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Department of Medicine, New York University School of Medicine, New York, NY 10016
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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11 nM and 13
µM, respectively) but not PMA. Wortmannin also inhibited
FMLP-stimulated adhesion of neutrophils to human endothelial cell
monolayers, suggesting a common signaling pathway for homotypic and
heterotypic adhesion. Neither CD11b/CD18 expression nor expression of
an activation-specific epitope of CD11b/CD18 was affected by wortmannin
in FMLP-stimulated cells. Moreover, wortmannin also inhibited the
aggregation of egranulate neutrophil cytoplasts that lack the capacity
for CD11b/CD18 up-regulation. Although wortmannin inhibited neutrophil
lysosomal enzyme release, it had no effect on FMLP-stimulated
up-regulation of CD35 in intact neutrophils, suggesting discrepant
signaling pathways for specific granule degranulation and secretory
vesicle release. Aggregation of human neutrophils is associated with
activation of the mitogen-activated protein kinases Erk1 and -2, and
Erk is activated in response to PI 3-K in some cell types. However,
wortmannin inhibited FMLP stimulation of neutrophil Erk only at
concentrations (IC50 
1 µM) inconsistent with an effect
on PI 3-K. Our data indicate that PI 3-K mediates neutrophil adhesion
by a mechanism independent of CD11b/CD18 up-regulation, suggesting that
PI 3-K acts either parallel to, or downstream of, Erk. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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FMLP and other chemoattractants engage G protein-linked, seven-transmembrane-domain receptors, leading to the activation of phospholipases C and D, generation of inositol trisphosphate and diacylglycerol, activation of protein kinase C, and calcium influx (10). However, the role(s) of these signals in adhesion is neither well established nor likely to constitute the entire pathway for CD11b/CD18 activation. For instance, we have recently observed that FMLP and other chemoattractants stimulate mitogen-activated protein kinases Erk1 and Erk2 in human neutrophils in a manner consistent with a role for Erk in homotypic aggregation (11). It is likely that other molecules in neutrophils will also be found to participate in adhesion signaling.
Phosphatidylinositol 3-kinase (PI 3-K)3 is a heterodimeric enzyme (consisting of an 85-kDa regulatory and a 110-kDa catalytic subunit) that phosphorylates the D-3 position of phosphoinositide and phosphoinositide phosphates (12). Although little is known about the downstream effectors of 3-phosphorylated phosphoinositides, PI 3-K has been implicated in a variety of responses in noninflammatory cell types, including membrane ruffling (13), DNA synthesis (14), and the sorting and transport of lysosomal proteins (15, 16). PI 3-K activation has been best studied in systems in which activated, autophosphorylated protein tyrosine kinase receptors (PTKR) interact with SH2 domains on the p85 regulatory subunit of PI 3-K, leading to phosphorylation of p85 and enzymatic activity of the p110 subunit (17, 18). PI 3-K may also be activated by other receptor systems, including G protein-linked receptors. In at least some systems, G proteins may interact directly with, and activate, an alternative form of PI 3-K that lacks a regulatory subunit (19). PI 3-K has been identified in neutrophils, and its activity has been shown to be stimulated by chemoattractants in a manner independent of tyrosine phosphorylation events (18, 20, 21, 22).
Wortmannin is a fungal metabolite and a specific inhibitor of PI 3-K
activity (23, 24). In neutrophils, wortmannin partially inhibits PI 3-K
at very low concentrations (IC50 5 nM) (20, 25, 26). At
concentrations in the range of 5 nM, wortmannin binds specifically and
irreversibly to the p110 catalytic subunit of human neutrophil PI 3-K
(27). At slightly higher concentrations (
100 nM), wortmannin also
binds to two apparently related p110 isoforms with a molecular mass of
108 and 112 kDa (27) and completely inhibits stimulated PI 3-K activity
in neutrophils exposed to FMLP or other agonists (20, 25, 26, 28, 29).
At higher concentrations (100 nM), wortmannin may inhibit non-PI 3-K
kinases such as myosin light chain kinase and PI 4-kinase (25, 30, 31).
