (Figures 4 and 6B).
According to 2-body collision theory, as shear rate is increased, cells
in suspension collide more frequently and with shorter encounter duration, which could limit adhesion by constraining the number of
integrin-ICAM-1 bonds formed. Alternatively, the predominant effect of
increased shear rate may be the accompanying increase in fluid drag and
translation of tensile force to the intercellular contact region. This
may independently limit capture efficiency by quickly rupturing the
bonds initially formed during transient contact. To examine the effects
of an increase in shear stress, we raised the buffer viscosity by
adding Ficoll. The increase in viscosity from 0.7 to 1.7 cp is expected
to result in approximately a 2.5-fold increase in shear stress at a
given shear rate.
Plots of peak heterotypic aggregation versus shear stress assumed
similar patterns in low- and high-viscosity buffers. Aggregation remained constant to a threshold level of shear stress of approximately 5 dyne/cm2 for Ihigh and approximately 2.5 dyne/cm2 for Ilow (Figure
7). The predominant influence of stress on
adhesion was revealed above these respective thresholds, where adhesion to each cell line steadily decreased as stress was increased. The
influence of shear rate was revealed by the significantly increased
aggregation of suspensions in the high-viscosity as compared with the
low-viscosity buffer, as the latter was sheared approximately 2.5 times
faster at a given shear stress.
View larger version (23K):
[in this window]
[in a new window]
| Fig 7.
The effect of shear stress on neutrophil adhesion to
ICAM-1.
Heterotypic aggregation of neutrophils (1 × 106
cells/mL) and 300.19 cells (2 × 106 cells/mL) was
measured as described previously in normal HEPES buffer of viscosity
0.7 cp (filled symbols) or in buffer augmented with 6% Ficoll to
increase viscosity to 1.7 cp (open symbols). Peak heterotypic
aggregation at 1 minute after stimulation with 1 µmol/L fMLP and
initiation of shear is plotted versus shear stress for Ilow
(circles) and Ihigh (squares). *P < .05
compared with maximum value on the same curve. Data are presented as
mean ± SEM from 3 to 6 separate experiments.
|
|
Strength of adhesion through CD11a/CD18- and CD11b/CD18-ICAM-1
bonds
The effects of increased stress on aggregates formed through
CD11a/CD18 or CD11b/CD18 were examined by blocking each with antibody.
Plots of peak aggregation versus shear stress indicated that either
CD11a/CD18 or CD11b/CD18 can support adhesion at stresses less than 4 dyne/cm2 (Figure 8A). At
stresses above this threshold, adhesion in low-viscosity buffer through
either subunit alone decreased at similar rates, reaching background by
7 dyne/cm2. In high-viscosity buffer, adhesion through
CD11a/CD18 was potentiated and supported 3-fold more aggregation than
CD11b/CD18 at stresses above 4 dyne/cm2. Analysis of
adhesion efficiency indicated that CD11a/CD18-mediated capture of
ICAM-1 cells was actually more efficient in high-viscosity as compared
with low-viscosity buffer (0.37 versus 0.22 at 90 s1). Together the data suggest that shear stress can
limit 2-integrin-ICAM-1 bond formation and that
CD11a/CD18 can support adhesion at 3-fold higher stress than
CD11b/CD18.
View larger version (22K):
[in this window]
[in a new window]
| Fig 8.
Strength of adhesion through CD11a/CD18 and CD11b/CD18.
Neutrophils (1 × 106 cells/mL) were preincubated
with anti-CD11a ( ) or anti-CD11b ( ) for 10 minutes
at saturating concentrations and then mixed with 300.19 Ihigh cells (2 × 106 cells/mL).
Suspensions were stimulated with 1 µmol/L fMLP and sheared in the
cone-plate viscometer over a range of shears. Peak extent of
heterotypic aggregation in the presence of anti-CD11b or anti-CD11a was
measured and plotted at each shear stress. Aggregation is compared for
experiments performed in (A) normal HEPES buffer of viscosity 0.7 cp or
(B) buffer of viscosity 1.7 cp augmented with 6% Ficoll. Data are
presented as mean ± SEM from 3 to 6 separate
experiments.
|
|
| |
Discussion |
The major findings of this study are as follows: (1) Neutrophils
constitutively adhered to ICAM-1 cells through CD11a/CD18 in isolated
suspensions and in whole blood; (2) CD11a/CD18 accounted for most of
the capture efficiency under shear conditions, whereas CD11b/CD18
supported stable adhesion over minutes of chemotactic stimulation; and
(3) chemotactic stimulation increased the efficiency of
CD11a/CD18-dependent adhesion by 4-fold. This efficiency was optimal at
the lowest applied shear and decreased as shear was increased. Thus, we
provide evidence that cooperation between CD11a/CD18 and CD11b/CD18 is
based at least in part on CD11a/CD18 functioning as a tether to sustain
contact duration sufficient for CD11b/CD18 binding.
