|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 5 (September 1), 1998:
pp. 1626-1638
Hydrodynamic Shear Shows Distinct Roles for LFA-1 and Mac-1 in
Neutrophil Adhesion to Intercellular Adhesion Molecule-1
By
Sriram Neelamegham,
Andrew D. Taylor,
Alan R. Burns,
C. Wayne Smith, and
Scott I. Simon
From the Section of Leukocyte Biology, Baylor College of Medicine,
Houston; Institute of Biosciences and Bioengineering, Rice University,
Houston, TX; and the Department of Chemical Engineering, State
University of New York, Buffalo, NY.
 |
ABSTRACT |
The binding of neutrophil  2 integrin to intercellular
adhesion molecule-1 (ICAM-1) expressed on the inflamed endothelium is
critical for neutrophil arrest at sites of tissue inflammation. To
quantify the strength and kinetics of this interaction, we measured the
adhesion between chemotactically stimulated neutrophils and
ICAM-1-transfected mouse cells (E3-ICAM) in suspension in a cone-plate
viscometer at shear rates typical of venular blood flow (100 s 1 to 500 s1). The kinetics of
aggregation were fit with a mathematical model based on two-body
collision theory. This enabled estimation of adhesion efficiency,
defined as the probability with which collisions between cells resulted
in firm adhesion. The efficiency of
2-integrin-dependent adhesion was highest (~0.2) at
100 s1 and it decreased to approximately zero at 400 s1. Both LFA-1 and Mac-1 contributed equally to adhesion
efficiency over the initial 30 seconds of stimulation, but adhesion was
entirely Mac-1-dependent by 120 seconds. Two hydrodynamic parameters
were observed to influence integrin-dependent adhesion efficiency: the
level of shear stress and the intercellular contact duration. Below a
critical shear stress (<2 dyn/cm2), contact duration
predominantly limited adhesion efficiency. The estimated minimum
contact duration for 2-integrin binding was
approximately 6.5 ms. Above the critical shear stress (>2 dyn/cm2), the efficiency of neutrophil adhesion to E3-ICAM
was limited by both the contact duration and the tensile stress. We
conclude that at low shear, neutrophil adhesion is modulated
independently through either LFA-1 or Mac-1, which initially contribute
with equal efficiency, but differ over the duration of chemotactic stimulation.
© 1998 by The American Society of Hematology.
| |
INTRODUCTION |
THE ATTACHMENT of neutrophils to the
vascular endothelium and their passage to sites of inflammation
involves a sequence of events mediated by at least three classes of
adhesion molecules: selectins, integrins, and members of the Ig gene
superfamily.1 Attachment has been modeled as a multistep
process involving neutrophil tethering, rolling, and firm
adhesion.2,3 While the tethering and rolling of neutrophils
on the endothelium is mediated by members of the selectin
family,4-6 cell arrest is dependent on two members of the
2-integrin family, LFA-1
( L2, CD11a/CD18) and Mac-1 (M2, CD11b/CD18).7,8
Intercellular adhesion molecule-1 (ICAM-1, CD54), a member of the Ig
gene superfamily expressed on the vascular endothelium, has been
identified as a major ligand for both of the CD18
integrins.9,10 The interaction between the 2
integrins and ICAM-1 accounts for 50% to 70% of the neutrophil adhesion to endothelial cells.10,11 The remaining adhesion has been attributed to Mac-1 binding unknown ligands and to non-CD18 integrin-dependent adhesion mechanisms.7,11,12
Currently, little is known about the dynamics of
2-integrin-dependent neutrophil adhesion under defined
shear flow. For example, what are the critical hydrodynamic parameters
(shear rate, intercellular contact duration, and tensile stress) that
modulate the rate of neutrophil recruitment onto the endothelium? Also,
what are the relative contributions of LFA-1 and Mac-1 to the strength
and binding kinetics of neutrophil adhesion with time after
stimulation? To investigate these issues, we developed an assay in
which cell suspensions are sheared in a cone-plate viscometer and
aggregation rates are quantitated by fluorescence flow cytometry. This
technique offers two major advantages over the conventional
parallel-plate flow chamber assay used to quantitate leukocyte
recruitment on substrates of endothelial cell monolayers or purified
ligands.8,13 (1) Cone and plate viscometry of cell
suspensions enables estimation of the number of cell-cell collisions
based on two-body collision theory. This enables computation of the
efficiency with which cell-cell collisions result in aggregate
formation.14,15 In contrast, in the parallel-plate flow
chamber, it is difficult to determine the flux of cells interacting
with the planar substrate, since it depends not only on the bulk cell
concentration, but also on the distance from the chamber entry point
and the shear rate applied.16 (2) The amount of time that a
leukocyte is activated before entry into the parallel plate flow
chamber is difficult to determine due to its dependence on the
residence time of the cells in the tubing before entry into the flow
chamber. In the suspension assay, the entire cell population is
activated at the same time, allowing the relationship between cell
adhesivity and time to be determined with a resolution of approximately
1 second.
The objective of this report is to examine the kinetics and strength of
formyl peptide (formyl-methionyl-leucyl-phenylalanine [FMLP])-stimulated neutrophil adhesion to ICAM-1-transfected mouse cells (E3-ICAM) under conditions where the shear rate, hydrodynamic forces, and encounter frequencies were precisely controlled. The experimental methodology combined with two-body collision theory allowed us to estimate adhesion efficiency under conditions in which
the functions of Mac-1 and LFA-1 were assessed. Although several recent
reports have investigated the biophysics of transient adhesion through
the selectins,17-19 this is the first study that examines
the kinetics and strength of adhesion through 2-integrin receptor bond formation independent of tethering through selectins. We
conclude here that at low shear, LFA-1 and Mac-1 contribute equally to
neutrophil adhesion initially, but their relative contributions differ
markedly over the time course of chemotactic stimulation.
| |
MATERIALS AND METHODS |
Materials.
Paraformaldehyde (FMLP) and Ficoll were purchased from Sigma Chemical
(St Louis, MO). Glutaraldehyde was obtained from Polysciences (Warrington, PA). Fluorescently labeled antibody to CD45
(CD45-fluorescein isothiocyanate [FITC]) was obtained from Becton
Dickinson Immunocytometry Systems (San Jose, CA), and nuclear acid
stain LDS-751 was purchased from Molecular Probes (Eugene, OR).
Anti-CD11a monoclonal antibody (MoAb) R3.1 (IgG1), anti-ICAM-1 domain
2 MoAb R6.5 (IgG2a), and anti-ICAM-1 domain 1 MoAb RR1/1 (IgG1) were
generous gifts from Dr Robert Rothlein (Boehringer-Ingelheim
Pharmaceuticals, Ridgefield, CT), and anti-ICAM-1 domain 3 MoAb
CBRIC1/7 (IgG1) was kindly provided by Dr Charles A. Parkos (Emory
University, Atlanta, GA). A humanized anti-CD11b MoAb 60.1 (denoted
h60.1, IgG1) was provided by Lora Whitehorse (Repligen, Cambridge, MA)
and L-selectin-blocking MoAb LAM1-3 was kindly supplied by Cell
Genesys (Foster City, CA). Fab fragments of MoAb R6.5, R3.1, and LAM1-3
were produced by digestion with papain and purified by passage over a
protein-A-Sepharose column using an ImmunoPure Fab preparation kit
from Pierce (Rockford, IL). In all of the adhesion experiments, unless
otherwise mentioned, the blocking MoAbs were added at saturating
concentrations: h60.1, RR1/1, and CBRIC1/7 were used at 20 µg/mL,
MoAb R6.5 Fab was used at 25 µg/mL, and R3.1 Fab and LAM1-3 Fab were
added at 30 µg/mL.
Cell preparation.
Fresh human blood was collected by venipuncture into a sterile syringe
containing 10 U/mL of heparin. Neutrophils were isolated using a
Ficoll-Hypaque density gradient (Mono-Poly resolving medium; Flow
Laboratories, McLean, VA) as previously described20 and kept at 4°C in Ca2+-free HEPES buffer for up to 3 hours
before the experiment. The purity of isolated neutrophils was more than
90% and the viability measured by trypan blue exclusion was more than
99%.
The parent mouse melanoma cell line B78H1 and the human ICAM-1
transfected cells (abbreviated by E3-ICAM) were generously provided by
Dr Lloyd H. Graf (University of Illinois, Chicago).21 These
cells were maintained in Dulbecco's modified Eagle media (D-MEM;
GIBCO, Grand Island, NY) with 10% fetal calf serum (FCS; Hyclone,
Logan, UT), 1% penicillin-streptomycin (GIBCO), and 10 mmol/L HEPES
(GIBCO). Transfected cells were selected in D-MEM media with 300 µg/mL Geneticin (GIBCO). Before each experiment, the B78H1 cells were
detached from the tissue culture substrate using Hanks' balanced salt
solution (HBSS; Sigma) containing 5 mmol/L EDTA (Sigma). Cells were
then pelleted by centrifugation (10 minutes at 250 g),
resuspended in HBSS buffer, and kept at 4°C. Viability for the mouse
cell line was determined to be approximately 95% by trypan blue
exclusion. Furthermore, fluorescent microscopy demonstrated that
E3-ICAM cells express a uniform distribution of ICAM-1 on their surface
(data not shown).
Fluorescent conjugation of MoAb and determination of ICAM-1-binding
sites.
The R6.5 Fab fragments were conjugated with a fluorescent cyanine dye,
Cy3, using the fluorolink-antibody Cy3 labeling kit from Amersham Life
Sciences (Pittsburgh, PA). To determine the equilibrium binding
constant, Kd, of R6.5 Fab, the labeled antibody at
various concentrations was added to the E3-ICAM cells at a concentration of 6 × 106 cells/mL. The mixture was
incubated at room temperature for 20 minutes before being washed once
and read on a FACScan flow cytometer (Becton Dickinson Immunocytometry
Systems, San Jose, CA). The MoAb equilibrium-binding constant,
Kd, was determined to be 0.94 µg/mL as described
earlier.22 R6.5 Fab conjugated with Cy3 was incubated with
the parent B78H1 cells in control experiments.
