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INTRODUCTION |
Neutrophils are among the first cells to respond to acute
inflammation through a multistep process of specific bond formation with adhesion molecules up-regulated on the surface of activated endothelium (1, 2). Endothelial and leukocyte selectins function to
capture leukocytes from the circulation by rapid bond formation.
However, selectins form transient bonds that last a second or less in
shear flow and only mediate cell capture and rolling on endothelium (3,
4). Unlike selectins, 
2 integrins (CD18) do not
constitutively recognize ligand but require cellular activation to form
stable shear-resistant bonds with endothelial ligands including
ICAM-1.1 Signaling of
integrin activation from the "inside-out" of leukocytes is
typically mediated through ligation of G-protein-coupled receptors (GPCR) such as the CXC chemokine receptors (CXCR1 and CXCR2) that bind
interleukin-8 (IL-8) (5-7). Chemokine binding to GPCR initiates a
cascade of pro-inflammatory cell responses. One of the earliest responses is intracellular calcium flux that signals cell shape change,
degranulation, integrin activation, adhesion, and cell motility
(8-11). This cascade of cell responses that hinge on activation
of CD18 is critical to neutrophil arrest on the endothelium as
evidenced by leukocyte adhesion deficiency (LAD) patients who manifest a diminished or absent immune response at sites of
inflammation (12, 13).
Recently, it was shown (14, 15) that leukocyte stimulation elicits a
conformational shift in LFA-1 that correlates with an increase in
affinity of binding to ICAM-1. An increase in avidity of LFA-1 on
lymphocytes has been shown to support cell rolling on a cellular
substrate of ICAM-1 in the shear field of a parallel plate flow chamber
(16). Published data also indicate that firm adhesion of neutrophils to
ICAM-1 involves a cooperative and sequential process of LFA-1
(CD11a/CD18)-dependent capture followed by Mac-1 (CD11b/CD18)-mediated stabilization (17). This occurs within seconds of
chemotactic stimulation of cell suspensions sheared in a cone-plate
viscometer over a range of stress typical of blood flow in the
microcirculation (~0.2-1.5 dynes/cm2) (17). Studies in
the microcirculation have corroborated the flow chamber data revealing
that LFA-1 is sufficient for the transition from leukocyte rolling to
arrest in the inflamed microcirculation of the mouse (18, 19).
Leukocyte stimulation by chemokines triggers a high affinity
conformation and an increase in the lateral mobility of CD18, resulting
in formation of receptor clustering on the membrane (20). Both of these
mechanisms can elicit the arrest of circulating lymphocytes (20). LFA-1
occurs at the I domain metal ion-dependent adhesion site (MIDAS) (21).
An allosteric shift in LFA-1 to a conformation that supports high
affinity ligand binding was recently shown to localize to a site
spatially distinct from the MIDAS, which is denoted the IDAS
(15, 22). This allosteric shift in the LFA-1 heterodimer resulted in a
6-fold increase in the Kd of binding of soluble
LFA-1 to ICAM-1 (15). Binding of a unique CD18 mAb 240Q was also found
to induce a high affinity conformation of LFA-1. A CD18 I-like domain
(I) mAb, 327C, was shown to report on the active conformation
induced by soluble ICAM-1, IDAS activating mutation or 240Q (14, 15, 22). The induction of the I domain 327C epitope was blocked by a
small molecule diarylsulfied inhibitor that binds to the IDAS (14).
Thus the IDAS conformation switch is conformationally linked to CD18
I domain conformation. In addition, regulation at the IDAS is
dominant to cellular activation of LFA-1 (15). Taken together the data
support a mechanism by which cellular activation of an LFA-1 high
affinity state that supports cell adhesion is regulated by a
conformational switch(s) at the IDAS and I domain (22, 23). Despite
a wealth of evidence indicating that conformational activation of
2 integrin plays a central role in neutrophil
recruitment during acute inflammation, the precise mechanisms that lead
from GPCR signaling to shear-resistant arrest are not entirely known.
For example, the relations between IL-8 receptor ligation, the rate of
activation of CD18, and the dynamics of neutrophil adhesion have not
been systematically studied. Yet, formation of bonds in sufficient
numbers may provide a critical gatekeeper in regulating the efficiency
of the transition from cell rolling to shear-resistant arrest, a
prerequisite for transendothelial migration (24).
In the current study, we examined neutrophil capture and firm adhesion
to ICAM-1 via LFA-1 and Mac-1 in response to stimulation with IL-8 and
allosteric activation by mAb 240Q. To examine the process of
collisional capture of neutrophils, a flow cytometric based assay was
employed to detect adhesion of fluorescent microbeads coated with
ICAM-1. The strength and stability of neutrophil adhesion to an
ICAM-1-expressing cellular substrate was imaged in a parallel plate
flow chamber as a function of applied shear stress. By correlating the
dynamics of adhesion with the expression and membrane distribution of
mAb 327C reporting on activated CD18, we show that IL-8 stimulation regulates neutrophil recruitment on ICAM-1 through changes in LFA-1
affinity and membrane mobility.
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EXPERIMENTAL PROCEDURES |
Isolation of Human Neutrophils and PBMC--
Whole blood was
obtained from healthy adult individuals by venipuncture into sterile
syringes with heparin (10 units/ml of blood, Elkins-Sinn, Inc., Cherry
Hill, NJ) using the University of California, Davis, approved human
subjects protocol (Protocol Identification 993120). Neutrophils were
isolated from whole blood using a one-step Ficoll-Paque density
gradient (Mono-Poly Resolving medium, ICN, Aurora, OH, or PMN, Robbins
Scientific Corp., Sunnyvale, CA) following the manufacturers'
protocols. Peripheral blood mononuclear cells (PBMC) were isolated from
whole blood using a one-step sodium metrizoate/Ficoll density gradient
(Lympho Sep, ICN Biomedicals, Aurora, OH) following the manufacturer's
protocol with the following modification. Blood was diluted 2-fold with
PBS prior to layering on the separation media. Following isolation,
neutrophils were washed once with HEPES buffer (10 mM KCl,
110 mM NaCl, 10 mM glucose, 1 mM
MgCl2, and 30 mM HEPES, pH 7.4), and PBMC were
washed 3 times with PBS by centrifugation at 300 × g
for 8 min. The PBMC or neutrophils were resuspended in HEPES buffer
(~2.5 × 107 cells/ml). Endotoxin was removed from
the buffer by affinity chromatography over polymyxin B-Sepharose
(Pierce). Neutrophils and PBMC were maintained at room temperature in a
calcium-free HEPES buffer. Neutrophils were assessed by trypan blue
exclusion and deemed to be >98% viable. Following cell isolation,
neutrophils were found to remain unactivated at room temperature for
~4 h after separation. The criteria was that incubation for 20 min at
37 °C yielded less than 10% of Mac-1 up-regulation.
Cell Culture and Reagents--
Transfected L cells expressing
human ICAM-1, as described by Gopalan et al. (25), were
maintained in a modified RPMI media (Invitrogen) supplemented with 10%
fetal calf serum, 2 mM L-glutamine, 100 units/ml penicillin (Invitrogen), 100 units/ml streptomycin (Invitrogen), and 50× HAT solution (Sigma). These transfected L cells
expressed ~600 ICAM-1 sites/µm2.
Transfected Chinese hamster ovary cells expressing human Mac-1
(CHO-Mac-1), as described by Tim A. Springer (26) and generously provided by Celetta G. Callaway (Baylor College of Medicine, Houston, TX), were maintained in a modified Eagle's medium (Invitrogen) supplemented with 10% dialyzed fetal calf serum, 16 µM
thymidine, 0.1 µM methotrexate, 2 mM
glutamine, 100 units/ml penicillin, and 100 units/ml streptomycin.
CHO-K1 cells (ATCC CCL-61), also provided by Celetta G. Callaway, were
maintained in 50% Dulbecco's modified Eagle's medium, 50% Ham's
F-12 medium supplemented with 10% fetal calf serum, 100 units/ml
penicillin, and 100 units/ml streptomycin. Cells were detached by
incubation in 0.05% trypsin and 0.5 mM EDTA, washed, and
resuspended in HEPES buffer.