Efficiency of 2-integrin-dependent neutrophil
adhesion
Previous studies using rotational viscometry have found that the
efficiency of homotypic neutrophil adhesion was 0.1 at 100 s1 and increased to a peak level of 0.4 between 400 s1 and 800 s1.34 When
L-selectin was blocked with antibody, adhesion was solely dependent on
CD11a/CD18 and CD11b/CD18 and the efficiency was again 0.1 at 100 s1, but in this case it dropped steadily to 0 by 400 s1.40 In the present study, efficiency
of neutrophil adhesion to 300.19-Ilow was also
approximately 0.1 at 100 s1 and dropped to 0 by 400 s1. Efficiency of capture of
300.19-Ihigh at 100 s1 was increased to
approximately 0.3 and remained above 0 at 900 s1.
These data demonstrate that increases in ICAM-1 site density increase
neutrophil adhesion efficiency in the absence of selectin tethering.
Chemotactic stimulation boosts the efficiency of ICAM-1 capture
predominantly through CD11a/CD18
Neutrophils exhibited a capacity to adhere to 300.19-ICAM-1 cells
in the presence of shear and absence of chemotactic stimulation. Both
isolated neutrophils and those in whole blood constitutively bound
300.19-ICAM-1 cells over a range of shear. The binding of CD11a/CD18
alone was sufficient for neutrophil capture, as addition of anti-CD11b
produced minimal inhibition at most shears. Chemotactic stimulation
increased the efficiency of adhesion almost 4-fold from the
unstimulated case. This boost was attributed to CD11a/CD18 because
anti-CD11b again had little effect. However, total inhibition of
adhesion required blocking of both 2-integrins. A
contribution of CD11b/CD18 to aggregate stability was apparent within
several minutes of shear in the presence and absence of fMLP
stimulation. This pattern of 2-integrin-mediated
adhesion is different from that reported in a previous study that
described adhesion to a murine melanoma cell line expressing ICAM-1
(E3-ICAM-1).31,37 Neutrophil adhesion to the E3-ICAM-1
cells was supported equally by CD11a/CD18 binding of ICAM-1 and
CD11b/CD18 binding of undefined ligands. In contrast, adhesion to
300.19-ICAM-1 was almost entirely dependent on expression of ICAM-1,
with less than a 15% contribution of CD11b/CD18 binding to non-ICAM-1 ligands.
Chemotactic stimulation increases the avidity of CD11a/CD18 to
bind ICAM-1
Recently published data have shown that chemotactic stimulation can
induce activation of CD11a/CD18 on lymphocytes over a time scale of
seconds, resulting in a transition from rolling to firm arrest through
binding to ICAM-1.28 The current study provides the first
evidence of a rapid increase in the avidity of CD11a/CD18 on
neutrophils after chemotactic stimulation. The precise mechanisms
underlying this boost in avidity remain unknown but appear to involve a
rapid increase in the binding of CD11a/CD18 to ICAM-1 at the site of
collisional contact rather than diffusion of constitutive or newly
activated receptor. This hypothesis is based on the fact that CD11a
expression, which is comparable to CD11b on resting neutrophils,
remains unchanged after chemotactic stimulation. Moreover, the
predicted minimum contact duration at which adhesion to
300.19-Ihigh cells was detected is on the order of 4 milliseconds at 600 s1 (e.g., tcontact  2.6/shear rate).34,41 This interval is insufficient for
significant numbers of CD11a/CD18 sites to diffuse into the region of
cell-cell contact.42,43
Sequential recognition events characterize the kinetics and
stability of adhesion of 2-integrins to ICAM-1
Shear rate and ICAM-1 availability were the predominant parameters
regulating the formation and stability of heterotypic aggregates. At
low shear rate and high ICAM-1 expression, either CD11a/CD18 or
CD11b/CD18 alone supported adhesion to 300.19 cells. However, differences in function became apparent when these parameters were
varied to limit receptor-ligand availability and collisional contact
duration. CD11a/CD18 exhibited greater avidity in both stimulated and
unstimulated states and was sufficient for optimal capture of ICAM-1 at
high and low expression levels over a range of shears. Adhesion in the
presence of CD11a/CD18 alone, however, was not stable over time in
shear. Conversely, CD11b/CD18 was less than half as efficient as
CD11a/CD18 in capture but supported stable adhesion for up to 15 minutes of shear. When CD11b was blocked and shear was greater than 300 s1, adhesion was reversible within 2 minutes (Figure
5). These data suggest that within minutes of fMLP stimulation,
CD11a/CD18 undergoes a reversal in its ability to both form and
maintain bonds with ICAM-1 and that CD11b/CD18 stabilizes adhesion over
time. The mechanism underlying the cooperation between CD11a and CD11b
in mediating optimum adhesion to ICAM-1 remains unknown. One
possibility is that initial high-avidity binding of CD11a/CD18
facilitates subsequent engagement of lower-avidity CD11b/CD18 sites by
prolonging contact duration and increasing membrane contact area.
