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Blood, Vol. 92 No. 12 (December 15), 1998:
pp. 4819-4827
Adhesion-Dependent Release of Elastase From Human Neutrophils in a
Novel, Flow-Based Model: Specificity of Different Chemotactic Agents
By
G. Ed Rainger,
Andrew F. Rowley, and
Gerard B. Nash
From the Department of Physiology, The Medical School, The University
of Birmingham, Birmingham, UK; and the School of Biological Sciences,
University of Wales Swansea, Swansea, Wales.
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ABSTRACT |
Neutrophils must adhere to the vessel wall, migrate, and degranulate
in an ordered manner to perform their protective function. Disruption
of these processes may be pathogenic. Current knowledge of the
degranulation process is derived almost exclusively from studies on
neutrophils in suspension, in which priming with the nonphysiological
agent cytochalasin B is necessary to obtain elastase release in
response to activating agents. To avoid this, we have adopted a
different approach. Using a novel flow-based adhesion system, we have
been able to quantify the release of elastase from the primary granules
of activated neutrophils adherent to immobilized platelets or purified
receptors without priming. Comparing stimuli, formyl tripeptide (fMLP),
interleukin-8 (IL-8), activated complement fragment C5a, and
platelet-activating factor (PAF) all induced rapid conversion to
CD11b/CD18 (MAC-1) -mediated stationary adhesion when perfused over
neutrophils already rolling on platelet monolayers or purified
P-selectin. However, fMLP, C5a, and IL-8, but not PAF, induced release
of elastase from the adherent cells in minutes. Neutrophils stimulated
in suspension showed little degranulation. Treatment of neutrophils
with an inhibitor of 5-lipoxygenase-activating protein (MK886) and
thus synthesis of leukotrienes (LTs) or with an antagonist of the
LTB4 receptor (LY223982) blocked the release of elastase.
This indicated that endogenous synthesis of 5-lipoxygenase products
such as LTs and autocrine activation of neutrophils was required for
fMLP-driven elastase release. We hypothesize that the differential
ability of PAF and fMLP to induce elastase release from
surface-adherent neutrophils could arise from differential ability to
generate leukotrienes, such as LTB4, and would be an appropriate mechanism for the control of elastase release during inflammation in vivo, where it is important that cytotoxic agents are
not released until activated neutrophils have migrated into the
extravascular tissues.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
NEUTROPHILS MUST exit the circulation and
enter inflamed tissues to undertake phagocytosis. Invasive
micro-organisms are then destroyed through production of reactive
oxygen species (ROS) and release of degradative enzymes from preformed
granule stores. Granule enzymes, including elastase, are released not only intracellularly into phagosomes, but also into the extracellular environment, where they may have digestive roles.1,2
Ordinarily neutrophils (and their products) are localized to an
inflammatory locus by a series of adhesive and activating steps that
are closely regulated by endothelial cells lining the microvasculature
(for review, see Zimmerman et al,3 Imhof and
Dunon,4 and Springer5). Clearly, regulation
must extend to the control of neutrophil granule release during
recruitment from the blood. Indeed, disregulation of the recruitment
process can lead to inappropriate adhesion, ROS production, and
degranulation of neutrophils, which is implicated in the pathologies of
emphysema,6 venous disease,7 rheumatoid arthritis,8 organ failure in sepsis,9 and
reperfusion of ischemic tissue (for review, see Welbourne et
al10), including the heart, during myocardial infarction
(for review see Siminiak and Ozawa11). The mechanisms
controlling these responses are thus of great importance.
Products secreted by neutrophils are compartmentalized into at least
three distinct granule populations. Primary granules contain elastase,
myeloperoxidase, and lipases,1 which are generally
associated with tissue remodelling in inflammation and tissue damage in
pathology.1,2,6-11 Both secondary and tertiary granules
contain a number of metaloproteinases,1 which specifically degrade the proteins found in extracellular matrix (eg, collagenase) and may be required for efficient transit of neutrophils from vessel
lumen, through subendothelial matrix, and into tissues. Tertiary
granules also contain the preformed pool of the
 2-integrin heterodimer CD11b/CD18,2 which
must be rapidly mobilized upon neutrophil activation to allow prolonged
adhesion and migration.12-14 This raises the question of
how the undesirable release of primary granule contents is separated
from the secondary and tertiary granule mobilization required for the
early stages of attachment to the vessel wall and for migration.
In vitro studies have shown that agents such as bacterial peptide
analogues (formyl tripeptide [fMLP]), activated complement fragment
C5a, platelet-activating factor (PAF), the chemokine interleukin-8
(IL-8), and the eicosanoid, leukotriene B4
(LTB4), can promote rapid release of all neutrophil
granular stores in suspension when the cells have been pretreated with
the cytoskeleton-disrupting agent cytochalasin B.1,15,16
However, cytochalasin B may not be essential for the release of
secondary and tertiary granules17 and is not required for
de novo expression of CD11b/CD18. Thus, only primary granule release
may be mediated by a route requiring cytochalasin B in suspension.