Agonists, Inhibitors, and Antibodies--
Recombinant human
ICAM-1/Fc was produced at the ICOS Corp. (Bothell, WA), as described
previously (22). IL-8 (72 amino acid) was purchased from R & D Systems
(Minneapolis, MN). Adhesion blocking monoclonal antibodies, R15.7
(anti-CD18) and R3.1 (anti-LFA-1), were generously provided by Dr.
Robert Rothlein (Boehringer Ingelheim Pharmaceuticals, Ridgefield, CT).
2LPM19c (anti-Mac-1) was purchased through Dako (Carpenteria, CA). For
inhibition of adhesive function, blocking antibodies were used in the
following concentrations: R15.7 and R3.1 at 10 µg/ml and 2LPM19c at 6 µg/ml (27, 28). Activating antibody 240Q (ICOS Corp., Bothell, WA),
which binds CD18 and induces an active conformation (22), was used at
concentrations ranging from 0.03 to 5 µg/ml representing 3-30%
binding of total CD18, respectively. Adhesion blocking and activating
antibodies were preincubated for 10 min at room temperature with cells
unless otherwise noted. Antibody 327C (ICOS Corp., Bothell, WA) binds CD18 at the I domain and reports on a neoepitope expressed on the
active binding site and was used at 10 µg/ml (15). 327C was labeled
with Alexa-488 (Molecular Probes, Eugene, OR) following the
manufacturer's protocol. For immunofluorescence studies, we used
PE anti-human CD11a (BD PharMingen), anti-Mac-1 mAb 2LPM19c-RPE (Dako, Carpenteria, CA), or biotinylated 327C followed by neutravidin FITC (Pierce) at 1:75 dilution. To study the effect of CD18 mobility on
neutrophil adhesion, neutrophils were preincubated for 30 min at
37 °C with 0.1% Me2SO (vehicle control),
cytochalasin B (Alexis Corp., San Diego, CA), or wortmannin (Alexis
Corp.) or LY294002 (Alexis Corp.) diluted in 0.1%
Me2SO. Cytochalasin B disrupts the formation of F-actin,
whereas wortmannin and LY294002 inhibit PI(3)K activity.
Parallel Plate Flow Chamber Assay--
Neutrophil adhesion to
ICAM-1 was measured in a parallel plate flow chamber that produces a
uniform laminar flow field as described previously (29). Briefly, L
cells expressing ICAM-1 were grown to confluence on 35 mm untreated
glass cover slips, washed twice with Dulbecco's PBS containing 11 mM glucose, and mounted on the flow chamber as described
previously (25). Neutrophils (1.5 × 106) were
incubated for 10 min at room temperature with 240Q at indicated concentrations, incubated for an additional 2 min at 37 °C, then stimulated with IL-8 at indicated concentrations, and injected as a
bolus into the flow chamber. Neutrophils were allowed to sediment to
the monolayer in the absence of shear stress. Shear stress was
initially set at 0.1 dyne/cm2 for an additional minute to
promote interactions with the monolayer substrate and then ramped to 4 dynes/cm2 to assess the strength and stability of adhesion.
Image sequences were captured (in a phase separated field of view) for
30 s (4 frames/s) throughout the period of applied shear using a
Nikon TE200 inverted microscope equipped with a Plan Fluor 20×
objective and a cooled CCD camera (Dage-MTI, Michigan City, IN). Images were compiled directly into digital sequences using a frame grabber (Scion Corp., Frederick, MD) and then analyzed using Image Pro Plus 4.1 software (Media Cybernetics, Silver Spring, MD). The number of adherent
neutrophils per field of view was quantitated by identifying their
phase-bright appearance in the same focal plane as the monolayer (Fig.
1a). Adherent neutrophils were defined as those neutrophils
that moved less than one cell diameter in 30 s. This fraction of
arrested cells was normalized to the average of the total number of
neutrophils, both adherent and rolling, present in the field of views
of the first two time points (0.1 dyne/cm2 time samples).
Preparation of Latex Beads Presenting ICAM-1 on Their
Surface--
To provide a quantitative measure of
CD18-dependent capture of ICAM-1, fluorescent latex beads
(1 µm diameter, Fluospheres, Molecular Probes, Eugene, OR) coated
with protein A were derivatized with recombinant human ICAM-1/Fc. Beads
were washed three times with Dulbecco's PBS without calcium and
magnesium (Invitrogen), resuspended in HEPES buffer, and incubated in a
sonic bath for 1 h with a saturating concentration (15 µg/ml) of
ICAM-1/Fc to allow for both dispersion and coating of beads. In order
to block nonspecific adhesion, blocked (Molecular Probes, Eugene, OR)
was added to the bead solution (20% by volume) for 30 min with
sonication. The density of ICAM-1/Fc on the surface of the beads has
been determined previously (30) to contain ~25 binding
sites/µm2. Beads were added to the samples at 2 × 107 beads/ml (20 beads per neutrophil).
Flow Cytometric Detection of ICAM-1-coated Latex Beads--
To
examine specific adhesion via the 2 integrins,
neutrophils were mixed with fluorescent beads with ICAM-1/Fc
derivatized to their surface. Sample volumes of 250 µl (HEPES buffer,
1.5 mM CaCl2) contained 1 × 106 neutrophils/ml, 2 × 107 beads/ml,
LDS-751 (8 ng/ml, Molecular Probes, Eugene, OR) to label neutrophils,
and a small magnetic stir bar. Adhesion blocking antibodies and/or 240Q
were preincubated for 10 min with the cell suspension without beads.
For samples stimulated with IL-8, stimulus was added just after the
beads, and the suspension was then placed immediately on the sample
injection port of the FACScan flow cytometer (BD PharMingen). Samples
were maintained at 37 °C within a mixing chamber with a magnetic
motor as described previously (31). The magnetic motor coupled with the
magnetic stir bar created a shear field (shear stress ~1.0
dyne/cm2) within the test tube and initiated collisional
interactions of relatively brief contact duration (~25 ms).
Neutrophil capture of ICAM-1-coated beads was continuously monitored at
low flow. Neutrophils were distinguished by their characteristic
forward and right angle light scatter properties and gated in order to exclude unbound beads. Neutrophil-bead adhesion was quantitated by
green bead fluorescence on fluorescence histograms. Quantal increases
in fluorescence appeared as peaks in the fluorescence histogram
corresponding to populations of neutrophils binding increasing numbers
of beads (31). To distinguish relative levels of bead capture within
the stimulated neutrophil population, neutrophil-bead interactions were
quantitated as the average number of beads per neutrophil according to
Equation 1,
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(Eq. 1)
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where N represents the number of nonadherent
neutrophils, and NBi represents the number of
neutrophil-bead aggregates bound to between 1 and 6 or more beads.
Activation Epitope Recognized by mAb 327C--
mAb 327C has been
reported previously (15) to recognize the active conformation of CD18
associated with LFA-1 function in phorbol ester or T cell
receptor cross-link-stimulated primary human lymphocytes. To determine
the kinetics of CD18 activation, neutrophils and PBMC were mixed with
327C-Alexa and examined for 10 min with the flow cytometer under
conditions of stimulation by soluble ICAM-1/Fc (150 µg/ml), 240Q, or
IL-8 using identical stimulation protocols as the bead adhesion assay.
Additional experiments examining the transience of active CD18
expression after IL-8 stimulation were conducted where 327C-Alexa was
added to unfixed samples at indicated times after stimulation by 1 nM IL-8. All samples were washed, resuspended, and analyzed
on the FACScan flow cytometer. Mean fluorescence intensity (MFI) was
determined for the neutrophil population at indicated time points.
Receptor expression in terms of sites/cell was determined by comparing the MFI of bound 327C to Quantum Simply Cellular beads (FCSC, San Juan,
Puerto Rico) which contain five bead sets with increasing numbers of
goat anti-mouse-binding sites on their surface. From this analysis, a
linear relation between MFI and receptor expression was determined for
each directly conjugated antibody bound to cells and the calibration
bead set.