Detection of the total number of ICAM-1 sites on the E3-ICAM cells was
performed using whole R6.5 MoAb labeled with FITC using a QuickTag FITC
Conjugation Kit (Boehringer-Mannheim, Indianapolis, IN). The number of
cell-surface-binding sites was determined using Quantum Simply
Cellular microbead standards purchased from Flow Cytometer Standards
(Research Triangle Park, NC). These uniform microbeads have a
calibrated number of goat-antimouse IgG sites on their surface. Both
the calibrated microbeads and the E3-ICAM cells were labeled with
R6.5-FITC at saturating concentrations. The number of binding sites per
cell was then determined by quantifying the fluorescence intensity of
the labeled cells and translating this value to the number of bound
antibodies using the microbead standards.
Cone-plate viscometry.
Both the homotypic and heterotypic aggregation assays were performed in
a cone-plate viscometer (Ferranti Electric, Commack, NY). The device
consists of a stationary plate placed beneath a rotating cone
maintained at 37°C.14,15 A cone angle of 1° was used in
the current study, and the gap between the cone and plate ranged from
less than 10 µm at the center to 610 µm at the outside edge.
Neutrophil and E3-ICAM cell suspensions were placed in this gap before
the experiment, cells were stimulated with FMLP, and shear was
immediately applied. The geometry of the viscometer enables application
of a uniform and linear shear rate to the entire cell suspension.
The neutrophil E3-ICAM cell suspension behaves like a newtonian fluid
and its shear stress varies linearly with shear rate as follows: shear
stress = viscosity · shear rate. The viscosity of the HEPES buffer
was measured at 0.7 cp (or 0.007 poise) at 37°C in a Brookfield
(Stoughton, MA) cone-plate viscometer. A shear rate of 100 s1 in this system would thus correspond to a shear
stress of 0.7 dyn/cm2 (0.007 × 100). In some
experiments, the buffer viscosity was increased by addition of Ficoll
(400,000 molecular weight; Sigma), a neutral hydrophilic polymer of
sucrose commonly used in density gradients for cell separation.
Addition of 6% (wt/vol) Ficoll to the HEPES buffer increased media
viscosity to 1.7 cp at 37°C. A shear rate of 100 s1 in
these experiments corresponds to a shear stress of 1.7 dyn/cm2.
Homotypic aggregation assay.
Homotypic neutrophil aggregation experiments were performed as
previously described.15,23 Briefly, neutrophil suspensions were stimulated with 1 µmol/L FMLP and sheared in a cone-plate viscometer. Aliquots of 30 µL were taken at each sampling time point
for up to 3 minutes after stimulation and immediately fixed in 200 µL
of 2% glutaraldehyde. A FACScan flow cytometer was used to analyze the
particle distributions of fixed cell suspensions. Singlet neutrophils
and aggregates were resolved using autofluorescence due to
glutaraldehyde fixation, and aggregates were quantitated as integral
multiples of the singlet fluorescence channel. The particle
distribution of neutrophil aggregates were determined using the
histograms of fluorescence intensity. The extent of homotypic
aggregation was expressed as the fraction of singlets recruited into
larger aggregates:
where the concentration of neutrophil singlets or aggregates
with i cells is denoted by [Ni] (cell
mL1). [N6+] is the
concentration of sextuplets and larger aggregates that were grouped
since they could not be resolved by flow cytometric analysis. In the
results presented here, aggregates with six or more neutrophils
accounted for less than 5% of the total particles.
Heterotypic aggregation assay.
In the heterotypic aggregation assay, neutrophils and E3-ICAM (or
B78H1) cells were labeled with spectrally distinct fluorescent stains.
Neutrophils were labeled with 5 µg/mL anti-CD45-FITC for detection
in the green (FL1) fluorescence channel, and E3-ICAM (or B78H1) cells
were stained for 15 minutes at 25°C with the vital nucleic acid dye
LDS-751 (0.5 µg/mL) for detection on the red (FL3) fluorescence
channel. Independent experiments showed that neither of these
fluorescent reagents either altered (1) the expression level of
2 integrin on resting neutrophils, (2) the rate of
change of 2-integrin expression following stimulation, or (3) the adhesivity of 2 integrin (data not shown).
After labeling, excess LDS-751 label was removed by a brief (5 to 6 seconds at 3,000g) centrifugation of E3-ICAM (or B78H1) cells.
The two cell populations (typically between 3 × 106 to
6 × 106 cells/mL) were then mixed and incubated for 2 minutes in buffer containing 1.5 mmol/L Ca2+. The combined
sample was stimulated with 1 µmol/L FMLP and sheared in a cone-plate
viscometer as described earlier.14,15 Cell-suspension aliquots of 40 µL were taken at desired time points and fixed in 100 µL of 0.5% cold paraformaldehyde to avoid autofluorescence interfering with dual-color fluorescence discrimination.
Labeling the cell populations with spectrally fluorescent dyes allowed
the quantitation of homotypic neutrophil aggregates, as well as
heterotypic neutrophil-E3-ICAM (or B78H1) aggregates by flow cytometric
analysis (Fig 1). The neutrophil and
E3-ICAM (or B78H1) population was isolated by gating on their
characteristic forward versus side scatter. Aggregation between
neutrophils and E3-ICAM (or B78H1) cells was quantitated by analyzing
the dot plot between the green (due to CD45-FITC) and red (due to
LDS-751) fluorescence channels (Fig 1C). Neutrophils are denoted by
N, while the E3-ICAM (or B78H1) cells are denoted by I. This technique allowed us to resolve the homotypic neutrophil aggregate
population of doublets (denoted by [N2]), and
aggregates of three or more cells (denoted by
[N3+]). Both flow cytometric
detection and light microscopy observations showed that neither the
B78H1 cells nor the transfected E3-ICAM cells aggregate homotypically
following application of shear. However, these cells formed heterotypic
aggregates with neutrophils. The population of E3-ICAM (or B78H1) cells
was resolved into singlets and into aggregates composed of a single
melanoma cell bound to either one, two, or more than two neutrophils.
The concentrations of these particles is represented by
[I], [IN1],
[IN2], and
[IN3+], respectively. Typically, less
than 2% of the ICAM-1 cells appear off scale in the dot plot. Most of
the experiments in this study (except Fig 1E) were performed in the
presence of onefold excess E3-ICAM cells, ie, 3 × 106
neutrophils/mL were stimulated and sheared with 6 × 106
E3-ICAM cells/mL. Because of this, a majority of neutrophils were
recruited into [IN1] (Fig 1C) and the number of
aggregates in [IN2] and
[IN3+] were relatively low.
[IN3+] typically accounted for less
than 5% of the heterotypic aggregates formed. The percentage of
neutrophil recruitment into heterotypic aggregates was computed as
follows:
|
|
![= <FENCE><FR><NU>[<IT>IN</IT><SUB>1</SUB>] + 2[<IT>IN</IT><SUB>2</SUB>] + 3[<IT>IN</IT><SUB>3</SUB><SUP>+</SUP>]</NU><DE>[<IT>N</IT><SUB>1</SUB>] + 2[<IT>N</IT><SUB>2</SUB>] + 3[<IT>N</IT><SUB>3</SUB>] + [<IT>IN</IT><SUB>1</SUB>] + 2[<IT>IN</IT><SUB>2</SUB>] + 3[<IT>IN</IT><SUB>3</SUB><SUP>+</SUP>]</DE></FR></FENCE> · 100 (2)](/content/vol92/issue5/fulltext/1626/img004.gif)
|
|
View larger version (43K):
[in this window]
[in a new window]
| Fig 1.
Flow cytometric detection of heterotypic aggregation
kinetics. neutrophils (3 × 106 cells/mL) were labeled
green (CD45-FITC) and E3-ICAM cells (6 × 106
cells/mL) were stained red (LDS-751) for 15 minutes at room
temperature. Excess LDS-751 label was removed by centrifugation, cell
populations were equilibrated in 37°C buffer containing 1.5 mmol/L
Ca2+ for 2 minutes, stimulated with 1 µmol/L FMLP, and
sheared in a cone-plate viscometer at a shear rate of 200 s1. Samples were withdrawn at indicated time points,
fixed with 0.5% cold paraformaldehyde, and analyzed on a flow
cytometer. (A) Neutrophil and E3-ICAM gated on their characteristic
forward versus side scatter. (B) Initial particle distribution at time
zero. (C) Heterotypic aggregate distribution 90 seconds after
stimulation and application of shear. (D) Kinetics of
neutrophil-E3-ICAM aggregation for the experiment depicted in A
through C. Dotted and solid lines denote the percentage of neutrophils
in heterotypic and homotypic aggregates, respectively. (E) Kinetics of
aggregation for a representative experiment where 3 × 106
neutrophils/mL were stimulated and sheared with 1.5 × 106
E3-ICAM cells/mL. (F) Adhesion efficiency (±SEM) for
neutrophil-neutrophil and neutrophil-E3-ICAM collisions calculated
from three independent experiments described in D and E.
|
|
Adhesion efficiency.