Wortmannin, along with LY294002, a chromone derivative that
specifically inhibits PI 3-K through a different mechanism of action
(32), has been used to study the role of PI 3-K in neutrophil
responses. At concentrations consistent with PI 3-K inhibition,
wortmannin and/or LY294002 inhibit specific and azurophilic granule
degranulation (33, 34), filamentous actin formation (20) and
O2
generation (20, 25, 27, 28, 29, 35, 36) but not chemotaxis (37) in
FMLP-stimulated human neutrophils. In platelets and other cell types,
wortmannin and/or LY294002 have been shown to inhibit stimulated
adhesion (38, 39). In contrast, the role of PI 3-K in neutrophil
cell-cell adhesion has not been systematically examined. Although
several studies have addressed the role of PI 3-K in neutrophil
adhesion to noncellular substrates, they obtained conflicting results.
Whereas Knall et al. observed inhibition of IL 8-stimulated neutrophil
adherence to plastic only at relatively high concentrations of
wortmannin (IC50 50 nM) (34), Vlahos et al. found no
inhibition by LY294002 of FMLP-stimulated neutrophil adherence to
plastic coated with keyhole limpet hemocyanin (36). In contrast,
Metzner et al. observed wortmannin and LY294002 inhibition of
neutrophil adhesion to fibrinogen-coated plastic in response to
FMLP (40).
We employed wortmannin and LY294002 to study the role of PI 3-kinase in neutrophil cell-cell adhesion. Our data indicate that wortmannin and LY294002 inhibit homotypic aggregation of neutrophils in response to FMLP and other chemoattractants at concentrations consistent with PI 3-kinase inhibition. Wortmannin also inhibited the heterotypic adhesion of FMLP-stimulated neutrophils to HUVEC monolayers. In contrast, wortmannin had no effect on FMLP-stimulated up-regulation of either the integrin CD11b/CD18 or CD35, a marker for the mobilization of secretory vesicles from the cytosolic compartment to the neutrophil plasma membrane (41). Finally, although PI 3-kinase has been shown to mediate the G protein-dependent activation of the mitogen-activated protein kinase Erk in some systems, wortmannin and LY294002 had no effect on FMLP-stimulated Erk activity. Our data demonstrate a specific and critical role for PI 3-K activity in neutrophil signaling for adhesion, suggesting that PI 3-K and Erk regulate adhesion by separate and parallel pathways.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Except as stated, all materials were purchased from Sigma
Chemical Co. (St. Louis, MO). Accuprep was from Accurate Scientific
(Westbury, NY). Dextran T500 was from Pharmacia LKB Biotechnology
(Piscataway, NJ). Myelin basic protein peptide (MBPp; APRTPGGRR)
and Ab to the p85 subunit of PI 3-K were from Upstate Biotechnology
(Lake Placid, NY). ATP and anti-CD11b mAb (anti-macrophage
(clone 44)) were from Boehringer Mannheim (Indianapolis, IN). FACS
lysing solution was from Becton Dickinson (San Jose, CA).
[
-32P]ATP was from Amersham (Arlington Heights, IL).
Phosphocellulose circles were from Fisher (Pittsburgh, PA). LY294002
was from Calbiochem (La Jolla, CA). Anti-CD35 mAb was from Serotec
(Washington, DC). Silica gel plates (with 1% potassium oxalate) were
from Analtech (Newark, DE).
Neutrophil isolation and cytoplast preparation
Neutrophils were isolated from heparinized venous blood of normal volunteers by Ficoll/Hypaque (Accuprep) centrifugation, dextran sedimentation, and hypotonic lysis of residual erythrocytes as described previously (42) and maintained in cell buffer (150 mM NaCl, 5 mM KOH, 1.3 mM CaCl2, 1.2 mM MgCl2, 10 mM HEPES, pH 7.4). This preparation yielded >98% neutrophils. Cytoplasts were prepared according to the method of Roos et al. (43). Neutrophil viability was determined by the release of lactate dehydrogenase, as previously described (44).