Alternatively, CD11a/CD18 binding may initiate intracellular signaling,
leading to a shear-dependent increase in CD11b/CD18 avidity. It has
been reported that CD11a/CD18 directly signals the release of reactive oxygen species in adherent neutrophils.44
Shear stress and shear rate differentially influence adhesion
through CD11a/CD18 and CD11b/CD18
A distinct threshold in stress was evident for each 300.19-ICAM-1
clone (Figure 7). Maximal adhesion to Ihigh was maintained to 5 dyne/cm2, twice the level for Ilow. Drag
force exerted on an aggregate suspended in a newtonian fluid is
predicted to increase linearly with an increase in shear rate or
viscosity.36 At stresses above the respective threshold
values, peak aggregation decreased in a linear manner (Figure 7). In
contrast, the nonlinear relation between shear rate and collisional
contact duration correlated with an exponential decline in adhesion
efficiency as shear rate was increased (Figure 3).34,41 The
dominant influence of contact duration on adhesion is illustrated in
the peak aggregation versus shear stress plots of Figure 8. At a given
stress, cells in high-viscosity buffer were sheared at a rate
approximately 2.5 times slower than in low-viscosity buffer. The
resulting prolonged collisional contact duration in high-viscosity
buffer doubled the extent of aggregation. CD11a/CD18 supported
aggregation at stresses greater than 10 dyne/cm2, whereas
adhesion through CD11b/CD18 fell to background above 5 dyne/cm2. At comparable bond density, the greater strength
of adhesion through CD11a/CD18 may be attributed to greater tensile
strength of CD11a/CD18-ICAM-1 bonds than of CD11b/CD18-ICAM-1 bonds.
Alternatively, CD11a/CD18 may bind more rapidly and form more
individual bonds during collision. The latter hypothesis appears more
likely given the capacity of CD11a/CD18 to support adhesion at higher
shear rates and low ICAM-1 surface density.
On the basis of the kinematics of 2 spherical particles in a linear
shear field,41 we estimated the minimum contact duration necessary for neutrophil capture of 300.19 cells to be approximately 13 milliseconds for Ilow and less than 4 milliseconds for
Ihigh. These predictions do not account for cell
deformation, which could prolong collision duration. The implication
for neutrophil emigration is that an increase in ICAM-1 expression from
levels at rest to those in inflamed endothelium could effectively raise
the permissible shear rate (i.e., decrease the minimum encounter
duration) at which 2-integrin-mediated capture is
likely to occur. Kinematic analysis of a spherical cell colliding with
a planar substrate predicts that the contact duration in the absence of
subsequent adhesion is approximately 0.1/shear rate.45 This
is 25 times shorter than for 2 cells colliding in suspension. Flow
chamber studies of activated neutrophils arresting on purified ICAM-1 have indicated that adhesion occurred at shear rates less than 28 s1.46 This corresponds to a minimum
contact duration of approximately 4 milliseconds, a value in close
agreement with the current estimate for CD11a/CD18-supported capture of
300.19-Ihigh cells in suspension.
In this study, we demonstrate that CD11a/CD18 on neutrophils is
constitutively active and increases its avidity for ICAM-1 within
seconds of chemotactic stimulation. Optimum adhesion was dependent on
chemotactic activation of CD11a/CD18 and CD11b/CD18, which bind
sequentially to support neutrophil capture and stable adhesion.
Efficiency of capture was influenced separately by ICAM-1 density and
fluid shear rate and stress. The implication is that during ischemic or
inflammatory events in the microcirculation, the efficiency of
neutrophil capture and arrest may be regulated by hydrodynamic and
molecular parameters.
| |
Acknowledgments |
We would like to thank Dr Robert Rothlein and Dr Kei Kishimoto of
Boehringer-Ingelheim Pharmaceuticals for generously supplying the
antibody reagents used.
|
Top
Abstract
Introduction