The rate at which neutrophils are incorporated into aggregates (as
determined in equations 1 and 2) is not only dependent on the biologic
properties of the cell that modulate its adhesivity, but also on the
physical parameters of the system, which include the concentration and
radius of the cells, and the applied shear rate. To quantify neutrophil
adhesion under various experimental protocols, independent of the
physical parameters, we estimated an index termed as adhesion
efficiency (equation 3).15
|
(3)
|
Briefly, adhesion efficiency is defined as the fraction of
intercellular collisions that result in firm adhesion and is always  1. It was estimated by fitting the data from homotypic and
heterotypic aggregation experiments over the first 30 seconds after the
application of shear with a mathematical model based on two-body
collision theory. The total number of collisions (denominator) in
equation 3 is dependent on the cell concentration, applied shear rate, and cell radius. For example, in the case two of unequal sized cells of
radius ri and rj, at
concentrations Ci and Cj,
respectively, the collision frequency, fij (no. of
collisions per second), at a shear rate G (1/s) is given by
fij = 2/3(ri + rj)3
CiCjG. Therefore, for
the case of neutrophils (ri = 3.75 µm) and
E3-ICAM cells (rj = 6 µm) being sheared at a
relative cell concentration ratio, (Neutrophil)/(E3-ICAM) = 0.5, the
number of heterotypic collisions is expected to exceed the homotypic
collisions by approximately 4.4 times. The number of effective
collisions (numerator) is measured based on the experimental
aggregation kinetics. Adhesion efficiency estimated by this methodology
is solely a function of the intrinsic biologic properties of the cell
that determine its adhesivity. Important among these properties are the
number, affinity, and distribution of adhesive receptors expressed on
the cell surface, their response to applied shear, and the time after
stimulation. This technique has previously been applied to estimate the
efficiency of homotypic neutrophil aggregation.14,15 In the
current report, we have extended it to estimate heterotypic adhesion
efficiency of neutrophil-E3-ICAM collisions.
Transmission electron microscopy.
Neutrophil and E3-ICAM cell samples were prepared as described
previously.24 Briefly, cell suspensions were fixed in 2% glutaraldehyde at room temperature for 30 minutes and postfixed for 1 hour in phosphate-buffered saline (PBS) containing 1% osmium tetroxide. Cells were then dehydrated in a graded series of ethanol and
embedded in LX-112 (Ladd Research Industries, Burlington, VT). After
polymerization, ultrathin sections were obtained on an RMC 7000 ultramicrotome (RMC, Tucson, AZ) equipped with a diamond knife.
Sections were stained with uranyl acetate and lead citrate before being
viewed on a JEOL 200CX electron microscope. The perimeter of the cells
and the length of the intercellular contacts were measured using a
SummaSketch III tablet (Seymour, CT) and Bioquant software (R&M
Biometrics, Nashville, TN). The contact length index was defined as
follows: (intercellular contact length)/(mean perimeter of the adherent
cells). This index for homotypic aggregates was measured from 33 randomly chosen aggregates from three donors, while the contact length
index for neutrophil-E3-ICAM aggregates was obtained from 12 images
from a single donor.
Statistics.
Data were analyzed using analysis of variance (ANOVA). Posttests were
performed using the Student-Newman-Keuls test and P values less
than .05 were considered significant.
| |
RESULTS |
Kinetics of heterotypic aggregation of neutrophils and
ICAM-1-transfected cells.
In the absence of any stimulus, shearing cells in the cone-plate
viscometer did not lead to aggregation. Less than 5% of the neutrophils were observed to form homotypic aggregates and less than
2% of the E3-ICAM cells were incorporated into aggregates with
neutrophils (data not shown). However, when stimulated by 1 µmol/L
FMLP and sheared in a cone-plate viscometer, neutrophils were rapidly
incorporated both into homotypic aggregates and into heterotypic
aggregates with E3-ICAM transfectants (Fig 1). The aggregate size and
composition was measured by flow cytometry over the time course of
stimulation. Neutrophil and E3-ICAM transfectants were gated on their
characteristic forward versus side scatter (Fig 1A). At time zero, all
of the neutrophils were observed to elicit only green fluorescence
(CD45-FITC), while the E3-ICAM transfectants elicited red fluorescence
(LDS-751) (Fig 1B). Following stimulation and application of shear at
200 s1 for 90 seconds, homotypic and heterotypic
aggregates were observed in the red versus green fluorescence dot plot
(Fig 1C).
The rate and extent of aggregation is a function of the initial cell
concentration. Under conditions where stimulated neutrophils were
sheared with onefold excess of E3-ICAM cells ([N]/[I] = 0.5), the
frequency of heterotypic collisions is estimated to be approximately 4.4 times that of homotypic neutrophil collisions. Neutrophils were
more rapidly recruited by E3-ICAM cells than by other neutrophils. Following 60 seconds of stimulation, approximately 75% of the neutrophils were incorporated into heterotypic aggregates (Fig 1B and
D). Most aggregates were composed of single neutrophils with E3-ICAM
cells (~70%), while the rest of the E3-ICAM aggregates were attached
to two or more neutrophils. E3-ICAM cells did not form aggregates with
each other. The aggregation kinetics varied with neutrophil and E3-ICAM
cell concentrations. For example, when neutrophils were stimulated and
sheared with half as many E3-ICAM cells ([N]/[I] = 2, Fig 1E),
approximately 40% of the neutrophils formed homotypic aggregates,
while only approximately 20% were in heterotypic aggregates at the
30-second time point. Neutrophil homotypic aggregation peaked at 30 seconds, beyond which point they appeared to be recruited into
heterotypic aggregates by E3-ICAM cells.
To quantify the adhesion between neutrophils and E3-ICAM cells
independent of experimental parameters including cell concentration, aggregate size, and shear rate applied, we estimated adhesion efficiency. The kinetic data presented in Fig 1D and E were modeled over the first 30 seconds of stimulation, and adhesion efficiency was
computed. Efficiency estimated in this fashion was equivalent for both
the [N]/[I] cell ratios (Fig 1F). This further confirmed that
adhesion efficiency as computed was invariant to changes in cell
concentrations. At a shear rate of 200 s1, homotypic
collisions between neutrophils were approximately three times more
effective compared to collisions between neutrophils and E3-ICAM cells.
The efficiency of neutrophil-neutrophil collision was approximately
0.25, while that of neutrophil-E3-ICAM collisions was approximately
0.07. We have previously shown that neutrophil-neutrophil adhesion is
dependent on L-selectin and 2 integrin binding their counterligand on opposing neutrophils.14 Blocking
L-selectin function with MoAb, at low shear rates, results in purely
2-integrin-dependent homotypic aggregation. As will be
shown later (Fig 4), the difference in adhesion efficiency between
neutrophil-neutrophil and neutrophil-E3-ICAM can be abolished by
blocking L-selectin.
The fraction of E3-ICAM cells incorporated into heterotypic aggregates
did not change after the first 90 seconds of stimulation (Fig 1D and E)
and the majority of the aggregates remained stable for at least 3 minutes. We tested the strength and stability of these heterotypic
aggregates by allowing aggregate formation at 200 s1 for
30 seconds before diluting the cell suspension in 20-fold excess buffer
containing 1 µmol/L FMLP and 1.5 mmol/L Ca2+ (data not
shown). This abrupt reduction in cell concentration caused the
collision frequency to decrease by approximately
400-fold,15 without affecting the disaggregation kinetics.
We observed less than a 10% change in the aggregate size distribution
between 30 seconds and 180 seconds. This implies that heterotypic
aggregates formed in the first 30 seconds remained stable and resistant
to shear up to 180 seconds.
Molecular requirements for neutrophil-E3-ICAM aggregation.
Cells were preincubated with MoAb that have been shown to block
adhesion function to determine the relative contributions of Mac-1 and
LFA-1 on neutrophils, and ICAM-1 on transfectants to heterotypic
aggregate formation (Fig 2). In these
experiments, the percentages of neutrophils recruited into heterotypic
aggregates 10 seconds after the application of shear were compared. The
10-second time point was chosen, because this is the earliest sampling
point, and at this time less than 20% of the E3-ICAM cells have
neutrophils adherent to them. Heterotypic aggregate formation before 10 seconds is thus the result of single neutrophils binding directly to
the E3-ICAM cell surface. This is opposed to a situation in which neutrophils bind E3-ICAM when they collide with other neutrophils that
are already adherent on the E3-ICAM cell. The latter phenomenon may
play a significant role at times more than 30 seconds.
View larger version (28K):
[in this window]
[in a new window]
| Fig 2.
Adhesion of neutrophils to E3-ICAM transfectants.
Neutrophils (3 × 106 cells/mL) were mixed with either
E3-ICAM-transfected cells or B78H1 parent cells
(6 × 106 cells/mL), stimulated with 1 µmol/L FMLP,
and sheared at a shear rate of 200 s1. The percentage of
neutrophils in heterotypic aggregates 10 seconds after the application
of shear is compared on addition of a panel of blocking antibodies at
saturation concentration: R3.1 Fab (to LFA-1), h60.1 (to Mac-1), and
R6.5 Fab (to ICAM-1). *The fifth bar represents experiments where R6.5
Fab was added to bind 50% of the ICAM-1 receptors on E3-ICAM cells.
#Not statistically different. Error bars represent SEM from
at least three independent experiments.
|
|
We have previously reported that resting neutrophils express
approximately equal numbers of LFA-1 and Mac-1 receptors (~50,000) on
their cell surface.25 In comparison, the transfected
E3-ICAM cells were found to homogeneously express at least
370,000 ± 50,000 (SD) ICAM-1 sites per cell. Heterotypic
aggregation at a shear rate of 200 s1 was found to be
supported equally by LFA-1 and Mac-1, since addition of MoAbs
R3.1 Fab (to LFA-1) or h60.1 (to Mac-1) blocked aggregation by
approximately 55%. Simultaneous addition of both antibodies completely blocked aggregation, confirming that neutrophil-E3-ICAM adhesion was purely 2-integrin-mediated.
Aggregation studies were performed in the presence of a blocking
antibody to ICAM-1 (R6.5 Fab). This MoAb binds domain 2 of ICAM-1 and
has been previously shown to block both LFA-1-ICAM-1 and Mac-1-ICAM-1
interactions.10 Blocking 50% of the ICAM-1 sites by
addition of this antibody at the measured Kd (0.94 µg/mL) decreased the level of aggregation by only 15%. At saturating concentrations of R6.5 Fab, heterotypic aggregation was inhibited by
approximately 60%. Simultaneously, blocking both LFA-1 and ICAM-1 did
not have any additive effect, while blocking Mac-1 and ICAM-1 inhibited
aggregation to baseline levels.