Neutrophil and cytoplast homotypic aggregation
Neutrophils (1.25 x 107/ml) or cytoplasts (4 x 108/ml) were preincubated in cell buffer for 5 or 10 min at 37°C in the absence or presence of wortmannin, LY294002, or Ab directed against CD11b, as indicated in Results. Aggregation was observed as the change in transmission of light through stirred suspensions of neutrophils stimulated with FMLP or other agonists in a platelet aggregometer (Payton Scientific, Inc., Buffalo, NY) and quantitated as the areas under the aggregation curves in the first 2 min following stimulation (5 min in the case of stimulation by PMA).
Neutrophil adhesion to ECV304 cell monolayers
2 x 107 neutrophils/condition were incubated for 10 min at 37°C in the absence or presence of wortmannin, washed x 3 (1000 rpm x 5 min) in cell buffer and resuspended in RPMI, followed by incubation over monolayers of ECV304, a spontaneously transformed cell line derived from HUVEC (grown to confluence in RPMI medium on 15-mm coverslips placed in the wells of 24-well plates) for 20 min at 37°C. Nonadherent neutrophils were removed by washing and aspirating gently three times. Adherent neutrophils and ECV304 monolayers were fixed with 2% paraformaldehyde and stained (Diff-Quick, Baxter Diagnostics McGaw Park, IL), and neutrophil adherence was determined by light microscopy as the number of neutrophils adherent per 40x field (mean of 5 fields counted, all experiments done in duplicate).
FMLP-stimulated lysozyme release and CD11b/CD18 surface expression
Neutrophil lysosomal enzyme release was measured as lysozyme release into the extracellular fluid (7). 10 x 106 neutrophils/condition (4 x 106/ml) were incubated in cell buffer containing cytochalasin B (5 µg/ml) in the absence or presence of wortmannin for 10 min at 37°C, followed by incubation in the absence or presence of 10-7 M FMLP for 10 min. Cells were transferred to ice for 5 min, then pelleted by centrifugation (4°C for 5 min at 1500 rpm). Supernatants were collected and lysozyme activity determined spectrophotometrically as the ability of supernatants to lyse the cell walls of Micrococcus lysodeikticus as previously described (44).
Measurements of CD11b/CD18 and CD35 surface expression on neutrophils in whole blood were performed according to a modification of the method of Cronstein et al. (45). Heparinized blood from normal volunteers was centrifuged (all centrifugations at 1000 rpm x 5 min), and the supernatant, containing plasma and platelets, was removed. Pellets were incubated for 10 min at 37°C in the absence or presence of 1 µM wortmannin, followed by stimulation for 1 or 10 min with 10-7 M FMLP. Reactions were stopped by the addition of excess ice-cold cell buffer. After incubation for 5 min on ice, the pellets were divided into equal fractions, which were reconcentrated and incubated for 15 min at room temperature in the presence of anti-CD11b, anti-CD35 mAb, or MOPC (nonspecific control mAb), washed once in cell buffer, and incubated for 15 min with FITC-conjugated goat anti-mouse antiserum. Pellets were washed once with cell buffer, and the remaining RBCs were lysed with FACS lysing solution for 10 to 15 min at room temperature in the dark. The pellets were washed three times with cell buffer and fixed with 2% paraformaldehyde, followed by flow cytometric analysis gated for granulocytes (New York University/Skirball Institute of Biomolecular Medicine flow cytometry facility). Fluorescence values with control mAb were subtracted from values obtained with specific mAbs to determine actual fluorescence. For assays of neutrophil surface expression performed on the buffy coat, heparinized blood was centrifuged, and the pellet/supernatant interface harvested and washed three times with cell buffer. Preparation of the buffy coat resulted in a near total decline of plasma protein concentration and a reduction in RBC concentration of approximately 90%. After washing, the buffy coat volume was adjusted with cell buffer, incubated in the absence or presence of wortmannin or sodium salicylate (NaS), and stimulated with FMLP for 10 min at 37°C. CD11b surface expression, or expression of an activation-specific CD11b/CD18 neoepitope defined by binding of mAb CBRM1/5, was determined as described for whole blood assays.