Although such differential responses are suggestive regarding a
mechanism by which primary granule mobilization is separated from the
mobilization of other granule compartments, modelling the processes in
suspension is far removed from the physiological situation where
neutrophils are activated at the vessel wall. A previous study using
adherent neutrophils indicated that binding of CD11b/CD18 to albumin
was sufficient to allow the release of primary and secondary granules
in response to fMLP.18 Studies of flowing blood have also
demonstrated that the release of elastase from activated neutrophils in
the extracorporeal circulation during coronary bypass
surgery19 or during hemodialysis20 correlated
with the expression of 2-integrin or required adhesion via this receptor. However, detailed studies of the effect of adhesion
on neutrophil degranulation in circulatory models have not been
reported. Such studies might provide a better understanding of the
pathophysiology of degranulation and of the mechanisms that control its
localization.
We describe here a novel perfusion system that has allowed us to study
adhesion and primary granule release of neutrophils independent of
cytochalasin B priming. Flowing neutrophils formed rolling attachments
to P-selectin presented on a monolayer of activated platelets or
immobilized as a purified protein. Rolling cells were subsequently
superfused with activating agents so that they converted to stationary
adhesion via 2-integrin CD11b/CD18.14,21 When perfusate was collected and assayed for content of elastase, it
was evident that fMLP, C5a, and IL-8 caused adherent neutrophils to
release this enzyme, but that PAF did not. The flow system allowed
analysis of the kinetics of elastase release, which peaked within 2.5 minutes and then steadily decreased. We also found that endogenous
synthesis of LTB4 via the 5-lipoxygenase pathway was an
essential autocrine activation step for elastase release stimulated by
fMLP under these conditions. Thus, adhesion to substrate can remove the
requirement for cytochalasin B during induction of primary granule
release by chemotactic agents, and differential induction by activating
signals could be important in localizing the potentially pathological
aspects of neutrophil degranulation to the extravascular space.
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MATERIALS AND METHODS |
Monoclonal antibodies (MoAbs).
MoAbs against CD18 were R6.5E (kind gift of Martyn Robinson, Celltech,
Slough, UK), used at 24 µg/mL. Antibody against CD11b was KIM247
(gift of Martyn Robinson), used at 14 µg/mL. MoAb G1 against
P-selectin (kind gift of Rodger McEver, University of Oklahoma,
Oklahoma City, OK) was used at a concentration of 50 µg/mL. All antibodies were IgG1.
Isolation of neutrophils.
Blood was collected from healthy volunteers into citrate phosphate
dextrose adenine-1 (CPDA-1; Baxter Health Care Ltd, Thetford, UK) and
neutrophils were isolated using two-step density gradients of
Histopaque (Sigma Chemical, Poole, UK), as previously
described.14,22 Neutrophils were washed, counted using a
Coulter counter (Coulter Electronics Ltd, Luton, UK), and adjusted to
1 × 106/mL in phosphate-buffered saline
containing 1 mmol/L Ca2+ and 0.5 mmol/L Mg2+
(PBS; Sigma) and 0.15% bovine serum albumin (BSA fraction IV; Sigma)
(PBS/BSA).
Immobilization of platelet monolayers or P-selectin in microslides.
Microslides (CamLab, Cambridge, UK) are glass capillary tubes with a
rectangular cross-section of 0.3 × 3 mm, a length of 5 cm, and
good optical qualities. To provide a substrate that readily binds
platelets, microslides were coated with 3-aminopropyltriethoxysilane (APES; Sigma) as previously described.23 Blood was
collected into sodium heparin (CP Pharmaceuticals Ltd, Wrexham, UK),
and platelet-rich plasma (PRP) was removed after centrifuging the blood
at 400g for 5 minutes. Platelets were counted, adjusted to 2 × 108/mL with PBS, loaded into microslides, and
incubated at room temperature for 40 minutes to allow them to sediment,
adhere, and form a confluent monolayer.21,22 Unbound
platelets were removed by washing before assay. Experiments were always
conducted using autologous platelets and neutrophils. Platelet
monolayers prepared in this manner can capture and support the rolling
adhesion of neutrophils for up to 20 minutes without evidence of
platelet-mediated neutrophil activation.14
A 730 amino acid residue recombinant human P-selectin stop protein,
lacking the transmembrane and cytoplasmic domains (affinity-purified from stably transfected CHO cells; gift of Ian Collins, R&D Systems, Abingdon, UK; also commercially available) was dissolved in PBS at a
concentration of 5 µg/mL. The protein was immobilized by incubation
in an APES-treated microslide for 60 minutes at 37°C. Free
protein-binding sites were subsequently blocked with 1% BSA (fraction
IV) for 60 minutes at 37°C. BSA is a sufficient ligand to support
the 2-integrin-mediated adhesion and migration of neutrophils in our own experiments21 and in other
experimental systems,24-26 including a study of
adhesion-dependent degranulation in response to fMLP.18
Adhesion and elastase release by neutrophils under conditions of
flow.