Mac-1 has previously been shown to bind the third Ig-like domain of
ICAM-1.26 We performed heterotypic adhesion experiments to
determine if the inability of anti-ICAM-1 MoAb R6.5 to completely block adhesion was due to its inability to block domain 3 of ICAM-1, or
due the presence of ICAM-1-independent ligands for Mac-1 on the mouse
cell line. For these experiments, a blocking MoAb CBRIC1/7 against
domain-3 of ICAM-1 was added. This MoAb has been previously shown to
completely inhibit ICAM-1 binding to Mac-1 without significantly affecting binding via LFA-1.27 We observed that addition of CBRIC1/7 alone did not significantly reduce heterotypic aggregation from the levels seen in the absence of any MoAb. Furthermore, addition
of CBRIC1/7 along with R6.5 only partially inhibited (~60%) the
level of neutrophil-E3-ICAM binding (data not shown). E3-ICAM cells
apparently expressed a ligand for Mac-1 other than ICAM-1. This
unidentified ligand for Mac-1 was also constitutively expressed on the
parent B78H1 cells, since the extent of heterotypic aggregation of the
parent cell with neutrophils was similar to that of neutrophil-E3-ICAM
adhesion in the presence of saturating concentrations of R6.5 Fab.
Binding of neutrophils to B78H1 parent cells could be abolished by
addition of anti-Mac-1 MoAb (Fig 2). In our experimental system, LFA-1
on neutrophils apparently binds ICAM-1 on the transfectants, and Mac-1
binding to ICAM-1 could not be resolved. This may be due to Mac-1
binding to ICAM-1 with a lower affinity as compared with LFA-1, as
previously reported.10,11,28 Alternatively, the Mac-1
ligand on the melanoma cells may be expressed in mass excess or bind
with a higher affinity than ICAM-1.
Transient changes in the adhesivity of
2-integrin receptor- ligand
interactions.
Within seconds of chemotactic stimulation of neutrophils, a rapid
increase in 2-integrin-dependent adhesion has been
observed.14,29 Mac-1 is also upregulated by 100% within 2 minutes after stimulation with 1 µmol/L FMLP, while the expression
level of LFA-1 does not change.25,29 To examine how these
changes in adhesivity may be reflected in changes in adhesion
efficiency over time, we stimulated the neutrophil E3-ICAM cell
suspension for a defined time period before applying shear in the
cone-plate viscometer. Under these conditions, a lag period was
introduced when cells were stimulated and receptor adhesivity changed,
before shear was applied to initiate cell-cell collision.
The kinetics of aggregate formation of E3-ICAM cells with neutrophils
activated for 0, 30, 120, and 300 seconds before being sheared at a
rate of 200 s1 is shown in Fig
3A. Both the rate and maximum extent of
aggregation decreased with time following activation. We assessed the
contributions of Mac-1 and LFA-1 to the time-dependent decrease in
neutrophil adhesivity following FMLP stimulation by preblocking
adhesion receptors with MoAbs (Fig 3B). LFA-1-dependent adhesion was
studied by addition of an anti-Mac-1 blocking MoAb h60.1, while
Mac-1-dependent adhesion was investigated in the presence of
anti-ICAM-1 domain 1 antibody RR1/1. RR1/1 has been shown to behave
identically to anti-LFA-1 MoAb R3.1 Fab, since they both specifically
block the binding of LFA-1 to ICAM-1.10 We further verified
that incubation of E3-ICAM cells with RR1/1 and R3.1 Fab simultaneously
provided no more inhibition than either MoAb alone. Incubation with
h60.1 and RR1/1 completely blocked aggregation (data not shown). The data in Fig 3b are presented using RR1/1 rather than R3.1, in part, due
to reagent limitations in our laboratory. Also, treatment of E3-ICAM
cells with RR1/1 allows complete inhibition of LFA-1/ICAM-1-mediated adhesion without intervening with neutrophil function. We observed that
120 seconds after stimulation, neutrophil adhesion was almost completely Mac-1-dependent, since blocking with anti-Mac-1 MoAb alone
was sufficient to completely inhibit neutrophil-E3-ICAM adhesion (Fig
3B).
View larger version (25K):
[in this window]
[in a new window]
View larger version (24K):
[in this window]
[in a new window]
View larger version (24K):
[in this window]
[in a new window]
| Fig 3.
Contributions of LFA-1 and Mac-1 to time-dependent
changes in cell adhesivity. Neutrophils (3 × 106
cells/mL) were mixed with E3-ICAM transfectants
(6 × 106 cells/mL) and stimulated with 1 µmol/L FMLP
for fixed time periods (0, 30, 60, 120, 180, 240, or 300 seconds)
before application of shear at 200 s1. Cell aggregation
kinetics was measured. (A) Percent neutrophils in heterotypic
aggregates for experiments where shear was applied either 0, 30, 120, or 300 seconds after stimulation. (B) Percent neutrophils recruited in
heterotypic aggregation when shear was applied 120 seconds after FMLP
stimulation either in the absence of MoAb (LFA-1- and Mac-1-dependent
adhesion) or on addition of anti-ICAM-1 domain 1 MoAb RR1/1
(Mac-1-dependent adhesion) or anti-Mac-1 MoAb h60.1 (LFA-1-dependent
adhesion). Error bars in (A) and (B) represent SEM from three
independent experiments. (C) Adhesion efficiency of neutrophil-E3-ICAM
interactions with time at a shear rate of 200 s1 either
in the absence of MoAb, or upon addition of MoAbs to domain-1 of ICAM-1
(RR1/1) or Mac-1 (h60.1). Smooth lines represent curve fit to
experimental data with a first-order exponential decay function as
described in Results. Data are means from at least three independent
experiments. *P < .05 with respect to adhesion efficiency
estimated in the absence of MoAb and on addition of RR1/1.
|
|
The data were further analyzed over the entire time course of
chemotactic stimulation in terms of neutrophil-E3-ICAM adhesion efficiency (Fig 3C). Adhesion efficiency was highest immediately after
stimulation and it decreased with time thereafter. The decrease in
adhesion efficiency over time was fit by a first-order exponential function as previously described,15 E = E0e t (Table
1). In this equation,
E0 is the adhesion efficiency immediately after
stimulation (t = 0) and is the decay constant that
describes the time-dependent decrease in efficiency. Both LFA-1 and
Mac-1 appear to contribute equally (~30% to 40%) to neutrophil-E3-ICAM adhesion efficiency immediately following
stimulation. However, adhesion mediated by LFA-1 decreased faster than
that due to Mac-1. LFA-1-ICAM-1 binding did not contribute to adhesion by 120 seconds. The decrease in efficiency over time is quantitatively described by the decay constant for LFA-1, which is approximately fourfold greater than Mac-1-mediated adhesion (Table 1). Mac-1 accounted for most of the adhesion by 120 seconds. After this time
point, Mac-1-dependent adhesion was not significantly different from
that of the untreated control (with no MoAb addition). This is the
first demonstration that adhesion mediated through LFA-1 binding to
ICAM-1 is downmodulated within minutes of FMLP stimulation.
Kinetics and strength of the 2-integrin
receptor-ligand interaction.
As shear rate increases, the contact duration of cells interacting in a
linear shear field is predicted to decrease, and there is a concomitant
increase in the amount of cell deformation and tensile forces acting to
break intercellular bonds. A decrease in adhesion efficiency is thus
expected to result. We examined the kinetics and strength of
2-integrin receptor-mediated adhesion between
neutrophils and E3-ICAM cells over a range of shear rates from 100 s1 to 500 s1 (Fig
4). The rate of neutrophil recruitment by
E3-ICAM cells was greatest at the lower shear rates (100 to 200 s1), and it decreased at higher shear rates (Fig 4A).
The adhesion efficiency fit to these kinetics decreased approximately
linearly with increasing shear rate between 100 s1 and
300 s1, reaching approximately zero at 400 s1 (Fig 4B). The results were compared with homotypic
neutrophil aggregation experiments performed in the presence of
saturating concentrations of anti-L-selectin-blocking MoAb LAM1-3. We
have previously established that on addition of LAM1-3, neutrophil adhesion was supported entirely by 2-integrin activation
and binding.14 At a shear rate of 200 s1,
homotypic neutrophil adhesion efficiency was approximately 0.25 (Fig
1F). However, on addition of MoAb LAM1-3, this efficiency decreased to
approximately 0.11 (Fig 4B). At this shear rate, the efficiency of
neutrophil-E3-ICAM adhesion closely matched the efficiency of
homotypic neutrophil adhesion in the presence of L-selectin-blocking
antibody. In fact, efficiency for both the cases was closely matched
over the entire range of shear rates tested. In both cases, efficiency
decreased from approximately 0.17 at 100 s1 to zero at
400 s1.
View larger version (26K):
[in this window]
[in a new window]
View larger version (22K):
[in this window]
[in a new window]
View larger version (21K):
[in this window]
[in a new window]
View larger version (21K):
[in this window]
[in a new window]
| Fig 4.
Kinetics and strength of 2-integrin
receptor-ligand interactions. Neutrophils (3 × 106
cells/mL) and E3-ICAM (5 × 106 cells/mL) cells were
stimulated with 1 µmol/L FMLP and sheared in a cone-plate viscometer
over a range of shear rates from 100 to 500 s1 in normal
HEPES buffer (media viscosity, =0.7 cp, A and B) or on addition of
6% Ficoll to the HEPES buffer (media viscosity, =1.7 cp, C and D).
(A) Percent neutrophils in heterotypic aggregates in normal buffer. (B)
Adhesion efficiencies for neutrophil-E3-ICAM collisions were computed
from data presented in A, and compared with the adhesion efficiency for
neutrophil-neutrophil collisions in independent homotypic aggregation
experiments in the presence of 30 µg/mL anti-L-selectin MoAb, LAM1-3
Fab. (C) Percent neutrophils in heterotypic aggregates in HEPES buffer
with 6% Ficoll. (D) Adhesion efficiencies for neutrophil-E3-ICAM were
computed for the data in C and compared with neutrophil-neutrophil
aggregation in the presence of LAM1-3 in media with viscosity 1.7 cp.
Error bars represents SEM from three to eight independent experiments.