Immunofluorescence--
To observe colocalization of either
LFA-1 or Mac-1 with 327C, neutrophils (1 × 106/ml)
were stimulated with IL-8 or 240Q and incubated at room
temperature for 2 min with biotinylated 327C (10 µg/ml) and either
anti-LFA-1-PE or anti-Mac-1 (2LPM19c-RPE) according to manufacturer's
suggested staining procedure. Cells were fixed in 1% paraformaldehyde,
and excess antibody was removed by centrifugation. Following this wash,
cells were incubated with neutravidin-FITC for 20 min at room
temperature. Cells were centrifuged following staining and resuspended
in ice-cold HEPES buffer with 0.1% HSA followed by fixation with an
equal volume of freshly prepared 2% paraformaldehyde to a final
concentration of 1%. To determine the expression of active CD18 as a
function of the time after IL-8 stimulation, neutrophils (1 × 106/ml) were stimulated at time 0 with 1 nM
IL-8. Biotinylated 327C (10 µg/ml) was added 2 min prior to each time
point. At indicated times, 105 cells were removed and
immediately fixed in HEPES buffer containing 1% paraformaldehyde and
0.1% HSA. Following fixation, neutrophils were incubated with
neutravidin-FITC for 20 min at room temperature, washed, and
resuspended in 1% paraformaldehyde. The labeled cells were imaged
using a Nikon TE200 inverted microscope employing an oil immersion 60×
Plan-Apo objective and bandpass filters appropriate for FITC or PE labels.
Intracellular Calcium Flux--
Neutrophil intercellular calcium
flux was measured using Fluo-3-AM (Molecular Probes, Eugene, OR).
Neutrophils (2 × 106 cells) were added to a 1 µM Fluo-3-AM solution in HEPES buffer with 0.1%
Me2SO with no calcium or HSA and incubated at 37 °C for
45 min in the dark to allow for dye loading. Neutrophils were washed
and resuspended in indicator-free HEPES with calcium and HSA and then incubated at room temperature for 30 min in the
dark. Neutrophils stimulated with 240Q or R15.7 were allowed to
incubate with the noted concentrations of mAb for an additional 10 min at room temperature. Samples were incubated for 2 min at 37 °C, maintained at 37 °C with a water jacket, and read on the FACScan flow cytometer. After ~1 min of reading, the sample tube was removed from the FACScan; stimulant was added at the indicated concentrations (IL-8, 240Q, R15.7, or no additional stimulation), and the sample tube
was replaced on the sample injection port of the FACScan and read for
an additional ~3 min. The activated neutrophil population was gated
according to the increase in Fluo-3 fluorescence (FL-1) due to
intracellular calcium flux that corresponded to two standard deviations
above unstimulated neutrophils.
Statistical Analysis--
Data were independently collected for
each experiment, and a mean and S.E. were calculated for all
experiments. The Student's t test was used to determine
statistical significance between mean values. Statistical analysis was
performed using Prism 3 (GraphPad Software Inc., San Diego, CA). Values
were considered significant when p < 0.05.
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RESULTS |
Neutrophil Arrest on ICAM-1 in a Parallel Plate Flow
Chamber--
Neutrophils overcome the repulsive shear force of blood
flow during the process of arrest on inflamed endothelium through the
formation of sufficient numbers of CD18 bonds to ICAM-1. We directly
observed the dynamics of neutrophil adhesion to a monolayer of cells
expressing ICAM-1 in a parallel plate flow chamber at defined shear
stress, and we quantitated these dynamics over the time course of
activation with IL-8 and mAb 240Q. Neutrophil suspensions were
stimulated and then injected into the flow chamber and allowed to
settle onto the monolayer substrate at low shear for 1 min. Phase
contrast microscopy of neutrophils interacting with the ICAM-1-expressing monolayer at low shear enabled clear delineation of
those interacting that remained arrested for at least 30 s (Fig.
1a). Analysis of neutrophil
capture efficiency, defined as the transition from collisional
interactions such as cell rolling or tumbling motion to shear-resistant
arrest, was limited to relatively few cells in a single field of view
following injection in the flow chamber over the low shear interval
(Fig. 1b). Ramping up the shear stress for the duration of
the experiment allowed enumeration of a greater number of neutrophils
that remained arrested as a function of time as measured on five
separate fields of view. A significant increase in capture from the
base-line level required stimulation above a threshold dose of IL-8
(~0.1 nM) (Fig. 1b). Neutrophil arrest
increased with both dose of stimulus and time at low shear, reaching a
peak of ~75% of interacting neutrophils at 1 nM IL-8
stimulation, which did not increase at a 5 nM dose (data
not shown). At the high shear stress a steady drop in the fraction of
neutrophils remaining arrested was observed beyond the 1st min of
stimulation over the dose range of IL-8. Even at a saturating dose of 5 nM IL-8, cell arrest approached base line by 5 min of flow.
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Fig. 1.
Neutrophil arrest on ICAM-1 in a parallel
plate flow chamber. Recruitment of activated neutrophils to an
ICAM-1-expressing L-cell monolayer was assessed in the parallel plate
flow chamber under defined shear stress. Neutrophils in suspension
(106/ml) were activated with either IL-8 or mAb 240Q and
introduced as a bolus into the flow chamber. Following injection,
neutrophils were allowed to sediment to the ICAM-1 substrate in the
absence of flow. Shear stress was then ramped up to 0.1 dyne/cm2 to facilitate interaction between neutrophils and
the ICAM-1 substrate. To assess adhesion strength, shear stress was
subsequently ramped up to 4 dynes/cm2 (vertical
dashed line) for an additional 5 min. a, video
micrographs of representative fields of neutrophils interacting and
arrested (phase white PMNs and dark L-cell monolayer) at 0.1 dyne/cm2 (left) and 4 dynes/cm2
(right). b, time course of the fraction of
neutrophil arrested as a function of the dose of IL-8 or 240Q. Plotted
on the ordinate is the ratio of arrested neutrophils,
defined as those moving less than one cell diameter in 30 s, to
the total number interacting with the monolayer at 1 min.
c, costimulation of neutrophils with IL-8 (50 pM) and 240Q (0.125 µg/ml). The percent of arrested
neutrophils following costimulation was evaluated after 5 min of high
flow. * denotes p < 0.05 compared with 240Q alone.
Data represent mean PMN arrested ± S.E. from 3 to 5 separate
experiments.
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In contrast to IL-8 stimulation, direct activation of 2
integrin-dependent adhesion through binding of mAb 240Q was
less efficient at eliciting initial capture over a range of binding up
to receptor saturation at 5 µg/ml (data not shown). However, activation of just 5% of surface-expressed CD18, corresponding to
0.125 µg/ml of 240Q bound to the IDAS, was sufficient to trigger adhesion at low flow and effected a steady increase in neutrophil arrest that was sustained for up to 6 min of flow at the high shear
stress. Interestingly, higher doses of mAb 240Q up to saturation of
binding to CD18 (5 µg/ml) elicited neither a higher initial capture
rate nor a greater extent of adhesion at 6 min than observed with 0.125 µg/ml of 240Q (data not shown). We next examined neutrophil recruitment in response to costimulation with a low dose of IL-8 (50 pM) and 240Q (0.125 µg/ml). Costimulation did not
significantly boost the initial capture of cells under low flow
conditions, as compared with either 240Q or IL-8 alone. However, the
number of neutrophils remaining arrested on ICAM-1 after 5 min at high flow was significantly increased above that induced by 240Q at any dose
(Fig. 1c). These flow chamber studies reveal an increase in
the arrested fraction of neutrophils with increasing dose of IL-8,
above a threshold required to initiate capture. Neutrophil arrest
signaled by chemotactic factor was transient. However, stabilizing as
few as 5% of surface CD18 in an active conformation sustained
shear-resistant adhesion in virtually the entire cell population.