Kinase activity assays
PI 3-K activity was assessed in p85 immunoprecipitates from lysates of neutrophils incubated in the absence or presence of wortmannin (10 nM) or LY294002 (10 µM) according to the method of Ding et al. (29), except that in some experiments a noncommercial anti-p85 Ab was employed. Neutrophil Erk activity was assayed as previously described (11). Briefly, neutrophils (2 x 108/ml) were incubated in the absence or presence of wortmannin or LY294002 for 5 min at 37°C, followed by incubation in the absence or presence of 100 nM FMLP or C5a for 1 min. Reactions were stopped by the addition of lysis buffer (20 mM Tris, pH 7.4, 1 mM NaEGTA, 2 mM sodium vanadate, 25 mM sodium fluoride, 0.5% Triton X, 2 mM PMSF, 10 trypsin inhibitor units/ml of aprotinin, and 10 µg/ml each of chymostatin, antipain, and pepstatin). After a 15-min incubation on ice, lysates were centrifuged (14,000 x g for 10 min at 4°C). The supernatants were recovered and incubated for 15 min at 37°C in a buffer (25 mM Tris, pH 7.4, 12.5 mM MgCl2, 125 mM NaEGTA, 1.25 mM sodium fluoride, 2 mM DTT 220 µM ATP, and 25 µCi/ml [32P]ATP) containing 500 µM MBPp, a peptide containing the specific amino acid sequence (PRTP) phosphorylated on myelin basic protein (MBP) by Erk. Reactions were stopped by addition of formic acid (6% v/v final concentration). The lysates were spotted onto phosphocellulose papers that were then washed thoroughly with distilled water and quantitated by scintillation counting. Duplicate assays in the absence of MBPp were performed to determine non-Erk background kinase activities. The specificity of this assay was confirmed by the ability of anti-Erk1/Erk2 antisera to immunodeplete >95% of FMLP-stimulated Erk activity.4
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Preincubation of neutrophils with a mAb directed at CD11b reduced
neutrophil aggregation in response to FMLP and other chemoattractants,
confirming previous reports that neutrophil aggregation is at least
partly CD11b/CD18 dependent (Table I
).
Wortmannin alone (0.1–1000 nM) had no effect on neutrophil
aggregation. Preincubation with wortmannin inhibited FMLP-stimulated
homotypic aggregation in a dose-dependent manner (Fig. 1A; maximum inhibition 50
± 6%). The IC50 (10 nM) was similar to that previously
reported for wortmannin inhibition of PI 3-K in neutrophils (20, 25, 26). Moreover, the IC50 was consistent with values
previously reported for wortmannin inhibition of other neutrophil
functions including
O2
generation and lysosomal enzyme release (20, 27). LY294002 also reduced
FMLP-stimulated homotypic aggregation at concentrations
(IC50 10 µM) consistent with previously reported
values for PI 3-K inhibition in neutrophils (26) and in PI 3-K purified
from bovine brain (36) (Fig. 1B). Moreover,
preincubation of neutrophils with concentrations of wortmannin (10 nM)
or LY294002 (10 µM) approximating their observed IC50
values for inhibition of aggregation, as well as their reported
IC50 for inhibition of PI 3-K activity in neutrophils and
other cells (20, 25, 26, 28), resulted in 57 ± 17% and 66
± 8% inhibition of FMLP-stimulated PI 3-K activity, respectively.
LY303511, an analogue of LY294002 that does not inhibit PI 3-K (32),
failed to inhibit FMLP-stimulated aggregation at concentrations similar
to those of LY294002 (Fig. 1B). Wortmannin and
LY294002 were solubilized in DMSO; however, DMSO at concentrations
equal to those in wortmannin- and LY294002-treated samples had no
effect on FMLP-stimulated homotypic aggregation (not shown).
Concentrations of wortmannin or LY294002 that were required to inhibit
homotypic aggregation had little or no effect on neutrophil viability
as measured by LDH release in the absence or presence of FMLP
stimulation, confirming that inhibition of aggregation by these agents
is selective and not due to a general toxic effect.
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). Under these
conditions, wortmannin inhibited FMLP-stimulated aggregation to degrees
and at concentrations similar to those observed in unwashed cells
(IC50 30 nM). In contrast, cells treated with LY294002
before washing showed no inhibition of FMLP-stimulated aggregation.
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). As in intact
neutrophils, wortmannin inhibited FMLP-stimulated cytoplast aggregation
at concentrations consistent with PI 3-K inhibition (IC50
10 nM), and the degree of this effect resembled that observed in
intact neutrophils.