*P < .05 with respect to neutrophil-E3-ICAM adhesion in the
absence of Ficoll.
|
|
We next examined whether the decrease in efficiency with shear rate was
primarily due to a decrease in the intercellular contact duration
during collision, which in turn limits the number of adhesion bonds
formed. Another mechanism that could account for the decrease in
adhesion efficiency with increased shear is the concomitant increase in
stresses acting to disrupt intercellular bonds.30 To
differentiate between these two effects, experiments were performed in
which buffer viscosity was increased by the addition of 6% Ficoll. At
37°C, normal HEPES buffer had a viscosity of 0.7 cp, while the
addition of 6% Ficoll increased the buffer viscosity to 1.7 cp.
Increasing the viscosity at the same shear rate caused a approximately
2.5-fold (=1.7/.7) increase in shear stress exerted on aggregates,
without affecting the intercellular contact duration, which varies
inversely with shear rate. We have previously shown that addition of
Ficoll to the buffer did not itself activate neutrophils.14
Cells remained in an unactivated spherical state. Moreover, Ficoll did
not inhibit the amount of Mac-1 upregulation and L-selectin shedding
characteristic of neutrophil stimulation with FMLP. At a low shear rate
of 100 s1, increasing viscosity did not alter the
kinetics of neutrophil-E3-ICAM adhesion (Fig 4A and C). However, at
shear rates  200 s1, the increase in shear stress
decreased the rate and extent of neutrophil-E3-ICAM adhesion. This
effect is clearly evident in comparing the efficiency of
neutrophil-E3-ICAM adhesion versus homotypic neutrophil adhesion in
the presence of anti-L-selectin MoAb (Fig 4D). While increasing shear
stress did not affect the linear decrease of efficiency with shear rate
for homotypic neutrophil adhesion, it decreased the efficiency of
neutrophil-E3-ICAM adhesion by 70% at a shear stress of approximately
2 dyn/cm2 (ie, on addition of 6% Ficoll at 200 s1). Taken together, the data indicate that the
predominant factor limiting homotypic aggregation over a range of shear
between 100 s1 and 400 s1 was
the decrease in the intercellular contact duration, rather than the
concomitant increase in shear stress up to approximately 5 dyn/cm2. In contrast, neutrophil adhesion to E3-ICAM cells
was markedly less efficient at shear stresses more than 2 dyn/cm2.
We next compared the relative contributions of LFA-1 and Mac-1 to the
efficiency and strength of adhesion with increased shear stress.
Experiments were performed in the presence of anti-LFA-1 MoAb
(Mac-1-dependent adhesion) and anti-Mac-1 MoAb (LFA-1-dependent adhesion) at a shear rate of 200 s1, both in normal
buffer and in high viscosity buffer with 6% Ficoll (Fig 5A and
B). Blocking either LFA-1 or Mac-1 caused
an approximately 50% decrease in heterotypic aggregation in the first
30 seconds after neutrophil stimulation, consistent with the MoAb
blocking data of Fig 2. However, over the time course of stimulation,
blocking Mac-1 was significantly more effective in inhibiting
aggregation. This implies that Mac-1, rather than LFA-1, is more
effective in sustaining neutrophil adhesion. Blocking both the integrin subunits simultaneously abrogated heterotypic aggregation over the
entire time course of the experiment. Increasing shear stress with
Ficoll decreased the rate and extent of heterotypic aggregation for
both integrin subunits (Fig 5B). Adhesion efficiency supported by
either integrin subunits was inhibited by approximately 50% on
increasing the shear stress (Fig 5C). The results suggest that LFA-1
and Mac-1 bonds formed in the first 30 seconds of stimulation mediate
adhesion with comparable kinetics and strength, but their contributions
differ markedly by 120 seconds at which time Mac-1 supports all the
adhesion.
View larger version (26K):
[in this window]
[in a new window]
View larger version (28K):
[in this window]
[in a new window]
View larger version (22K):
[in this window]
[in a new window]
| Fig 5.
Strength of LFA-1- and Mac-1-mediated adhesion.
Neutrophils (3 × 106 cells/mL) and E3-ICAM cells
(5 × 106 cells/mL) were stimulated with 1 µmol/L FMLP
and sheared in a cone-plate viscometer at 200 s1 either
in the absence of any MoAb (LFA-1- and Mac-1-dependent adhesion) or
in the presence of anti-LFA-1 MoAb R3.1 Fab (Mac-1-dependent
adhesion), anti-Mac-1 MoAb h60.1 (LFA-1-dependent adhesion), or on
addition of both R3.1 and h60.1 (LFA-1- and Mac-1-independent
adhesion): (A) in normal HEPES buffer (media viscosity, 0.7 cp), or (B)
in buffer containing 6% Ficoll (media viscosity, 1.7 cp). The percent
neutrophils in heterotypic aggregates is reported in the 2 panels. (C)
Adhesion efficiencies for the experiment described in (A) and (B).
Error bars represent SEM from at least three independent experiments.
*P < .05 with respect to the same treatment in the absence of
Ficoll.
|
|
Intercellular contact areas of neutrophil-E3-ICAM aggregates.
The strength of adhesion between apposing cells has been shown to be
dependent on the area and topography over which molecular bonds are
distributed.31,32 The adhesion efficiency computed for
neutrophil homotypic and neutrophil-E3-ICAM adhesion as demonstrated in
Fig 4 was remarkably comparable over the range of shear rates in the
low viscosity buffer. However, on increasing media viscosity, the
adhesion efficiency of neutrophil-E3-ICAM adhesion was markedly lower
than that due to neutrophil homotypic aggregation. We examined if these
differences were due to differences in the relative contact areas of
neutrophils engaged in heterotypic and homotypic aggregation (Fig
6). At the time point of maximum
aggregation, at a shear rate of 200 s1, samples were
fixed and prepared for transmission electron microscopy. In the absence
of stimulation, neutrophils appeared as singlets with a round
morphology (data not shown). E3-ICAM cells were also spherical with an
average radius twice that of neutrophils. After stimulation and
application of shear for 90 seconds, homotypic neutrophil aggregates
were adherent either at multiple contact sites on their planar membrane
or along continuous segments of the neutrophil surface (Fig 6A). The
nature of the contacts in terms of the level of membrane
interdigitation for homotypic and neutrophil-E3-ICAM aggregates were
qualitatively similar (Fig 6B). We quantitatively compared the contact
regions for the two types of aggregates by measuring the lengths of
intercellular contacts in the plane of the electron micrograph sections
as described in Materials and Methods. The contact length index for
neutrophil-neutrophil aggregates (0.16 ± 0.01, n = 33) was not
significantly different from that of neutrophil-E3-ICAM aggregates
(0.14 ± 0.019, n = 12). This implied that the surface area over
which adhesive bonds were formed in the two cases was equivalent.
View larger version (106K):
[in this window]
[in a new window]
| Fig 6.
Neutrophils and E3-ICAM cells observed by transmission
electron microscopy. (A) Homotypic neutrophil aggregate formed 90 seconds after 1 µmol/L FMLP stimulation. (B) Neutrophil-E3-ICAM
aggregate 60 seconds after FMLP stimulation. Arrow heads indicate
regions of intercellular contact.
|
|
| |
DISCUSSION |
The dynamics of 2-integrin-dependent adhesion between
chemotactically stimulated neutrophils and an ICAM-1-transfected
murine melanoma cell line were examined. Cell adhesion was quantified under defined hydrodynamic shear conditions where the shear rate, hydrodynamic forces, and intercellular encounter frequencies were precisely controlled.
The neutrophil-E3-ICAM system.
The efficiency of homotypic neutrophil aggregation mediated by
L-selectin and 2 integrin was observed to be
approximately 0.25 at a shear rate of 200 s1. This is
approximately threefold higher than that of neutrophil-E3-ICAM aggregation under the same shear conditions. In the presence of a MoAb
that blocks L-selectin function, the efficiency of
2-integrin-mediated homotypic adhesion decreased to
approximately 0.07. In the absence of L-selectin tethering, the
kinetics and efficiency of adhesion were identical for neutrophils
adhering to each other or to E3-ICAM transfectants. LFA-1 and Mac-1
contributed equally to adhesion efficiency and adhesion strength over
the first minute of stimulation for both neutrophil-neutrophil and
neutrophil-E3-ICAM adhesion. It appears that the dynamics of adhesion
in this system is controlled primarily by the activation and binding of
2 integrin, rather than the availability of
counterstructures that differ in homotypic and heterotypic aggregation.
The adhesion of neutrophils to E3-ICAM cells was found to depend
entirely on LFA-1 and Mac-1. ICAM-1 accounted for all the adhesion via
LFA-1 at 200 s1, while Mac-1 binding to ICAM-1 was not a
requirement under these conditions of shear. Mac-1-dependent adhesion
of neutrophils to E3-ICAM cells was not significantly inhibited by
treatment with MoAbs that block 2-integrin binding to
domains 2 and 3 of ICAM-1. This may be attributed to Mac-1 binding
ICAM-1 with a low affinity as previously reported in adhesion
experiments under no shear conditions.10,11,28 These
studies have also demonstrated other differences between the nature of
Mac-1 and LFA-1 binding to their distinct domains on ICAM-1. Mac-1
recognition of ICAM-1 was more temperature sensitive than LFA-1-ICAM-1
interactions, and the former was influenced by the extent of N-linked
glycosylation of ICAM-1.10,26 An alternate explanation for
our experimental observations is that the endogenous Mac-1 ligand(s)
expressed on both the parent B7H81 and E3-ICAM murine cells may bind
Mac-1 with a higher affinity than ICAM-1. Regardless of the nature of the Mac-1 counterstructure in neutrophil adhesion to E3-ICAM-1, the
current model provides a quantitative comparison of the strength and
kinetics of LFA-1-ICAM-1 and Mac-1 binding under defined hydrodynamic shear. The presence of ligands for Mac-1 other than ICAM-1 is not
surprising, and has been observed in previous studies of neutrophil adhesion to human endothelial cell monolayers10,11 and in
vivo in animal models.12 Mac-1 is a promiscuous integrin
with respect to its repertoire of ligands, which includes C3b, ICAM-1,
factor X, heparan sulfate, fibrinogen, elastase, and denatured serum proteins, including albumin.33-35
2-integrin-mediated adhesion is limited
by intercellular contact duration.