Dynamics of Neutrophil Capture of ICAM-1 under Fluid Shear--
To
define further the relationship between the dynamics of CD18 activation
and the efficiency of ICAM-1 capture, we analyzed the collisional
interactions between neutrophils and ICAM-1-coated fluorescent latex
beads in real time by flow cytometry. Beads were coated with ICAM-1 at
a site density commensurate with that expressed on inflamed endothelium
(~25 sites/µm2) and mixed together with neutrophils in
a cytometry tube at physiological shear stress (~1
dyne/cm2). Under these conditions each neutrophil
experience on the order of 10 collisions/s with beads just upstream of
the flow cytometer providing continuous analysis at rates of up to 200 cells/s. Over the 1st min of IL-8 stimulation the rate of capture
increased by 20-fold from the base-line adhesion of 3 beads per 100 neutrophils in unstimulated cells and up to 60 beads per 100 neutrophils at maximum response (Fig.
2a). In the absence of
stimulation, this base-line adhesion remained constant over 10 min of
shear. Stimulation with IL-8 (1 nM) elicited bead capture
that increased by 4-fold over the 1st min, culminating in a 10-fold
rise in the average number of ICAM-1 beads firmly adherent to
neutrophils within 10 min. Direct activation of CD18 by addition of mAb
240Q also elicited capture of ICAM-1 beads but at one-half the rate
induced by IL-8 stimulation.
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Fig. 2.
Dynamics of neutrophil capture of
ICAM-1-coated fluorescent beads in sheared suspension by flow
cytometry. Fluorescent protein-A microbeads were coated with
ICAM-1/Fc at a density of ~25 sites/µm2 and sheared in
suspension with neutrophils in a cytometry test tube. Bead adhesion was
detected in real time on the flow cytometer by gating on neutrophils
capturing between 1 and 6+ green fluorescent beads. Beads/PMN represent
the average number of beads captured by neutrophils. a, time
course of ICAM-1 bead capture stimulated with optimal doses of IL-8 (1 nM) or 240Q (5 µg/ml). Molecular specificity of adhesion
determined by preincubating neutrophils with function blocking mAbs to
LFA-1 (CD11a/CD18), Mac-1 (CD11b/CD18), or CD18. Neutrophils were
stimulated with IL-8 (1 nM) (b) and 240Q (5 µg/ml) (c). Data are presented as the mean ± S.E.
from 3 to 5 separate experiments.
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We next examined the relative contributions of LFA-1 and Mac-1 to the
capture of ICAM-1 beads in response to either chemotactic or allosteric
activation. Treatment of neutrophils with anti-LFA-1 abrogated the
initial rapid boost in ICAM-1 capture triggered by IL-8, while
anti-Mac-1 inhibited the accumulation of beads only beyond 4 min of
stimulation (Fig. 2b). Blocking with anti-CD18 completely
abrogated bead adhesion. Together, the data indicate that LFA-1 is
sufficient for capture of ICAM-1, whereas Mac-1 accounts for ~30% of
ICAM-1 bead adhesion at later time points of IL-8 stimulation.
In contrast to GPCR signaling of adhesion, allosteric activation of
CD18 at a saturating dose of 240Q elicited capture solely supported by
LFA-1. Blocking Mac-1 function with mAb did not significantly reduce
ICAM-1 bead capture, as compared with 240Q alone (Fig. 2c).
ICAM-1 capture was entirely accounted for by activation of LFA-1, as
blocking with anti-LFA-1 abrogated 240Q-induced adhesion. We confirmed
that 240Q indeed bound to the Mac-1 receptor by testing binding to cell
lines expressing Mac-1 in either the active or inactive conformation
(data not shown). Additional studies were performed on neutrophils to
assess Mac-1 function in response to stimulation with both 240Q and
IL-8. Neutrophil capture of albumin-coated latex beads in the flow
cytometry assay has been shown previously (32) to be mediated entirely
by activation and binding of Mac-1, and was found to be blocked in the
presence of mAb 240Q binding to CD18 (data not shown). These data
confirmed that activated LFA-1 is sufficient for IL-8-stimulated or
240Q-induced capture of ICAM-1.
Relation between Stimulus Dose and Rate of Capture of
ICAM-1--
Recent studies (17, 30) have shown that neutrophil
adhesion elicited by chemotactic stimulation is most rapid over the initial seconds following addition of stimulus. Therefore, we computed
the rate of ICAM-1 bead capture following the 1st min of applied shear
and stimulation over a dose range of IL-8 or 240Q. A direct correlation
was observed between the dose of stimulus and the rate of ICAM-1 bead
capture above a threshold of 75 pM IL-8 necessary to induce
significant bead adhesion. The sensitivity of this assay was exhibited
by a detectable increase in rate and extent of adhesion as the IL-8
dose was incremented by just 100 pM (Fig.
3a). The effective
concentration to induce half-maximum capture (EC50) was 250 pM IL-8, which corresponds to ~25% IL-8 receptor
occupancy.
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Fig. 3.
Rate of ICAM-1 bead capture following
stimulation with IL-8 and 240Q. The rate of ICAM-1 bead capture
over the 1st min of activation in the flow cytometry assay was
determined for neutrophil suspensions activated over a dose range of
IL-8 (a) and 240Q (b). Plotted is the slope of
the beads/PMN time course (as depicted in Fig. 2) over the 1st min
following stimulation (c). Costimulation of neutrophils
with IL-8 (100 pM) and 240Q over a range of doses expressed
in terms of the site occupancy of surface CD18. Plotted is the increase
in rate of capture above activation with 240Q alone. Data are presented
as the mean ± S.E. from 3 to 8 separate experiments, and *
denotes a significant increase (p < 0.05) in capture
compared with activation by IL-8 or 240Q alone.
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Activation via the I domain and the conformationally linked IDAS
using mAb 240Q yielded significant adhesion at 0.125 µg/ml, a dose
that corresponds to ~5% site occupancy of surface-expressed CD18
(Fig. 3b). The rate of bead capture increased in direct
proportion to binding of 240Q up to 15% CD18 occupancy. A plateau in
the efficiency of ICAM-1 bead capture corresponded to occupancy of only
~30% CD18. At this dose of 240Q, IDAS-activated capture of ICAM-1
was less than one-half that elicited by maximal IL-8 stimulation (0.31 versus 0.64 beads/PMN/min; p < 0.05).
Commensurate with the parallel plate flow chamber data, these results
confirm that inside-out signaling via ligation of IL-8 is more
efficient at eliciting dose-dependent neutrophil capture on
ICAM-1 than via allosteric activation of CD18.
We examined further the ability of IL-8 to augment the efficiency of
ICAM-1 bead capture over a range of CD18 site occupancy by 240Q (Fig.
3c). At a dose of IL-8 (100 pM) just above the
threshold to induce adhesion, the rate of ICAM-1 bead capture was
amplified most profoundly at low CD18 site occupancy of 240Q. With only ~3% IDAS activation of CD18, IL-8 amplified the rate of capture by
over 3-fold above 240Q alone. However, bead capture induced by 240Q
doses exceeding 15% occupancy of CD18 was not augmented by addition of
IL-8. Taken together, the data demonstrate that capture efficiency
induced via allosteric activation of CD18 is limited, yet cellular
processes initiated through chemotactic signaling significantly amplify adhesion.
Intracellular Calcium Flux Signaled via IL-8 and 240Q--
In
light of recent evidence (33) linking an increase in intracellular
calcium with subsequent surface redistribution of LFA-1 into high
avidity clusters, we measured Ca2+ flux in neutrophils
stimulated with IL-8 and 240Q. In particular, we examined whether
costimulation would result in a potentiation of the Ca2+
flux, as was observed for neutrophil capture and arrest on ICAM-1. Neutrophils were loaded with the fluorescent calcium indicator Fluo-3,
and intracellular Ca2+ flux was detected by flow cytometry
over the time course of stimulation (Fig.