The assay system was a modification of that recently
described14,27 and is shown schematically in
Fig 1. A microslide containing adhesive
substrate was attached to the common outlet of a four-port tap
(Hamilton miniature valve; V.A. Howe Ltd, London, UK). Plastic, 2.5-mL
perfusion syringes containing either chemotactic agent, PBS/BSA, or a
suspension of neutrophils were connected to one of the three remaining
tap inlets via flexible silicone tubing. These syringes were mounted
back-to-back with identical water-filled slave syringes on a specially
engineered rig (Fig 1) that held them immobile. Expulsion of the
perfusate was driven at a controlled rate by a 50-mL master syringe on
a Harvard syringe pump that pushed flow into the slave syringes.
Selection between perfusates was achieved using two electronic valves
(Lee Products Ltd, Gerards Cross, UK) that switched the route of the
output from the master syringe to a chosen slave syringe. A constant
wall shear stress of 0.05 Pa (or 0.5 dyn/cm2) was
maintained in the microslide by choice of the appropriate flow rate.
This is at the low end of the physiological range of wall shear stress
in post capillary venules28 and was chosen so that large
numbers of adherent neutrophils could be established and concentration
of released substances maximized.
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| Fig 1.
Diagrammatic representation of the flow-based adhesion
assay incorporating an hydraulic-driven perfusion system allowing the
collection of buffer perfused over adherent cells. The master syringe
(driven by a Harvard syringe pump) expels water into one of three slave
syringes. Switching between the slave syringes is via electronic
valves. Each slave syringe is mounted back to back with and drives a
perfusion syringe containing a different perfusate (wash buffer, cell
suspension, or chemotactic agent). Output from the perfusion syringes
is selected via a 4-way teflon tap, the common outlet of which is
attached to a microslide containing the adhesive substrate. The
microslide sits on the stage of a video-microscope. Perfused medium for
analysis is collected from the distal end of the microslide into
micro-centrifuge tubes.
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Microslides were mounted on the stage of a microscope fitted with a
video camera, monitor, and recorder. The whole perfusion system was
maintained at 37°C. Neutrophils were perfused across adhesive
substrates at a concentration of 106/mL for 5 minutes.
Perfused cells that were close to the wall of the microslide adhered
via P-selectin and formed rolling attachments. The majority of
neutrophils remained in the bulk flow of the perfusing buffer and were
nonadherent. Nonadherent neutrophils were removed from the microslide
with a 2-minute perfusion of wash buffer, leaving a large population
(~1,500/mm2) of continuously rolling, adherent
neutrophils. A video record was made in at least five fields of view of
known dimensions to allow the number of adherent cells to be counted.
Continuously rolling neutrophils were then activated by the perfusion
of PBS/BSA containing either fMLP (10 7 mol/L;
Sigma), PAF (107 mol/L; Sigma), IL-8 (1 µg/mL; R&D
Systems), or 1% zymosan (Sigma) -activated plasma (ZAP) as a source of
C5a. ZAP was made by incubating 8 mg zymosan/mL of plasma for 30 minutes at 37°C, followed by centrifugation at 10,000g for
5 minutes and filtration through a sterile 0.2-µm filter. Upon
receipt of a chemotactic stimulus, neutrophils rapidly stopped rolling
and became stationary (~1 to 2 seconds), changed shape and spread
(~0.5 to 1 minute), and began to migrate (1 minute
onwards).14,21 Chemotactic agents were perfused
continuously for 10 minutes and perfusate was collected from the outlet
in 2.5-minute fractions for assay of elastase content. As controls,
PBS/BSA alone was flowed over continuously rolling neutrophils and
perfusate was collected in the same manner. Aliquots were centrifuged
at 10,000g for 1 minute to remove detached cells and stored at
 30°C until assay.
Adhesive requirements for neutrophil elastase release.
To investigate the adhesive requirements for elastase release, we
blocked the neutrophil integrins CD11b and CD18 and the platelet
receptor P-selectin. Antibodies to integrins were incubated with neutrophils for 15 minutes at room temperature. Because activated neutrophils rapidly mobilize granule stores of both CD18 and CD11b, it
was also necessary to include MoAb at the same concentrations in the
buffer containing fMLP to block newly expressed integrin molecules.