Neutrophil adhesion mediated by 2 integrin was shown to
decrease in a linear manner as shear was increased from 100 to 400 s1. Over this range of shear rates, adhesion efficiency
of neutrophil-E3-ICAM was virtually identical to that of
neutrophil-neutrophil adhesion, provided L-selectin was blocked by
MoAb. Below a critical threshold shear stress (~2
dyn/cm2), the primary factor affecting the kinetics of
neutrophil adhesion to E3-ICAM was the intercellular contact duration.
A mathematical analysis of uniform spheres tumbling in a linear shear
field has estimated that the average intercellular contact duration
during cell-cell collisions varies inversely with shear rate as
approximately 2.62/shear rate.14,36 Based on this relation
and the current observation that adhesion was abrogated at shear rates
more than 400 s1, it follows that a minimum contact
duration of 6.5 ms (~2.62/400 seconds) was required for
2 integrin to bind in sufficient numbers to support
neutrophil adhesion. Maximum adhesion was measured at a shear rate of
100 s1, corresponding to a contact durations of
approximately 25 ms.
This estimate of the duration of intercellular contact required for
2-integrin-mediated neutrophil adhesion is consistent with in vitro and in vivo observations. Parallel-plate flow chamber geometries are widely used to quantitate the recruitment of leukocytes from the flow stream to substrates expressing physiologic ligands. The
contact duration between a cell and the substrate in this chamber is
estimated to vary with shear rate as approximately 0.1/shear
rate.37 Based on our estimate of the minimum contact duration for adhesion in sheared cell suspensions, we estimate that
2-integrin-mediated adhesion in a flow chamber can
occur only at shear rates less than 15 s1 (~0.1/6.5
ms). This prediction is consistent with measurements of
phorbol myristate acetate (PMA)-stimulated neutrophils
becoming arrested on a planar bilayer expressing ICAM-1.8
In these studies, adhesion was observed only at shear stresses less
than 0.2 dyn/cm2 and corresponding shear rates of less than
approximately 28 s1.
The current data provides a quantitative framework to interpret the
molecular dynamics that regulate the transition from selectin-dependent rolling to integrin-mediated leukocyte arrest on activated endothelium. Selectin-mediated rolling velocities of neutrophils typically range
from 2 to 15 µm/s in vitro5,38 and 40 to 70 µm/s in vivo.39,40 In the multistep paradigm of leukocyte
recruitment to the vessel wall, cell rolling is thought to increase the
duration and extent of membrane contact on the endothelium, thereby
facilitating the binding of inflammatory mediators such as
platelet-activating factor (PAF) and interleukin-8
(IL-8). This in turn may enable activation and upregulation of
2-integrin affinity, a requirement of cell arrest and
transmigration.41,42 In recent in vivo studies, it was
estimated that during rolling, approximately 10% of the neutrophil
surface is in contact with the endothelium.43 Assuming a
maximum rolling velocity for neutrophils to be 70 µm/s, we estimate that the average contact duration between a rolling neutrophil and the
endothelium is approximately 40 ms (=cell circumference · fraction of surface in contact/rolling velocity). This interval exceeds our
estimate for the minimum time required for
2-integrin-mediated firm adhesion (~6.5 ms) by
approximately sixfold, and it corresponds to efficiencies that we would
observe at shear rates of approximately 50 s1 (>0.2).
Hence, we hypothesize that if endothelial ligand density is not
limiting, the distance that a neutrophil rolls before firm arrest in
the vasculature is primarily determined by the extent of inflammatory
mediator binding and the time required for signal transduction and
activation of 2 integrin, rather than the rolling velocity of the cell.
High shear stresses decrease neutrophil-E3-ICAM adhesion.
While the adhesion efficiency for neutrophil-neutrophil and
neutrophil-E3-ICAM aggregates was similar at low shear stress, the two
interactions were different at a shear stress more than 2 dyn/cm2. Above this critical shear stress, the efficiency
of neutrophil-E3-ICAM and not neutrophil-neutrophil adhesion decreased
on increasing viscosity. Apparently, neutrophil-E3-ICAM adhesion was
more susceptible to dissociation by tensile loading than homotypic
neutrophil adhesion. This behavior was not unique to either
-subunit, since the efficiency of both LFA-1- and Mac-1-mediated
adhesion decreased equally on increasing shear stress. There are two
possible explanations for this phenomenon. (1) The number of bonds
formed between neutrophils and E3-ICAM was lower, or the tensile
strength of these bonds was weaker than that between two neutrophils.
(2) The distribution of adhesion sites over the membrane contact area,
or the stress acting on these bonds was different in the two adhesion
systems. Several lines of evidence demonstrated that adhesion was not
limited by the number of ICAM-1 receptors. If adhesion was limited by the availability of ICAM-1, we would expect more than the 15% decrease
in adhesion observed when 50% of the ICAM-1 sites were blocked.
Furthermore, analysis of the intercellular contact area by transmission
electron microscopy (TEM) demonstrated that there was no apparent
difference between the length and interdigitation of the contact
regions between the neutrophil-neutrophil and neutrophil-E3-ICAM aggregates. The data suggest that the overall tensile strength of
intercellular 2-integrin bonds, including the effects of
bond number, distribution, and bond strength, is greater in the case of
neutrophil-neutrophil adhesion as compared with neutrophil-E3-ICAM adhesion. Furthermore, it also demonstrates that changing the level of
hydrodynamic shear may alter the critical parameter (intercellular contact duration or tensile stress) that controls neutrophil adhesion kinetics.
The lifetime for adhesion via LFA-1 and Mac-1.
Neutrophil adhesion is typically reversible over the time course of
chemotactic stimulation under conditions of shear. This is a
requirement for the transition from cell arrest to diapedesis and
chemotaxis at sites of tissue inflammation. The current data indicate
that the transience in neutrophil adhesivity was not due to a
downregulation in the number of integrin receptors on the cell surface,
since the expression levels of LFA-1 remained unchanged following
stimulation. In contrast, Mac-1 was upregulated by at least 100%
within 2 minutes of FMLP stimulation. Other possible explanations for
the decreased adhesivity include (1) downregulation in the affinity of
2 integrin; (2) an increase in the tensile forces acting
on each receptor over the time course of stimulation, which could be
achieved by diffusion of 2-integrin sites to a smaller
contact area on the neutrophil,44 and additionally by the
generation of active forces exerted through the
cytoskeleton45; and (3) activated Mac-1 sites may be
prevented from engaging ligand on the E3-ICAM cells by competitive
binding of endogenous ligands such as elastase released after
neutrophil stimulation.35
The decrease in neutrophil adhesivity following stimulation was
observed for both of the 2-integrin subunits. Adhesion
mediated by LFA-1-ICAM-1 bonds decreased to approximately zero by 120 seconds of chemotactic stimulation. In contrast, adhesion mediated via Mac-1 decreased approximately four times slower. One possible explanation for the differential behavior of the
2-integrin subunits may be due to the adhesive
contribution of the upregulated Mac-1 receptors, which are expressed on
the neutrophil surface following stimulation.46
Furthermore, receptor redistribution and/or clustering of LFA-1
and Mac-1 may also contribute to their differential contribution to
cell adhesion. It has been recently reported that 2
integrins (LFA-1 and Mac-1) are expressed predominantly on the cell
body and not on the microvilli of resting polymorphonuclear
neutrophils.47,48 Upon activation, Mac-1 expression is
rapidly upregulated and it appears on the microvilli, cell surface
ruffles, and on the cell body. The effect of surface distribution and
the dynamics of redistribution to neutrophil adhesion is currently an
issue being addressed in our laboratory. We have measured an identical
decrease in LFA-1 adhesivity in a previous study of homotypic
neutrophil aggregation where LFA-1 bound ICAM-3 on an apposing
neutrophil (Taylor et al, in preparation). In this
study, the exponential decay constant, measured for
LFA-1-dependent and Mac-1-dependent homotypic adhesion, was within
10% of the values reported in Table 1. This comparison suggests that
the dynamics of LFA-1 adhesion to either ICAM-1 during heterotypic
aggregation, or ICAM-3 during homotypic aggregation is dependent on the
activation state and binding affinity of LFA-1, rather than the nature
of the counterstructure. Although previous studies with lymphocytes
have shown that LFA-1 alters its activation state following stimulation
with either phorbol esters or antibody,49,50 this is the
first study to demonstrate that neutrophil LFA-1-ICAM-1 adhesivity
decreases following chemotactic stimulation.
In this study, we have introduced a new technique that combines
cone-plate viscometry and flow cytometry to reveal the distinct nature
of adhesion via LFA-1 and Mac-1. Consistent with published reports of
2-integrin-dependent neutrophil arrest on ICAM-1
bearing human umbilical vein endothelial cells, both
activated LFA-1 and Mac-1 contributed equally to adhesion within the
first minute of chemotactic stimulation.11 Furthermore, we
show here that with increased time, Mac-1 becomes the predominant
2 integrin mediating neutrophil adhesion. Regardless of
whether LFA-1 is binding to ICAM-1 in heterotypic interactions or
ICAM-3 in homotypic aggregation, its contribution to the efficiency of
capture and strength of adhesion rapidly decreases by 2 minutes of
chemotactic stimulation. One implication is that the distinct functions
attributed to LFA-1 and Mac-1 in mediating extravasation
(LFA-1-predominant) and secretory and phagocytic processes
(Mac-1-predominant)51 may be mediated by their
differential responses over time of stimulation and level of
hydrodynamic shear.
| |
FOOTNOTES |
Submitted January 7, 1998;
accepted April 22, 1998.
Supported by National Institutes of Health Grants No. AI23521,
5P50NS23327, AI31652, and HL42550. S.I.S. is an Established Investigator of the American Heart Association and a Fellow of the
Whitaker Biomedical Foundation. A.R.B. is the recipient of a grant from
the Methodist Hospital Foundation.