4, inset). Activation was
quantitated as the neutrophil fraction that responded to stimulus with
a rapid increase in fluorescence above the base-line value in
unstimulated cells (Fig. 4a). Ca2+ flux was
found to be the most sensitive assay to the onset of activation, as a
threshold dose of just 25 pM IL-8 elicited a significant
signal in ~10% of the neutrophil population (Fig. 4a).
The EC50 of Ca2+ flux was ~100 pM
IL-8, almost 3-fold lower than measured for ICAM-1 adhesion. Maximal
Ca2+ flux corresponded to 1 nM IL-8, which
elicited a response in 85% of neutrophils. These data verify the role
of Ca2+ flux as a faithful secondary messenger in
regulating neutrophil adhesion signaled through ligation of chemotactic
receptor.
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Fig. 4.
Intracellular release of Ca2+ in
response to stimulation with IL-8 and 240Q. Intracellular calcium
flux was quantitated by flow cytometry and expressed as the percentage
of neutrophils immediately responding to stimulation above a threshold
level of Fluo-3 fluorescence, set at two standard deviations above the
unstimulated mean fluorescence intensity. a,
neutrophils stimulated over a dose range of IL-8. Inset
shows time course of Fluo-3 fluorescence for a cell suspension
stimulated with IL-8 (0.1 nM). b,
costimulation with IL-8 and 240Q (0.125 µg/ml) on calcium flux.
Anti-CD18 mAb R15.7 was used as a control. Data are presented as the
mean ± S.E. from three separate experiments. * denotes
significant increase in Ca2+ flux (p < 0.05) compared with IL-8 alone or unstimulated.
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Allosteric activation of neutrophils with mAb 240Q, but not a control
anti-CD18 (mAb R15.7), at doses that saturate receptor binding failed
to induce significant Ca2+ flux (Fig. 4b). In
contrast, costimulation with both 240Q (5% CD18 site occupancy) and
IL-8 (100 nM) resulted in a 32% potentiation above IL-8
alone in signaling Ca2+ flux. This boost was not observed
when IL-8 was combined with mAb R15.7, which binds to CD18 with
comparable affinity to 240Q. These data suggest that allosteric
activation of CD18 as induced by mAb 240Q may also function in
receptor-mediated signaling from the outside-in.
Activation of CD18 Detected by Binding of Monoclonal Antibody
327C--
Lymphocyte adhesion to ICAM-1 mediated by LFA-1 was recently
shown to correlate with a shift in binding affinity regulated at the
IDAS (15, 22). Previous studies have demonstrated that 327C faithfully
reports on the active conformation of LFA-1 high affinity. In contrast
to the binding of mAb24 which also reports on CD18 activation, 327C
binding to LFA-1 on lymphocytes does not require ICAM-1 binding at the
I domain (14, 34). We first examined expression of 327C on neutrophils
and PBMC in response to soluble ICAM-1/Fc (150 µg/ml). Consistent
with Lupher et al. (15), ICAM-1/Fc induced a 4-fold increase
in 327C binding from base-line-unstimulated mononuclear cells (Fig.
5). This represented a shift to an active
conformation in ~50% of LFA-1 expressed on PBMC. In contrast,
base-line expression of 327C was higher in purified neutrophils and was
not up-regulated in response to incubation with ICAM-1/Fc. It appears
that neutrophils do not undergo ligand-induced activation of LFA-1,
suggesting that significant differences exist in the regulation of
LFA-1 function between neutrophils and PBMC.
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Fig. 5.
Expression of activated CD18 on neutrophils
and peripheral blood mononuclear cells in the presence of soluble
ICAM-1. Receptor expression of LFA-1 and the active ligand binding
conformation of CD18 was assessed on neutrophils and PBMC following
addition of soluble ICAM-1/Fc (150 µg/ml). LFA-1 was detected with a
directly conjugated mAb, and expression of activated CD18 was reported
by mAb 327C that recognizes a neoepitope previously shown to
up-regulate in response to cell stimulation (14). Mean fluorescence
intensities of antibody binding was converted to receptor site numbers
using calibration beads with known receptor site densities as described
under "Experimental Procedures." Data are presented as the
mean ± S.E. from three separate experiments. * denotes
significant increase (p < 0.05) in activated CD18
compared with 327C alone.
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Next we used mAb 327C to elucidate the kinetics of CD18 activation in
real time following the addition of IL-8 or mAb 240Q. Binding of
fluorescently conjugated 327C to neutrophil CD18 was continuously
measured by flow cytometry as cells were stimulated with either IL-8 or
240Q. In the absence of stimulus, neutrophils expressed a background
level of 327C-binding sites that did not increase significantly over 10 min of shear at 37°C (Fig.
6a). Stimulation elicited a
rapid up-regulation of active CD18 sites in which the rate and extent
was commensurate with the dose of IL-8. Similar to the kinetics of
ICAM-1 capture, expression of the CD18 activation epitope was most
rapid over the 1st min of stimulation regardless of IL-8 dose.
Up-regulation of 327C increased 10-fold over a dose range of IL-8, from
a base-line detection of ~10,000 sites up to 100,000 sites/neutrophil
(Fig. 6b). The dose-response curve of CD18 activation was
similar in shape to that observed for adhesion function, exhibiting a
threshold dose of 100 pM and an EC50 of ~300
pM IL-8 (Fig. 6b).
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Fig. 6.
Dynamics and dose dependence of CD18
activation of neutrophils. Up-regulation of activated CD18 was
examined as a function of IL-8 and 240Q dose in the presence of
fluorescently conjugated 327C. a, the time course of CD18
activation in response to addition of a high and low dose of IL-8 and
saturating concentration of 240Q was quantitated by the increased
binding of 327C-Alexa as measured in real time by flow cytometry. The
extent of active CD18 expression was assessed over a dose range of IL-8
(b) and 240Q (c). MFI values were expressed in
terms of active receptors using calibration beads with known receptor
site densities. Data are presented as the mean ± S.E. from 3 to 5 separate experiments. * denotes significant increase in active CD18
sites (p < 0.05) compared with unstimulated
control.
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To assess the kinetics and extent of 327C up-regulation in response to
allosteric activation of CD18, neutrophils were pretreated for 10 min
at room temperature with 240Q and then continuously measured on the
flow cytometer at 37 °C. Preincubation with 240Q induced a higher
base-line expression of 327C as compared with IL-8 stimulation.
Up-regulation of active CD18 with incubation was most rapid over the
1st min of incubation and increased at a rate comparable with low dose
IL-8 stimulation (Fig. 6a). As with adhesion to ICAM-1, IDAS
activation of CD18 exhibited a 40% narrower dynamic range than for
IL-8 stimulation, increasing from a base line of 15,000 sites up to
only 60,000 sites (Fig. 6c).
This comparable difference in efficacy of CD18 activation via GPCR and
the IDAS also correlated with a difference in ICAM-1 capture efficiency
between the two modes of stimulation. For example, the threshold dose
of 240Q (5% CD18 occupancy) that induced activation of ~23,000 CD18
sites/PMN corresponded to a rate of ICAM-1 capture of ~0.1
beads/PMN/min. By comparison this number of active CD18 was elicited by
~150 pM IL-8, which corresponded to greater than 0.2 beads/PMN/min. This trend also held at higher doses of stimulus, whereas 0.5 nM IL-8 and 240Q at 1 µg/ml (20% CD18
occupancy) both elicit about the same number of active sites (~40,000
sites); IL-8 activated a 3-fold greater rate of ICAM-1 capture.
Additionally, although costimulation at low doses of IL-8 and 240Q
elicited significant increases in adhesive function, no significant
increases were detected for 100 pM IL-8 and 0.03-0.125
µg/ml 240Q (data not shown).
Immunofluorescence Detection of Active CD18 on the Neutrophil
Surface--
The above data are consistent with the hypothesis that
CD18-adhesive function in neutrophils is acutely dependent on factors in addition to a shift from a low to high affinity conformation. For
example, it has been demonstrated that lymphocyte adhesion to ICAM-1
may be regulated by an increase in the lateral diffusion of integrins
that supports the formation of high avidity clusters of receptors (33).