Because pretreatment of platelet monolayers with antibody against
P-selectin abolished all adhesion,22 we used a different strategy. We perfused the anti-P-selectin MoAb with the fMLP over a
population of rolling neutrophils. This permitted neutrophil capture
from flow and rolling adhesion before the delivery of fMLP but blocked
P-selectin interactions after the initiation of
2-integrin-mediated adhesion. We have successfully used
this strategy in a previous study to block P-selectin-mediated signals that modify the migration rate of activated neutrophils.21
Perfusion of the antibody with fMLP does not affect the number of
neutrophils that become stationary adherent.21
Enzyme-linked immunosorbent assay (ELISA) for neutrophil elastase.
Elastase concentrations in perfusates were determined using a
commercial, colorimetric immunoassay (Merck Immunoassay PMN elastase;
Merck, Lutterworth, UK) according to the manufacturer's instructions.
The assay measures the concentration of neutrophil elastase complexed
with  1-antitrypsin. Because neither plasma nor serum was present in
our assay, samples were first incubated for 30 minutes at 37°C with
100 µg/mL purified 1-antitrypsin (Sigma), which ensured a large
molar excess over elastase. Reducing or increasing the 1-antitrypsin
concentration by a factor of 10 did not influence the sensitivity of
the assay (data not shown). The assay was calibrated against known
concentrations of purified elastase-1-antitrypsin complex provided
with the kit and elastase concentration was normalized for the number
of cells present in each microslide (nanograms per 106
neutrophils). For comparison, neutrophils in suspension at known concentration were stimulated with the same agents for 10 minutes, and
the supernatant was isolated after centrifugation at 10,000g for 1 minute. The supernatant was assayed in the same manner as the
perfusate. Total elastase content of neutrophils was also determined by
lysing known numbers with 0.5% sodium dodecyl sulphate (SDS; Sigma)
and isolation of the supernatant after centrifugation at
10,000g for 5 minutes.
Inhibition of 5-lipoxygenase and blockade of LTB4
receptor.
In some experiments, neutrophils were treated with MK886 (a kind gift
from Dr P. Vickers, MerckFrosst Centre for Therapeutic Research, Pointe
Dorval, Canada), an inhibitor of 5-lipoxygenase activating protein
(FLAP),29 at concentrations of 50 or 500 nmol/L for 10 minutes before experimentation. On other occasions, the
LTB4 receptor antagonist LY223982 (Lilly Research
Laboratories, Indianapolis, IN)30 was preincubated with
neutrophils at a concentration of 100 µmol/L for 10 minutes. The
agents were also included in all perfusates to which the neutrophils
were exposed. Both reagents were solubilized in dimethyl sulfoxide
(DMSO), and, after dilution, carrier concentrations were
0.1% or less. Control experiments using carrier alone showed no effect
on degranulation in response to fMLP (data not shown).
Enzyme immunoassay for LTB4.
Perfusate from neutrophils exposed to fMLP or PAF in the presence or
absence of MK886 were collected and stored at 70°C before analysis. LTB4 was quantified using a commercially
available enzyme immunoassay (Amersham Life Science, Little Chalfont,
UK) with a reported sensitivity of 6 pg/mL.
Statistical analysis.
Concurrent effects of time and different treatment group were tested
using analysis of covariance, followed by one-way analysis of variance
(ANOVA) for effect of time or dose on a single treatment where
appropriate. Comparison of individual treatments or conditions at
single time points was made using the paired t-test or unpaired (Student's) t-test as appropriate. All tests were made using
the computer program Minitab (Minitab Inc, State College, PA).
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RESULTS |
Elastase release from neutrophils adherent to platelet monolayers.
Neutrophils perfused over immobilized activated platelets at a wall
shear stress of 0.05 Pa were captured from flow by P-selectin. The
great majority of adherent cells were rolling (>85%) with a velocity
of 1.9 ± 0.2 µm/sec (mean ± SEM of 60 cells from 3 experiments). The remaining cells spontaneously activated on the platelet monolayer and were stationary adherent. After superfusion of
each chemotactic stimulus (fMLP, C5a, IL-8, or PAF), all neutrophils rapidly stopped rolling, spread, and migrated across the platelet monolayer. These responses have been described in detail
elsewhere.14,21
Neutrophils in suspension released little elastase when activated with
fMLP, PAF, C5a, or IL-8, although the latter two agents did induce
greater release than unstimulated control
(Fig 2). However, when these agents were
superfused over neutrophils that were rolling on platelets, there was a
marked release of elastase, except in the case of PAF, which did not
induce elastase release (Fig 2). We also observed a small but
significant increase in elastase release when untreated neutrophils
rolling on platelets were compared with those in suspension (Fig 2).
This increase may represent release from those neutrophils that were
spontaneously activated while in contact with the platelet monolayer.
As a marker for activation, less than 2% of neutrophils in the
original suspension showed pseudopod formation, but 13% ± 2% of
neutrophils adherent to platelets were distorted in shape (mean ± SEM from 7 experiments). Total elastase content of neutrophils was 1.4 ± 0.2 µg/106 cells (mean ± SEM of 3 experiments).