Address reprint requests to Scott I. Simon, PhD,
Section of Leukocyte Biology, 1100 Bates, Room 6014, Houston, TX
77030-2600.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We acknowledge Dr J. David Hellums for the use of his laboratory
facilities, Lisa Thurmon for assisting with the cell culture, and
Evelyn Brown for preparing the TEM samples.
| |
REFERENCES |
1.
Springer TA:
Traffic signals on endothelium for lymphocyte recirculation and leukocyte emigration.
Annu Rev Physiol
57:827,
1995[Medline]
[Order article via Infotrieve]
2.
Lawrence MB,
Bainton DF,
Springer TA:
Neutrophil tethering to and rolling on E-selectin are separable by requirement for L-selectin.
Immunity
1:137,
1994[Medline]
[Order article via Infotrieve]
3.
von Andrian UH,
Chambers JD,
McEvoy LM,
Bargatze RF,
Arfors K-E,
Butcher EC:
Two step model of leukocyte-endothelial cell interaction in inflammation: Distinct roles for LECAM-1 and the leukocyte beta-2 integrins in vivo.
Proc Natl Acad Sci USA
88:7538,
1991[Abstract/Free Full Text]
4.
Smith CW,
Kishimoto TK,
Abbassi O,
Hughes BJ,
Rothlein R,
McIntire LV,
Butcher E,
Anderson DC:
Chemotactic factors regulate lectin adhesion molecule 1 (LECAM-1)-dependent neutrophil adhesion to cytokine-stimulated endothelial cells in vitro.
J Clin Invest
87:609,
1991
5.
Jones DA,
Abbassi O,
McIntire LV,
McEver RP,
Smith CW:
P-selectin mediates neutrophil rolling on histamine-stimulated endothelial cells.
Biophys J
65:1560,
1993[Medline]
[Order article via Infotrieve]
6.
Abbassi O,
Kishimoto TK,
McIntire LV,
Anderson DC,
Smith CW:
E-selectin supports neutrophil rolling in vitro under conditions of flow.
J Clin Invest
92:2719,
1993
7.
Smith CW,
Rothlein R,
Hughes BJ,
Mariscalco MM,
Schmalstieg FC,
Anderson DC:
Recognition of an endothelial determinant for CD18-dependent human neutrophil adherence and transendothelial migration.
J Clin Invest
82:1746,
1988
8.
Lawrence MB,
Springer TA:
Leukocytes roll on a selectin at physiologic flow rates: Distinction from and prerequisite for adhesion through integrins.
Cell
65:859,
1991[Medline]
[Order article via Infotrieve]
9.
Dustin ML,
Springer TA:
Lymphocyte function associated antigen-1 (LFA-1) interaction with intercellular adhesion molecule-1 (ICAM-1) is one of at least three mechanisms for lymphocyte adhesion to cultured endothelial cells.
J Cell Biol
107:321,
1988[Abstract/Free Full Text]
10.
Diamond MS,
Staunton DE,
de Fougerolles AR,
Stacker SA,
Garcia-Aguilar J,
Hibbs ML,
Springer TA:
ICAM-1 (CD54): A counter-receptor for Mac-1 (CD11b/CD18).
J Cell Biol
111:3129,
1990[Abstract/Free Full Text]
11.
Smith CW,
Marlin SD,
Rothlein R,
Toman C,
Anderson DC:
Cooperative interactions of LFA-1 and Mac-1 with intercellular adhesion molecule-1 in facilitating adherence and transendothelial migration of human neutrophils in vitro.
J Clin Invest
83:2008,
1989
12.
Doerschuk CM,
Winn RK,
Coxson HO,
Harlan JM:
CD18-dependent and independent mechanisms of neutrophil emigration in the pulmonary and systemic microcirculation of rabbits.
J Immunol
144:2327,
1990[Abstract]
13.
Lawrence MB,
McIntire LV,
Eskin SG:
Effect of flow on polymorphonuclear leukocyte/endothelial cell adhesion.
Blood
70:1284,
1987[Abstract/Free Full Text]
14.
Taylor AD,
Neelamegham S,
Hellums JD,
Smith CW,
Simon SI:
Molecular dynamics of the transition from L-selectin- to 2-integrin-dependent neutrophil adhesion under defined hydrodynamic shear.
Biophys J
71:3488,
1996[Medline]
[Order article via Infotrieve]
15.
Neelamegham S,
Taylor AD,
Hellums JD,
Dembo M,
Smith CW,
Simon SI:
Modeling the reversible kinetics of neutrophil aggregation under hydrodynamic shear.
Biophys J
72:1527,
1997[Medline]
[Order article via Infotrieve]
16.
Munn LL,
Melder RJ,
Jain RK:
Analysis of cell flux in the parallel plate flow chamber: Implications for cell capture studies.
Biophys J
67:889,
1994[Medline]
[Order article via Infotrieve]
17.
Lawrence MB,
Kansas GS,
Kunkel EJ,
Ley K:
Threshold levels of fluid shear promote leukocyte adhesion through selectins (CD62L,P,E).
J Cell Biol
136:717,
1997[Abstract/Free Full Text]
18.
Finger EB,
Purl K,
Alon R,
Lawrence MB,
von Andrian UH,
Springer TA:
Adhesion through L-selectin requires a threshold hydrodynamic shear.
Nature
379:266,
1996[Medline]
[Order article via Infotrieve]
19.
Alon RD,
Hammer A,
Springer TA:
Lifetime of the P-selectin-carbohydrate bond and its response to tensile force in hydrodynamic flow.
Nature
374:539,
1995[Medline]
[Order article via Infotrieve]
20.
Simon SI,
Burns AR,
Taylor AD,
Gopalan PK,
Lynam EB,
Sklar LA,
Smith CW:
L-selectin (CD62L) crosslinking signals neutrophil adhesive functions via the Mac-1 (CD11b/CD18) 2-integrin.
J Immunol
155:1502,
1995[Abstract]
21.
Graf LH,
Rosenberg CD,
Mancino V,
Ferrone S:
Transfer and co-amplification of a gene encoding a 96-kDa immune IFN-inducible human melanoma-associated antigen: Preferential expression by mouse melanoma host cells.
J Immunol
141:1054,
1988[Abstract]
22.
Neelamegham S,
Simon SI,
Yednock TA,
McIntyre BW,
Zygourakis K:
Induction of homotypic lymphocyte aggregation: Evidence for a novel activation state of the 1-integrin.
J Leuk Biol
59:872,
1996[Abstract]
23.
Simon SI,
Chambers JD,
Sklar LA:
Flow cytometric analysis and modeling of cell-cell adhesive interactions: The neutrophil as a model.
J Cell Biol
111:2747,
1990[Abstract/Free Full Text]
24.
Burns AR,
Simon SI,
Kukielka GL,
Rowen JL,
Mendoza LH,
Brown ES,
Entman ML,
Smith CW:
Chemotactic factors stimulate CD18-dependent canine neutrophil adherence and motility on lung fibroblasts.
J Immunol
156:3389,
1996[Abstract]
25.
Simon SI,
Chambers JD,
Butcher E,
Sklar LA:
Neutrophil aggregation is 2-integrin- and L-selectin-dependent in blood and isolated cells.
J Immunol
149:2765,
1992[Abstract]
26.
Diamond MS,
Staunton DE,
Marlin SD,
Springer TA:
Binding of the integrin Mac-1 (CD11b/CD18) to the third immunoglobulin-like domain of ICAM-1 (CD54) and its regulation by glycosylation.
Cell
65:961,
1991[Medline]
[Order article via Infotrieve]
27.
Parkos CA,
Colgan SP,
Diamond MS,
Nusrat A,
Liang TW,
Springer TA,
Madara JL:
Expression and polarization of intercellular adhesion molecule-1 on human intestinal epithelia: consequences for CD11b/CD18-mediated interactions with neutrophils.
Molec Med
2:489,
1996[Medline]
[Order article via Infotrieve]
28.
Lo SK,
Van Seventer GA,
Levin SM,
Wright SD:
Two leukocyte receptors (CD11a/CD18) mediate transient adhesion to endothelium by binding to different ligands.
J Immunol
143:3325,
1989[Abstract]
29.
Kishimoto TK,
Jutila MA,
Berg EL,
Butcher EC:
Neutrophil Mac-1 and MEL-14 adhesion proteins inversely regulated by chemotactic factors.
Science
245:1238,
1989[Abstract/Free Full Text]
30.
Tees DFJ,
Coenen O,
Goldsmith HL:
Interaction forces between red cells agglutinated by antibody. IV. Time and force dependence of break-up.
Biophys J
65:1318,
1993[Medline]
[Order article via Infotrieve]
31.
Capo C,
Garrouste F,
Benoliel A-M,
Bongrand P,
Ryter A,
Bell GI:
Concanavalin-a-mediated thymocyte agglutination: A model for a quantitative study cell adhesion.
J Cell Sci
56:21,
1982[Abstract/Free Full Text]
32.
Tozeren A:
Cell-cell conjugation: Transient analysis and experimental implications.
Biophys J
58:641,
1990[Medline]
[Order article via Infotrieve]
33.
Diamond MS,
Alon R,
Parkos CA,
Quinn MT,
Springer TA:
Heparin is an adhesive ligand for the leukocyte integrin mac-1 (CD11b/CD18).
J Cell Biol
130:1473,
1995[Abstract/Free Full Text]
34.
Diamond MS,
Springer TA:
A subpopulation of Mac-1 (CD11b/CD18) molecules mediates neutrophil adhesion to ICAM-1 and fibrinogen.
J Cell Biol
120:545,
1993[Abstract/Free Full Text]
35.
Cai T-Q,
Wright SD:
Human leukocyte elastase is an endogenous ligand for the intergrin CR3 (CD11b/CD18, Mac-1, M2) and modulates polymorphonuclear leukocyte adhesion.
J Exp Med
184:1213,
1996[Abstract/Free Full Text]
36.
Bartok W,
Mason SG:
Particle motions in sheared suspensions: V. Rigid rods and collision doublets of spheres.