We employed two-color immunofluorescence to examine the surface
topography of mAb 327C relative to LFA-1 or Mac-1 on neutrophils
following stimulation with IL-8 and/or 240Q (Fig.
7 and Table
I). In the resting state LFA-1 and Mac-1 were uniformly distributed on the cell surface as revealed by the
circumferential red fluorescence on unstimulated neutrophils (Fig. 7,
a and b). The absence of 327C green fluorescence
indicates the absence of detectable levels of activated CD18.
Stimulation with IL-8 induced a redistribution of LFA-1 into punctate
clusters of red fluorescence circumferentially distributed on the
plasma membrane (Fig. 7c). Image analysis revealed that 75%
of the LFA-1 colocalized with active CD18 as indicated by clusters and
larger patches of 327C-associated yellow fluorescence (Table I). Mac-1 expression increased with IL-8 stimulation but remained uniformly distributed, and only ~35% colocalized with clusters of 327C (Fig. 7d and Table I). Activation of CD18 in the presence of 240Q
again resulted in colocalization of membrane LFA-1 with 327C defined active CD18 (Fig. 7e). In contrast to IL-8 stimulation that
elicited a wide distribution of active sites, 240Q induced tight
clusters of yellow fluorescence recruiting a smaller fraction of
activated LFA-1 (Table I). Surface distribution of Mac-1 sites was not altered in the presence of 240Q (Fig. 7f).
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Fig. 7.
Colocalization of LFA-1 and Mac-1 with
the active conformation of CD18. The topography of CD18 subunits
and the activation epitope defined by 327C was visualized using
fluorescence microscopy before (a and b) and
after stimulation with IL-8 (1 nM) (c and
d) and 240Q (0.125 µg/ml) (e and f).
Activated sites of CD18 (green) were imaged using
biotinylated mAb 327C in conjunction with an avidin-FITC secondary
label. LFA-1 and Mac-1 were imaged using fluorescently labeled
anti-LFA-1-PE and anti-Mac-1-PE monoclonal antibodies (red).
Micrographs are representative of 15-25 neutrophil observations in two
separate experiments. Cell images were obtained by phase contrast
microscopy and overlaid using Image Pro Plus 4.5 image analysis
software.
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Table I
Colocalization of LFA-1, Mac-1, and mAb 327C defined active CD18
Data represent percent of fluorescent LFA-1 or Mac-1 associated with
327C as determined by immunofluorescence. These data correlate to
representative images depicted in Fig. 7 and fractional colocalization
analyzed by image analysis of 15-25 cells for each condition from two
separate experiments.
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Kinetic Analysis of CD18 Topography following Activation by
IL-8--
The dynamics of CD18 activation and surface distribution
were examined by correlating 327C topography by immunofluorescence with
the time course of expression by flow cytometry. 327C expression increased most rapidly over the initial 30 s of IL-8 addition, increasing from a base line of ~10,000 sites up to 70,000 sites/PMN (Fig. 8a). This peak in
expression was maintained over the first minutes of stimulation before
dropping back toward base line by 20 min, clearly revealing a shift in
CD18 conformation back to the resting state. This time course of 327C
expression would appear to be at odds with the kinetics depicted in
Fig. 5, which increased to a peak level that was maintained over
10 min. This apparent discrepancy highlights an interesting feature of
mAb 327C in that binding to CD18 stabilized the active conformation of
CD18 under conditions in which it is present during stimulation and
flow cytometric analysis.
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Fig. 8.
Redistribution of active CD18 following
stimulation with IL-8. The expression and topography of active
CD18 was quantitated on 327C-labeled samples over the time course of
stimulation. a, expression dynamics of active CD18 were
quantitated in terms of 327C site number following IL-8 (1 nM) stimulation. Neutrophil suspensions in the presence of
327C-Alexa were stimulated, washed, and analyzed for 327C-Alexa binding
using flow cytometry. b, binding of 327C to active CD18
on stimulated neutrophils was imaged at specific time points following
stimulation with IL-8 (1 nM). Samples were labeled with
biotinylated mAb 327C and then rapidly fixed with 1% paraformaldehyde,
secondarily labeled with avidin-FITC, and imaged by phase contrast
fluorescence microscopy. Each image is representative of at least 15 separate observations. Data are presented as the mean site density of
327C/PMN ± S.E. from three separate experiments.
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The kinetics of CD18 activation and down-regulation correlated
temporally with the decrease in firm adhesion to ICAM-1 under shear as
observed in the parallel plate flow chamber. This prompted us to
examine the dynamics of receptor redistribution immediately following
activation with IL-8. Fluorescence microscopy of neutrophils fixed and
labeled with 327C revealed the formation of both small punctate
clusters (<1 µm2) and large caps (~3
µm2) within 30 s of stimulation (Fig.
8b). Within 2 min of stimulation, these caps dissipated into
numerous smaller proximal clusters. By 10 min the smaller clusters had
decreased in number, and the overall surface fluorescence was
diminished. In contrast, treatment with 240Q induced small clusters of
active CD18 that remained intact for over 10 min of observation (data
not shown). Taken together, the immunofluorescence revealed the rapid
mobility of 327C into aggregates of active CD18, which formed and
subsequently dissipated over a time course that mirrored the rise and
fall of ICAM-1 capture and adhesion stability in the flow chamber.
Inhibition of CD18 Mobility and Neutrophil Capture of ICAM-1
Beads--
It has been reported recently (35) that chemotactically
stimulated neutrophil adhesion to ICAM-1, but not expression of activated 2 integrin, is dependent upon PI(3)K activity.
It has also been reported that chemokine-triggered PI(3)K activity
regulates the rapid lateral mobility of LFA-1 in lymphocytes
(20). Although the mechanism for this phenomenon is not clearly
understood, it is thought that PI(3)K generates lipid secondary
messengers including inositol 1,4,5-trisphosphate and Ca2+
flux that in turn regulate the formation of complexes between integrins
and the cytoskeleton (36). Based on these findings, we examined the
effect of inhibitors of PI(3)K and F-actin polymerization on the
surface distribution and adhesive function of active LFA-1 following
stimulation. Treatment with wortmannin did not affect the expression of
LFA-1 but significantly inhibited the extent of clustering and capping
of active CD18 stimulated by IL-8 (Fig. 9a). In contrast, wortmannin
had no effect on the formation of punctate clusters of 327C induced by
240Q (data not shown).
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Fig. 9.
Neutrophil activation in presence of
inhibitors of PI(3)K and actin polymerization. The role of the
cytoskeleton and PI(3)K signaling activity on activation of CD18 was
examined using wortmannin, LY294002, or cytochalasin B in conjunction
with IL-8 or 240Q stimulation. a, topography of LFA-1
and active CD18 on neutrophils and stimulated with IL-8. Neutrophils
were fixed 2 min after IL-8 (1 nM) stimulation and compared
with those preincubated with wortmannin (100 nM). Images
are representative of 20 observations. b,
conformational activation of CD18 in the presence of inhibitors of
PI(3)K and actin polymerization. Expression of 327C was quantitated by
flow cytometry as described under "Experimental Procedures."
Neutrophils were preincubated with cytochalasin B (0.3 mg/ml),
wortmannin (100 nM), or LY294002 (20 mM) and
then stimulated with either IL-8 (1 nM) or 240Q (5 µg/ml)
for 10 min. c, ICAM-1 bead capture in the presence of
inhibitors of actin and PI(3)K. Neutrophils were preincubated with
inhibitors and stimulated as described above. Activation of CD18 is
plotted as percentage of stimulated vehicle control samples (0.1%
MeSO). Data are presented as the mean ± S.E. from three separate
experiments. * denotes significant change (p < 0.05)
in CD18 function compared with vehicle control.