Elastase released by surface-adherent neutrophils activated with fMLP,
C5a, or IL-8 over 10 minutes (Fig 2) equated to 20% to 25% of total
neutrophil elastase.
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| Fig 2.
Elastase release from unstimulated neutrophils (Con) or
neutrophils treated with chemotactic agents (107 mol/L
PAF, 107 mol/L fMLP, 1% ZAP as a source of C5a, or 1 µg/mL IL-8) either in suspension ( ) or while adherent to platelet
monolayers ( ). Data are the mean ± SEM from three to eight
experiments. ANOVA showed significant effect of treatment and of
adhesion on release of elastase. Paired t-test showed a
significant increase (#P < .05) in elastase released from
neutrophils in suspension activated by C5a and IL-8 compared with
control; a significant increase (**P < .01) in elastase
released from surface-adherent neutrophils activated by fMLP, C5a, or
IL-8 compared with control; a significant increase (+P < .05) in elastase released from rolling unstimulated control neutrophils
compared with unstimulated control neutrophils in suspension.
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We followed the kinetics of elastase release by assaying perfusate
collected at 2.5-minute intervals. Figure 3
demonstrates that release was maximal within the first 2.5 minutes for
fMLP, C5a, or IL-8. Elastase release then decreased slowly over the next 7.5 minutes, but was still greater than levels from unstimulated adherent cells that gave a steady low level of release over the 10 minutes (Fig 3).
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| Fig 3.
Time course of elastase release from neutrophils adherent
to platelets either (A) unstimulated and continuously rolling () or
stimulated with 107 mol/L fMLP (); (B) stimulated
with 1% ZAP as a source of C5a; or (C) stimulated with 1 ng/mL IL-8.
Data are the mean ± SEM of least four experiments. Analysis
of covariance showed that there was a significant effect of time and
treatment on elastase release (P < .01) and that treatments
with chemotactic agents were significantly different from control
(P < .01), but not different from each other. One-way ANOVA
showed that elastase release from control cells did not vary with time
but that there was a significant effect of time on release for
individual treatments with chemotactic agents (P < .05 in
each case).
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Elastase release from neutrophils adherent to purified
P-selectin and albumin.
In a separate series of experiments, release of elastase from
neutrophils adherent to platelets or adherent to purified P-selectin coimmobilized with albumin was compared. Superfusion of PAF did not
induce an increase in release of elastase for either type of surface
(Fig 4). However, fMLP induced release of
elastase with equal efficiency on platelets or immobilized adhesion
receptors (Fig 4). Adhesion per se was therefore required for
neutrophil elastase release, but platelets were not essential.
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| Fig 4.
Elastase release from neutrophils that were unstimulated
and continuously rolling (), stimulated with 107
mol/L PAF ( ), or stimulated with 107 mol/L fMLP ()
while adherent to platelet monolayers (Platelets) or 5 µg/mL purified
recombinant P-selectin coimmobilized with 1% BSA (P-sel/Alb). Data are
the mean ± SEM of three experiments. ANOVA showed that there was a
significant effect of treatment on release of elastase but that
adherent substrate did not affect release. Paired t-test showed
that elastase release from fMLP-but not PAF-treated cells was
significantly increased (**P < .01) compared with rolling
unstimulated control cells on either adhesive substrate.
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Adhesive requirements for neutrophil elastase release.
Using adhesion blocking MoAbs, we investigated the role of the
neutrophil integrin CD11b/CD18 and the platelet rolling receptor P-selectin in elastase release from neutrophils activated with fMLP on
platelet monolayers. In the presence of antibodies against CD11b or
CD18 neutrophil, elastase release was significantly reduced by between
50% and 60%. However, blockade of P-selectin at the time of delivery
of fMLP had no effect on elastase release
(Fig 5).
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| Fig 5.
Elastase release from neutrophils adherent to platelet
monolayers in the presence of 107 mol/L fMLP (Control)
and an MoAb against either P-selectin (Anti-P-sel), CD18 (Anti-CD18),
or CD11b (AntiCD11b). Data are the mean ± SEM from three experiments.
Paired t-test showed that elastase release from cells in the
presence of CD18 or CD11b antibodies was significantly lower
(*P < .05) than in untreated control cells.
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Role of leukotrienes in elastase release.
MK886, a FLAP inhibitor and hence an inhibitor of leukotriene
generation, and the LTB4 receptor antagonist LY223982 were
tested for their effects on neutrophil elastase release. MK886 at 50 nmol/L inhibited approximately 40% of elastase release and at 500 nmol/L elastase release was reduced by about 80%
(Fig 6). LY223982 used at 100 µmol/L also
essentially abolished the release of elastase from neutrophils in
response to fMLP (Fig 6).