J Colloid Sci
12:243,
1957
37.
Benoliel A-M:
Use of hydrodynamic flows to study cell adhesion
, in Bongrand P
(ed):
Physical Basis of Cell-Cell Adhesion.
Boca Raton, FL, CRC
, 1988
, p 125
38.
Lawrence MB,
Smith CW,
Eskin SG,
McIntire LV:
Effect of venous shear stress on CD18-mediated neutrophil adhesion to cultured endothelium.
Blood
75:227,
1990[Abstract/Free Full Text]
39.
Perry MA,
Granger DN:
Role of CD11/CD18 in shear rate-dependent leukocyte-endothelial cell interactions in cat mesenteric venules.
J Clin Invest
87:1798,
1991
40.
Gaboury JP,
Johnston B,
Niu XF,
Kubes P:
Mechanisms underlying acute mast cell-induced leukocyte rolling and adhesion in vivo.
J Immunol
154:804,
1995[Abstract]
41.
Detmers PA,
Lo SK,
Olsen-Egbert E,
Walz A,
Baggiolini M,
Cohn ZA:
Neutrophil-activating protein 1/interleukin 8 stimulates the binding activity of the leukocyte adhesion receptor CD11b/CD18 on human neutrophils.
J Exp Med
171:1155,
1990[Abstract/Free Full Text]
42.
Zimmerman GA,
McIntyre TM,
Mehra M,
Prescott SM:
Endothelial cell-associated platelet-activating factor: A novel mechanism for signaling intercellular adhesion.
J Cell Biol
110:529,
1990[Abstract/Free Full Text]
43.
Damiano ER,
Westheider J,
Tözeren A,
Ley K:
Variation in the velocity, deformation, and adhesion energy density of leukocytes rolling within venules.
Circ Res
79:1122,
1996[Abstract/Free Full Text]
44.
Hughes BJ,
Hollers JC,
Crockett-Torabi E,
Smith CW:
Recruitment of CD11b/CD18 to the neutrophil surface and adherence-dependent cell locomotion.
J Clin Invest
90:1687,
1992
45.
Schmid-Schonbein GW:
Biophysical analysis of neutrophil motility
, in Akkas N
(ed):
in NATO-ASI Series.
Berlin, Germany, Springer-Verlag
, 1989
, p 429
46.
Vedder NB,
Harlan JM:
Increased surface expression of CD11b/CD18 (Mac-1) is not required for stimulated neutrophil adherence to cultured endothelium.
J Clin Invest
81:676,
1988
47.
Erlandsen SL,
Hasslen SR,
Nelson RD:
Detection and spatial distribution of the beta 2 integrin (Mac-1) and L-selectin (LECAM-1) adherence receptors on human neutrophils by high-resolution field emission SEM.
J Histochem Cytochem
41:327,
1993[Abstract]
48.
Berlin C,
Bargatze RF,
Campbell JJ,
von Andrian UH,
Szabo MC,
Hasslen SR,
Nelson RD,
Berg EL,
Erlandsen SL,
Butcher EC:
4 Integrins mediate lymphocyte attachment and rolling under physiologic flow.
Cell
80:413,
1995[Medline]
[Order article via Infotrieve]
49.
Van Kooyk Y,
Weder P,
Hogervorst F,
Verhoeven AJ,
Van Seventer GA,
te Velde AA,
Borst J,
Keizer GD,
Figdor CG:
Activation of LFA-1 through a Ca2+-dependent epitope stimulates lymphocyte adhesion.
J Cell Biol
112:345,
1991[Abstract/Free Full Text]
50.
Lollo BA,
Chan KWH,
Hanson EM,
Moy VT,
Brian AA:
Direct evidence for two affinity states for lymphocyte function-associated antigen 1 on activated T cells.
J Biol Chem
268:1,
1993[Abstract/Free Full Text]
51.
Lu H,
Smith CW,
Perrard J,
Bullard D,
Tang L,
Shappell SB,
Entman ML,
Beaudet AL,
Ballantyne CM:
LFA-1 is sufficient in mediating neutrophil emigration in Mac-1 deficient mice.
J Clin Invest
99:1340,
1997[Medline]
[Order article via Infotrieve]
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
Z. Xiao, G. P. Visentin, K. M. Dayananda, and S. Neelamegham
Immune complexes formed following the binding of anti-platelet factor 4 (CXCL4) antibodies to CXCL4 stimulate human neutrophil activation and cell adhesion
Blood,
August 15, 2008;
112(4):
1091 - 1100.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Liang, C. Fu, D. Wagner, H. Guo, D. Zhan, C. Dong, and M. Long
Two-dimensional kinetics of {beta}2-integrin and ICAM-1 bindings between neutrophils and melanoma cells in a shear flow
Am J Physiol Cell Physiol,
March 1, 2008;
294(3):
C743 - C753.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Zirlik, C. Maier, N. Gerdes, L. MacFarlane, J. Soosairajah, U. Bavendiek, I. Ahrens, S. Ernst, N. Bassler, A. Missiou, et al.
CD40 Ligand Mediates Inflammation Independently of CD40 by Interaction With Mac-1
Circulation,
March 27, 2007;
115(12):
1571 - 1580.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. N. Mundhekar, D. C. Bullard, and D. F. Kucik
Intracellular heterogeneity in adhesiveness of endothelium affects early steps in leukocyte adhesion
Am J Physiol Cell Physiol,
July 1, 2006;
291(1):
C130 - C137.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. J. Slattery, S. Liang, and C. Dong
Distinct role of hydrodynamic shear in leukocyte-facilitated tumor cell extravasation
Am J Physiol Cell Physiol,
April 1, 2005;
288(4):
C831 - C839.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. W. Calderwood, J. M. Williams, M. D. Morgan, G. B. Nash, and C. O. S. Savage
ANCA induces {beta}2 integrin and CXC chemokine-dependent neutrophil-endothelial cell interactions that mimic those of highly cytokine-activated endothelium
J. Leukoc. Biol.,
January 1, 2005;
77(1):
33 - 43.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Jadhav and K. Konstantopoulos
Fluid shear- and time-dependent modulation of molecular interactions between PMNs and colon carcinomas
Am J Physiol Cell Physiol,
October 1, 2002;
283(4):
C1133 - C1143.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. F. H. Lum, C. E. Green, G. R. Lee, D. E. Staunton, and S. I. Simon
Dynamic Regulation of LFA-1 Activation and Neutrophil Arrest on Intercellular Adhesion Molecule 1 (ICAM-1) in Shear Flow
J. Biol. Chem.,
May 31, 2002;
277(23):
20660 - 20670.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. L. Dunne, C. M. Ballantyne, A. L. Beaudet, and K. Ley
Control of leukocyte rolling velocity in TNF-alpha -induced inflammation by LFA-1 and Mac-1
Blood,
January 1, 2002;
99(1):
336 - 341.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. M. Seo, L. V. McIntire, and C. W. Smith
Effects of IL-8, Gro-alpha , and LTB4 on the adhesive kinetics of LFA-1 and Mac-1 on human neutrophils
Am J Physiol Cell Physiol,
November 1, 2001;
281(5):
C1568 - C1578.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. B. Henderson, L. H.K. Lim, P. A. Tessier, F. N.E. Gavins, M. Mathies, M. Perretti, and N. Hogg
The Use of Lymphocyte Function-associated Antigen (LFA)-1-deficient Mice to Determine the Role of LFA-1, Mac-1, and {alpha}4 Integrin in the Inflammatory Response of Neutrophils
J. Exp. Med.,
July 16, 2001;
194(2):
219 - 226.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. Piccardoni, R. Sideri, S. Manarini, A. Piccoli, N. Martelli, G. de Gaetano, C. Cerletti, and V. Evangelista
Platelet/polymorphonuclear leukocyte adhesion: a new role for SRC kinases in Mac-1 adhesive function triggered by P-selectin
Blood,
July 1, 2001;
98(1):
108 - 116.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. I. Simon, Y. Hu, D. Vestweber, and C. W. Smith
Neutrophil Tethering on E-Selectin Activates {beta}2 Integrin Binding to ICAM-1 Through a Mitogen-Activated Protein Kinase Signal Transduction Pathway
J. Immunol.,
April 15, 2000;
164(8):
4348 - 4358.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Neelamegham, A. D. Taylor, H. Shankaran, C. W. Smith, and S. I. Simon
Shear and Time-Dependent Changes in Mac-1, LFA-1, and ICAM-3 Binding Regulate Neutrophil Homotypic Adhesion
J. Immunol.,
April 1, 2000;
164(7):
3798 - 3805.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Stefanec
Endothelial Apoptosis: Could It Have a Role in the Pathogenesis and Treatment of Disease?
Chest,
March 1, 2000;
117(3):
841 - 854.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. R. Hentzen, S. Neelamegham, G. S. Kansas, J. A. Benanti, L. V. McIntire, C. W. Smith, and S. I. Simon
Sequential binding of CD11a/CD18 and CD11b/CD18 defines neutrophil capture and stable adhesion to intercellular adhesion molecule-1
Blood,
February 1, 2000;
95(3):
911 - 920.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
Abstract
Introduction
Abstract
![= <FENCE><FR><NU>2[<IT>N</IT><SUB>2</SUB>] + 3[<IT>N</IT><SUB>3</SUB>] + 4[<IT>N</IT><SUB>4</SUB>] + 5[<IT>N</IT><SUB>5</SUB>] + 6[<IT>N</IT><SUB>6</SUB><SUP>+</SUP>]</NU><DE>[<IT>N</IT><SUB>1</SUB>] + 2[<IT>N</IT><SUB>2</SUB>] + 3[<IT>N</IT><SUB>3</SUB>] + 4[<IT>N</IT><SUB>4</SUB>] + 5[<IT>N</IT><SUB>5</SUB>] + 6[<IT>N</IT><SUB>6</SUB><SUP>+</SUP>]</DE></FR></FENCE> · 100 (1)](/content/vol92/issue5/fulltext/1626/img002.gif)