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We next examined the effects of pharmacological inhibition on
expression and function of CD18 assessed by fluorescence flow cytometry. Cytochalasin B was applied to disrupt the F-actin
polymerization, whereas PI(3)K activity was specifically inhibited by
pretreatment with wortmannin or LY294002. Blocking PI(3)K activity or
F-actin formation resulted in nominal inhibition in expression of
active CD18 induced by either IL-8 or 240Q (Fig. 9b).
Interestingly, treatment with cytochalasin B augmented expression of
327C sites in response to either mode of CD18 activation. Capture of
ICAM-1 beads in response to IL-8 stimulation was significantly
diminished in the presence of either inhibitor of PI(3)K, but not by
treatment with cytochalasin B (Fig. 9c). In contrast, ICAM-1
bead adhesion activated by 240Q was not diminished in the presence of
PI(3)K inhibitors. Treatment with cytochalasin B did not significantly inhibit ICAM-1 bead capture stimulated through either mode of activation. The data are consistent with a mechanism in which signaling
via IL-8 triggers conformational activation of LFA-1, which
subsequently redistributes into patches through a
PI(3)K-dependent process that is independent of F-actin
formation. These steps appear to be critical for optimum capture
efficiency of ICAM-1 as allosteric activation of LFA-1 in the presence
of 240Q was less efficient at mediating capture of ICAM-1 but was not
affected by PI(3)K blockers.
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DISCUSSION |
Neutrophils constitute greater than 70% of circulating leukocytes
and are the first to accumulate within inflamed microcirculation arresting within a few cell diameters of the site of transmigration (37). Recruitment of neutrophils is a tightly regulated process in
which the efficiency of conversion from transient to shear-resistant adhesion is influenced by the extent of cellular stimulation, which in
turn controls the coordinated expression of activated receptor and
vascular ligand (38). Several lines of evidence indicate that the
gatekeeper in neutrophil recruitment is binding of activated
2 integrins to ICAM-1 and ICAM-2 on endothelium (39). In
the current study we investigated the following two modes of CD18
activation in order to elucidate the molecular dynamics of neutrophil
capture in shear flow: stabilization of the active conformation through
binding of mAb 240Q, and chemotactic signaling via the IL-8 receptors,
CXCR1 and CXCR2. By using this strategy we demonstrated the following.
1) A shift in LFA-1 conformation is necessary and sufficient for
neutrophil adhesion to surface-expressed ICAM-1 at physiological levels
of shear stress. 2) IL-8 at 75 pM, corresponding to
ligation of no more than ~100 receptors/PMN, is sufficient to trigger
neutrophil capture to ICAM-1. 3) Capture efficiency and expression of
activated CD18 increased in direct proportion to IL-8 over a log range
in dose. 4) Allosteric activation of only 5% of surface LFA-1 via 240Q
was sufficient to trigger adhesion to ICAM-1 but at half the efficiency
stimulated by IL-8. 5) the boost in capture efficiency involves a rapid
redistribution of activated LFA-1 into membrane clusters via a
signaling pathway involving PI(3)K but not F-actin assembly. 6) Stable
adhesion is reversible within minutes of IL-8 activation and involves a progressive shift from high to low affinity CD18, which could be
reversed by 240Q stabilization of the IDAS.
The adhesion kinetics observed in the parallel plate flow chamber
mirrored the initial boost and subsequent drop in expression of the
activation epitope of CD18 defined by 327C. Allosteric activation of
CD18 by binding of 240Q was less effective at mediating capture over
the initial low shear interval in the flow chamber even at a saturating
dose of 5 µg/ml, corresponding to activation of ~30% of expressed
CD18. However, increased numbers of neutrophils transitioned to stable
adhesion with time of exposure to high shear stress. Occupancy of just
5% of surface CD18 with 240Q was sufficient to stabilize ~20,000
receptors per cell in an active state, resulting in arrest of ~60%
of interacting neutrophils. Interestingly, the arrested fraction did
not increase with dose of 240Q; however, costimulation with low dose
IL-8 (0.05 nM) and 240Q (5% CD18 occupancy) resulted in
nearly the entire neutrophil population remaining arrested for up to 10 min of high shear stress. This synergistic response with costimulation
suggests that adhesion is regulated by a shift in CD18 conformation in
addition to other cell responses elicited by GPCR stimulation.
Supporting this was the observation that allosteric activation, and not
simply binding of antibody to CD18, potentiated intracellular
Ca2+ flux stimulated by IL-8. This result suggests that
conformational activation of CD18 itself may provide an important
outside-in signal. Adhesion strengthening with costimulation was
determined primarily by the number of LFA-1 remaining in the active
conformation over time, as Mac-1 adhesion function was blocked in the
presence of bound 240Q. In this regard it has recently been reported
that LFA-1 provides an important cosignal in activation of neutrophils. For example, superoxide secretion is defective in LFA-1 knockout mice
(40). Moreover, LFA-1 is necessary and sufficient for transmigration at
sites of inflammation as indicated in studies of LFA-1 and Mac-1
knockout mice (41, 42).
Lymphocyte adhesion via LFA-1 can be signaled by phorbol ester or
treatment with divalent cations such as Mg2+ (43), which
was found to correlate with a shift in binding affinity regulated at
the IDAS (15). Expression of 327C on lymphocytes correlated with the
ligand-binding state of LFA-1 but was not dependent upon ICAM-1 binding
(14). Consistent with previous reports, ICAM-1/Fc induced an
active conformation in ~50% of available LFA-1 in PBMCs resulting in
a 4-fold increase in binding of 327C (15). This was not observed in a
donor-matched population of neutrophils that constitutively expressed
higher levels of active CD18 but exhibited no activation in the
presence of excess ICAM-1/Fc. Another difference between LFA-1-mediated
adhesion of neutrophils and PBMCs is the respective dependence on
F-actin. We found that neutrophil capture of ICAM-1 beads was not
affected in the presence of cytochalasin B. Others have reported (44)
that lymphocyte adhesion to ICAM-1 is facilitated by disruption of
F-actin. These provide the first data showing conformational activation
of LFA-1 may be differentially regulated on neutrophils as compared
with PBMCs.
Flow cytometry provided a real time assay of the rate of adhesion
between neutrophils and fluorescent beads coated with recombinant ICAM-1 at a site density commensurate with that measured in inflamed venules (~25 sites/µm2) (30). This assay proved to be
twice as sensitive as the parallel plate flow chamber in detecting the
threshold dose of IL-8 (75 pM) sufficient to activate
neutrophil capture of ICAM-1. Incrementing IL-8 dose by as little as
100 pM elicited a detectable increase in the rate and
extent of ICAM-1 bead capture. Adhesion function closely followed
stimulus dose, increasing by 6-fold over a log range in IL-8
concentration from ~0.1 to 1 nM. Peak capture efficiency at 1 nM corresponded to the reported Kd
of IL-8 binding, ~50% receptor occupancy. A threshold dose of 0.1 nM IL-8 was also found to be sufficient to trigger binding
of 327C, which reported on ~20,000 active CD18 (~100
sites/µm2). This dose of stimulus also corresponded to
the minimum activation that triggered capture of ICAM-1 beads.
Moreover, the dose response between IL-8 stimulation and 327C
expression was nearly identical to the curve describing ICAM-1 bead
adhesion. An important difference was that maximal expression of 327C
at a dose of 5 nM IL-8 exceeded by 5-fold that required to
activate optimal ICAM-1 adhesion. Thus, maximal IL-8 stimulation
corresponded to activation of ~100,000 CD18 or ~50% of total
membrane-expressed 2 integrin. However, half-maximum
capture of ICAM-1 corresponded to ~0.2 nM IL-8 and activation of only 30,000 CD18, of which no more than ~15,000 LFA-1
participated in adhesion. Thus, with increasing dose of IL-8 stimulus,
up-regulation of activated CD18 exceeds by as much as 3-fold that
necessary to trigger adhesion function. Taken together, the data reveal
a wide dynamic range coupling stimulus and response such that small
increments in fractional occupancy of IL-8 above the threshold dose
elicit an exponential increase in signaling of adhesion.