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| Fig 6.
Elastase release from neutrophils adherent to platelet
monolayers in the presence of 107 mol/L PAF or
107 mol/L fMLP and either 50 or 500 nmol/L of the FLAP
inhibitor MK886 or 100 nmol/L of the LTB4 receptor
antagonist LY223982 (LTB4 ra). Data are expressed relative to values
obtained from paired samples stimulated with fMLP alone (Con). Data are
the mean ± SEM of six experiments using PAF or LY223982 and of three
experiments for each intervention with MK886. Paired t-test
showed that elastase release was significantly lower (**P < .01) than fMLP control for cells treated with PAF or cells treated with
fMLP and LY223982 or 500 nmol/L MK886.
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We also analyzed neutrophil production of LTB4 by enzyme
immunoassay (EIA). Neutrophils in suspension released no detectable LTB4 with or without the addition of PAF (3 experiments;
signal below detection limit of EIA), but, when stimulated by fMLP
production of LTB4, was detected, but at low concentrations
(23.5 ± 3.1 pg/106 cells; mean value ± SEM, n = 5).
When surface adherent neutrophils were activated with fMLP,
LTB4 was detectable in perfusate (41.7 ± 6.8 pg/106 cells; mean value ± SEM, n = 5). When PAF was
used to activate surface adherent neutrophils in three paired
experiments, LTB4 was undetectable in the perfusate in two
of the three experiments. Pretreatment of surface adherent neutrophils
with the FLAP inhibitor, MK886, followed by challenge with fMLP
resulted in a reduction in LTB4 generation to levels less
than the detection limit of the assay. Finally, we established that
LTB4 could induce elastase release. In two experiments,
neutrophils adherent to platelet monolayers and stimulated with
107 mol/L LTB4 generated 168 ± 14 ng
of elastase/106 cells, whereas unstimulated control
neutrophils released only 63 ± 2 ng of elastase/106
cells.
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DISCUSSION |
For the first time we have investigated the control of elastase release
from the primary granules of neutrophils in a flow-based model of the
circulation. When neutrophils were perfused with fMLP, C5a, or IL-8
while rolling on P-selectin presented by a platelet monolayer, they
became immobilized and showed marked release of elastase.
Interestingly, although PAF also induced activation and immobilization
of rolling neutrophils, it was an insufficient stimulus to promote
elastase release. When compared with freely suspended cells, elastase
release was amplified greatly by adhesion via CD11b/CD18 to a substrate
of platelets or purified albumin. Release was comparable on platelet-
or protein-coated surfaces, so that agents derived from platelets, such
as eicosanoids or platelet activating factor,31 were not
essential requirements. Elastase release was maximal within 2.5 minutes
after chemotactic stimulation, but significant release was maintained
for at least 10 minutes and totalled approximately 25% of elastase
stores. Neutrophils activated with fMLP released low levels of
LTB4. An inhibitor of 5-lipoxygenase-activating protein
(MK 886) and an LTB4 receptor antagonist (LY223982) each
reduced the release of elastase markedly. Thus, eicosanoids and,
specifically, LTB4 generated via the 5-lipoxygenase pathway
in neutrophils acted in an autocrine manner to promote elastase
release.
These studies were made possible by the development of a novel
perfusion system. Hydraulically driven syringes allowed sequential perfusion of neutrophils, wash buffer, and chemotactic agents over an
adhesive substrate and timed collection of perfusate for analysis of
released compounds. This approach has several advantages over assays of
neutrophil degranulation using suspensions. It more closely models the
in vivo situation, in which degranulation occurs mostly in adherent
cells, and obviates the need to artificially prime the neutrophils with
cytochalasin B, an essential step for the release of primary granules
in suspension.1,15,16 We used an adhesive surface of
activated platelets (that might resemble the situation in thrombotic
vessels) or purified P-selectin and albumin, but other substrates might
be chosen to allow receptor-specific modulation of degranulation to be
studied. Flow-based adhesion assays are well adapted to follow rapid
changes in adhesive behavior of activated neutrophils14,21
and here allowed the secretory and/or synthetic responses of
neutrophils to be monitored continually. Currently, perfusate was
collected at intervals of 2.5 minutes (perfused volume, ~0.5 mL)
because of initial uncertainty regarding the concentration of released
substances. In fact, collected samples required dilution 10-fold for
elastase assay and were sufficient for repeated assays of this or other
compounds. Thus, for a plentifully released compound such as elastase,
greater resolution of the kinetics of release could have been achieved
by collecting smaller samples of the perfusuate more frequently.