In comparison to GPCR signaling, allosteric activation of
2 integrin was less potent at eliciting capture of
ICAM-1. However, only 5% occupancy of CD18 by 240Q, an estimated
~5,000 LFA-1 receptors or ~25 sites/µm2, was
sufficient to trigger ICAM-1 adhesion above base line in the bead
capture and parallel plate adhesion assays. Efficiency of bead capture
increased with binding of 240Q but over a relatively narrow dose range
and at one-half the efficiency signaled by IL-8 stimulus. A limitation
in eliciting optimum adhesion via the IDAS correlated with a diminished
capacity of 240Q to bind and activate greater than ~25% of membrane
CD18, as compared with the 50% CD18 activation elicited by IL-8.
Because 240Q activates ICAM-1 adhesion through LFA-1, and not Mac-1,
this corresponds to activation of less than 15% of CD18 at maximum
allosteric activation. Binding kinetics of 327C to stimulated
neutrophil suspensions confirmed what was observed for ICAM-1 bead
capture, that CD18 activation occurred most rapidly over the 1st min of
stimulation regardless of IL-8 dose. In contrast, binding of 327C to
neutrophils maximally activated with 240Q simply revealed the kinetics
of recognition for CD18 already converted to the active state. These
dynamics highlight the extraordinary efficiency by which IL-8 binds its receptor and converts CD18 to the active conformation over the initial
seconds of stimulation. Thus, conformational activation of CD18 did not
appear to be the rate-limiting step in ICAM-1 capture in the presence
of IL-8.
Immunofluorescence of 2 integrin expression on resting
and activated neutrophils revealed little 327C binding to unstimulated cells. IL-8 stimulation of neutrophils transduced rapid activation of
CD18 as adhesion was measured within seconds, and subsequent deactivation of sites and disaggregation of clusters occurred within
minutes. This transience in the active conformation of CD18 could only
be detected by 327C labeling of samples that were removed from the test
tube over the time course of stimulation, as 327C in excess over the
course of stimulation appeared to stabilize the active conformation
(Figs. 8a versus 6a). After only
30 s of IL-8 stimulation, a 7-fold increase in CD18 activation was detected, and a majority of active sites redistributed into submicron clusters and larger patches several µm2 in area.
Two-color fluorescence showed that ~75% of LFA-1 colocalized with
active CD18, as compared with less than 40% of Mac-1, despite the fact
that Mac-1 expression was up-regulated severalfold with chemotactic
stimulation. After only 2 min of stimulation membrane patches began to
disperse with only small clusters remaining, and by 10 min practically
all clusters had dispersed, and the number of active receptors dropped
by ~50%. This transience of both active CD18 and LFA-1 cluster
formation corresponded temporally with the reversibility of firm
adhesion in the parallel plate flow chamber. Transience in the avidity
of LFA-1 has been reported previously (30) for adhesion to
ICAM-1-expressing cells and in adhesion to ICAM-1/Fc on beads. In
contrast, 240Q-treated neutrophils remained adherent at a shear stress
of 4 dynes/cm2. Because 240Q was also shown to lock CD18 in
an active conformation, this suggests that a conformational shift to
low affinity was responsible for the transience in adhesion.
Treatment with 240Q resulted in formation of small punctate clusters of
active CD18 by a mechanism that did not necessarily require outside-in
signal transduction. Supporting this was the observation that binding
of 240Q did not itself signal intercellular calcium flux. The
possibility exists that cluster formation was a consequence of CD18
dimerization upon bivalent binding of 240Q. However, this is probably
not the case as Fab fragments of 240Q have been reported (14) to be
equally effective at conformational activation of CD18.
Antibody-induced aggregation of LFA-1 into clusters is an unlikely
mechanism as discrete clusters of 327C formed in response to binding of
just 1-5% of CD18 receptors by 240Q. Clustering of LFA-1 has been
reported in response to binding of its physiological ligand, ICAM-3,
which activated strong LFA-1/ICAM-1 adhesion in T-cells (45).
Chemokine stimulation can initiate signaling via distinct pathways that
trigger a high affinity binding state and an increase in the lateral
mobility of LFA-1, and both mechanisms activate the arrest of
circulating lymphocytes (20, 33, 46). Enhanced membrane mobility of
LFA-1 can be mediated by calcium-dependent activation of
calpain, which dissociates LFA-1 from the cytoskeleton by proteolysis
of talin links to the actin cytoskeleton (47, 48). Rapid lateral
mobility of LFA-1 has been shown to be a critical factor in the
efficiency of chemokine-triggered lymphocyte arrest to ICAM-1, and this
process requires PI(3)K activity (20). Less information has been
available regarding the mechanism of neutrophil capture on ICAM-1;
however, it was recently reported (35) that CD18-mediated adhesion is
dependent upon PI(3)K activation. We found that two inhibitors of
PI(3)K activity, wortmannin and LY294002, significantly diminished the
efficiency of ICAM-1 bead capture stimulated by IL-8 but had no effect
on adhesion activated via the I domain or IDAS conformational shift.
IL-8 stimulated a rapid clustering of conformationally active LFA-1
that was essential to optimum capture efficiency of ICAM-1. Blocking
PI(3)K decreased, but did not eliminate, clustering of 327C while
leaving intact the ability of CD18 to undergo conformational activation
in response to IL-8 or 240Q. Depleting cytoplasmic F-actin by
pretreatment with cytochalasin actually increased expression of active
CD18. These data support a model of IL-8-stimulated neutrophil capture on ICAM-1 that is critically dependent on an increase in LFA-1 mobility
over a time frame of less than 1 s. The transition to stable
shear-resistant adhesion was associated with the rapid formation
(~seconds) of patches of active LFA-1. The involvement of PI(3)K on
both mobility and patching of LFA-1 suggests that it may be involved in
the initial release of cytoplasmic attachments of LFA-1 to 
-actinin
(47). Our data also show that Ca2+ flux, a signal
downstream of PI(3)K activation, is important in the amplification of
the adhesion. Stimulation with only 25 pM IL-8,
corresponding to as few as 10 GPCR receptors bound per neutrophil,
elicited Ca2+ flux in a significant fraction of cells. A
remarkable finding was the boost in Ca2+ flux in response
to costimulation with 240Q and IL-8. The fact that 240Q alone was
sufficient to induce small clusters of LFA-1 but not a Ca2+
flux suggests that GPCR-signaled events are critical to optimal adhesion efficiency. Increased lateral or rotational diffusion and
formation of clusters of active LFA-1 could serve to amplify the
probability of ligand binding and promote efficient capture. Allosteric
activation at the IDAS via 240Q also elicited distinct clusters of
active CD18, but this redistribution was not dependent on PI(3)K, and
capture of ICAM-1 was less than one-half as efficient as that elicited
by GPCR signaling.
In this study, we examined how the efficiency of neutrophil capture and
firm adhesion on ICAM-1 is dynamically regulated by GPCR-signaled
alterations in LFA-1 affinity and membrane distribution. The
implication for neutrophil recruitment at acute sites of inflammation is that, like selectin bonds, capture may be mediated by attachment of
few activated LFA-1 receptors elicited by as few as 10 GPCR. This
notion has been confirmed recently in the microcirculation of the
mouse, and Dunne et al. (19) has found that LFA-1 supports slow rolling of leukocytes and the efficient transition to cell arrest.
In addition, our study has implications for the use of small molecule
inhibitors of the active conformation of LFA-1. Recent data (14, 49,
50) suggest that statins as well as other I domain inhibitors act as
potent antagonists of adhesion function. Our results suggest that
suppression of recruitment at the site of inflammation will require
tight binding of such inhibitors and occupancy of greater than 90% of
active CD18. We conclude that the efficiency of neutrophil capture is
tightly regulated by the dose of G-protein-linked stimuli and its
effect on the site density and subsequent mobility of active LFA-1.
Clustering of LFA-1 to high avidity sites supports adhesion
strengthening leading to neutrophil arrest, but these adhesive bonds
exist for only minutes thereby facilitating cell motility and
transendothelial migration.
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ABSTRACT
INTRODUCTION