Elastase release occurred with equal efficiency on immobilized
activated platelets or on purified recombinant P-selectin coimmobilized with albumin. This implied that the adhesion-dependent signal acted
through a neutrophil P-selectin ligand and/or the neutrophil 2-integrin CD11b/CD18 that supports activation-dependent
adhesion to platelets14,21,32 or immobilized
albumin.21 When we used MoAbs to block neutrophil adhesion
to platelets, we found that anti-CD18 or anti-CD11b but not
anti-P-selectin blocked elastase release. This agrees closely with
data from a static adhesion system in which primary granule release
from fMLP-stimulated neutrophils adherent to albumin was blocked by
MoAb against CD18 or CD11b.18 Interestingly, increased
expression of CD11b/CD18 on neutrophils, which is indicative of
neutrophil activation, correlated with elastase release during
extracorporeal circulation of blood,19 whereas
antibody-blockade of CD18 inhibited neutrophil elastase release induced
by exposure of blood to hemodialysis membranes.20 CD11b/CD18 binding to albumin was also required for the degranulation of another member of the granulocyte lineage, the eosinophil, in
response to granulocyte-macrophage colony-stimulating factor or
PAF.33 Thus, there is now compelling evidence to suggest that the release of neutrophil elastase from primary granules is an
activation-driven process that requires adhesion via the glycoprotein
CD11/CD18.
Activation-dependent adhesion via 2-integrins was
essential for the efficient release of elastase from neutrophils.
Release of elastase from rolling adherent unactivated neutrophils or
from neutrophils activated in suspension was minimal. However, whereas activation-dependent adhesion was essential for primary granule release, it was not necessarily sufficient, because PAF was unable to
induce release of elastase. The inability of PAF to induce neutrophil
elastase release was not due to the modulatory effects of agents
generated by activated platelets. Although activation of flowing
neutrophils by platelet-derived stimuli such as LTB4 and
PAF has been previously reported,31 we could demonstrate no
qualitative or quantitative differences in neutrophil responses to
stimulation with fMLP or PAF when experiments were conducted on
platelets or on a substrate of purified adhesion receptors. Thus, any
platelet-derived agents did not modify the response to fMLP or PAF.
However, the low levels of stationary adhesion and elastase release
observed here on platelet monolayers without exogenous stimulation
suggest the presence of platelet-derived stimuli, although presumably
PAF did not cause release of elastase.
The route by which adhesive and chemotactic signals are integrated to
control degranulation remains uncertain. It is possible that elastase
release is influenced by changes in the cytoskeleton of the cell
dependent on adhesion and not linked to signaling from an integrin
receptor. Activation-dependent adhesion is generally associated with
dynamic rearrangement of the actin cytoskeleton.13 Moreover, cytoskeletal disruption by cytochalasin B allows neutrophil primary granule release in suspension,1,15,16 although the release of other granule compartments may occur via a
cytochalasin-insensitive route.17 Cytochalasin B has been
suggested to remove a mechanical barrier to granule translocation to
the outer membrane by the degradation of F-actin (for review, see
Smolen1). Thus, cytoskeletal remodelling may provide a
common mechanism allowing primary granule mobilization in both
surface-adherent and cytochalasin-primed neutrophils. An alternative
route by which cytochalasin B could enhance degranulation is via
increased production of diacylglycerol leading to protein kinase
C-dependent neutrophil priming.34,35 Increased mobilization
of this second messenger in cytochalsin B-treated and surface-adherent
neutrophils could also provide a common route to primary granule
release.
Studies with the FLAP-inhibitor, MK 886, and LTB4 receptor
antagonist LY223982 and measurements of released LTB4
strongly indicated that LT synthesis was required for elastase release. The levels of LTB4 in perfusate were near the limit of
detection by EIA and were equivalent to about 5 to 10 pg/mL. Previous
studies have shown that, in neutrophils stimulated with C5a,
LTB4 can be retained within the cell and operate as an
intracellular second messenger rather than a secreted chemotactic
agent.36 This may account for the evident response at the
low levels of this eicosanoid found in perfusates. Moreover, autocrine
stimulation of degranulation by newly synthesized LTB4 has
previously been demonstrated in human neutrophils activated by the
Ca2+ ionophore A23187.37 There are several
other reports that LTB4 can cause degranulation in
mammalian neutrophils,38,39 and this response is augmented
in the presence of other lipid mediators such as PAF and
5(S)-hydroxyeicosatetraenoic acid.39 We further established that the addition of 107 mol/L
LTB4 induced elastase release in the current model.
Interestingly, neutrophils from patients with rheumatoid arthritis
treated with Tenidap sodium (a 5-lipoxygenase inhibitor) demonstrated a
reduction in ability both to synthesize LTB4 and to release
neutrophil elastase.40 The above-mentioned results indicate
that the inability of PAF to promote degranulation and to induce the
synthesis of LTB4 may be linked. The differential ability
of PAF and fMLP to promote LTB4 synthesis may arise because
the receptors for these agents can be coupled to different G proteins
and thus may signal via separate second messenger
cascades.41-44 Nevertheless, PAF has been shown to cause
elastase release from neutrophils in the presence of cytochalasin
B39,45 and has been shown to prime neutrophils adherent to
endothelium for enhanced elastase release in response to a secondary
challenge with fMLP.46
Based on the above-noted considerations and previous studies of
intracellular signaling pathways, we propose a model that explains
common adhesive, but divergent degranulation responses induced by PAF
and fMLP (Fig 7). On binding of PAF or
fMLP, specific G-protein-linked receptors activate both phospholipase
A2 (PLA2) and phospholipase C
(PLC).41,47,48 Activation of PLC then upregulates
2-integrin function,49-51 possibly by
controlling local Ca2+ concentration.52
PLA2 causes release of arachidonic acid (AA) that is
essential for de novo expression and activation (cycling) of
2-integrin CD11b/CD18 from the tertiary granule
stores48,53 and thus is important in prolonged neutrophil
adhesion.14,53 Continual binding of CD11b/CD18 acts as a
signal which promotes primary granule release but is itself
insufficient. An additional pathway arises through activation of
5-lipoxygenase and, thus, LTB4 synthesis induced by fMLP
but not PAF. LTB4 generated intracellularly or possibly
also via a transcellular route (pathway not shown; for review, see
Serhan54) binds in turn to a specific G-protein-linked receptor and promotes the production of phosphatidic acid (PA) by the
action of phospholipase D (PLD) on membrane
phospholipids.55 PA has been reported to be essential for
neutrophil primary granule release55 and acts as the second
signal integrated with that from integrin-binding to induce elastase
secretion. We have represented the uncertainty surrounding integration
of these two signals as a black box. Ultimately granules containing
elastase are translocated to and fuse with the external lipid membrane,
liberating their contents into the extracellular environment.
View larger version (25K):
[in this window]
[in a new window]
| Fig 7.
A model of the differential control of neutrophil
degranulation by the chemotactic agents fMLP and PAF. Both fMLP and PAF
induce PLA2 and PLC activation via G-protein-linked
serpentine receptors. PLC may regulate
2-integrin-mediated adhesion by modulating local
calcium fluxes. AA may be required for CD11b/CD18 cycling (de novo
expression and activation), a process necessary for prolonged
neutrophil adhesion and migration. Signaling via the fMLP receptor but
not the PAF receptor also mobilizes FLAP, which in turn activates five
lipoxygenase (5LO). LTB4 is synthesized by the action of
5LO on arachidonic acid. Details are not shown, but the intermediate
metabolite LTA4 may also be transformed by intercellular
pathways. The binding of LTB4 to a specific
G-protein-linked serpentine receptor activates PLD, the action of
which generates PA from membrane phospholipids. Signals from PA and
ligand-bound CD11b/CD18 are integrated in an unknown manner () that
results in granule mobilization, fusion with the outer cell membrane,
and release of contents into the extracellular environment (see text
for abbreviations).
|
|
Differential, adhesion-dependent response of neutrophils to chemotactic
agents may be physiologically important. The release of primary granule
contents is not appropriate for neutrophils suspended in blood or for
adherent cells in the process of migrating from the vessel lumen. PAF
is a neutrophil-activating agent ordinarily found on
cytokine-stimulated endothelium, which promotes immobilization of
neutrophils and their transendothelial migration.3,56 This processes requires the rapid mobilization of
CD11b/CD181,12,13 from tertiary granules,1 but
does not require potentially harmful primary granule release.
Therefore, PAF may promote neutrophil adhesion and migration across the
endothelial barrier of blood vessels without inducing primary granule
release. In contrast, activating agents ordinarily restricted to the
extravascular tissues during inflammation, for instance, bacterial
peptides and activated complement fragments, can induce primary granule
release as well as tertiary granule mobilization. Such responses would
be most suited to phagocytosis and destruction of invasive
micro-organisms in the extravascular tissues during inflammation. Thus,
we advance a paradigm in which transendothelial migration of
neutrophils occurs in response to agents such as PAF, while ensuring
that defense mechanisms such as the mobilization of primary granules and the release of their contents is only triggered in the tissues by
chemotactic agents concentrated there. Disruption of this model, for
instance, by delivery of inappropriate activating agents to the
vascular compartment, could be the basis of pathological degranulation of adherent neutrophils, whereas interventions directed against the
pathway acting through 5-lipoxygenase might be protective against
tissue damage.
| |
ACKNOWLEDGMENT |
The authors are grateful to Prof Charles Serhan for helpful comments
regarding the manuscript.
| |
FOOTNOTES |
Submitted December 3, 1997;
accepted August 5, 1998.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to G. Ed Rainger, PhD, Department
of Physiology, The Medical School, The University of Birmingham,
Birmingham B15 2TT, UK; e-mail: raingege{at}bham.ac.uk.
| |
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Abstract
Introduction