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To determine whether the effect of wortmannin on homotypic
aggregation was unique to FMLP we tested the ability of wortmannin to
inhibit neutrophil aggregation in response to other stimuli (Table I
).
Aggregation stimulated by the G protein-linked receptor agonists C5a
and leukotriene B4 (LTB4) was sensitive to
wortmannin. Moreover, the IC50 values for wortmannin on C5a
and LTB4 were 22 and 21 nM, respectively, similar to the
IC50 of wortmannin for FMLP-stimulated aggregation and
consistent with a role for PI 3-kinase inhibition. In contrast,
wortmannin and LY294002 have been reported to have no effect on
PMA-stimulated
O2
generation and degranulation in human neutrophils (20, 29, 36).
Consistent with these observations, wortmannin had no effect on
PMA-stimulated aggregation. Previous studies have indicated that
wortmannin and LY294002 do not affect calcium influx in stimulated
neutrophils (36). However, the effect of wortmannin on functions
stimulated by calcium has not been previously assessed. We therefore
tested the ability of wortmannin to inhibit neutrophil homotypic
aggregation in response to the calcium ionophores A23187 and ionomycin.
In contrast to its lack of effect on PMA-stimulated aggregation,
preincubation with wortmannin reduced neutrophil homotypic aggregation
in response to both A23187 and ionomycin. As in the case of G
protein-linked receptor agonists, the concentrations of wortmannin
required for inhibition of A23187- and ionomycin-stimulated aggregation
(IC50 12 nM and 13 nM, respectively) were consistent
with a role for PI 3-kinase inhibition. The maximal inhibitions
observed (26% for A21387, p = 0.0045, and 27% for
ionomycin, p = 0.0001) were similar for the two
ionophores but less than the inhibitory effects of wortmannin on the G
protein-linked receptor agonists. Consistent with its effects on intact
neutrophils, wortmannin had no effect on PMA-stimulated aggregation of
cytoplasts, whereas A23187-stimulated aggregation was reduced by about
25% (not shown).
Wortmannin inhibits FMLP-stimulated neutrophil adhesion to endothelial cell monolayers
Because neutrophil heterotypic adhesion to endothelium might be
regulated in a manner different from homotypic aggregation, we tested
the effect of wortmannin on the capacity of FMLP-stimulated neutrophils
to adhere to ECV304 monolayers (Fig. 4).
Consistent with previous reports, stimulation of neutrophils with 100
nM FMLP resulted in a 68 ± 19% increase in adherence of
neutrophils to ECV304 monolayers. As in the case of homotypic
aggregation, preincubation of neutrophils with wortmannin resulted in a
dose-dependent inhibition of FMLP-stimulated heterotypic adhesion to
ECV304. The IC50 for this effect (3 nM) was
consistent with PI 3-K inhibition. This effect was independent of any
effect of wortmannin on ECV304 cells, as neutrophils were washed
thoroughly to remove excess wortmannin before their addition to ECV304
monolayers.
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We employed FACS analysis to test the effect of wortmannin on
CD11b/CD18 surface expression after 1 or 10 min of stimulation with
FMLP. Our previous studies in purified neutrophils have demonstrated
only a small increase above background neutrophil cell-surface
CD11b/CD18 expression after 1 min of FMLP stimulation (7). Because
purification of neutrophils by standard methods results in increased
levels of CD11b/CD18 expression that can obscure small degrees of
agonist-stimulated up-regulation, we incubated whole blood preparations
in the absence or presence of wortmannin and stimulated them with FMLP
before fixation, indirect immunofluorescence staining for CD11b, and
flow cytometric analysis gated for granulocytes. The addition of 100 nM
FMLP to whole blood for 1 min, a time at which FMLP-stimulated
homotypic aggregation is half maximal, resulted in little or no
increase in granulocyte cell-surface CD11b/CD18 expression. Moreover,
wortmannin had no effect on the degree of CD11b/CD18 expression in the
absence or presence of FMLP (Fig. 5A). As previously
reported in isolated neutrophils (7), longer stimulation times were
required for CD11b/CD18 up-regulation: the addition of 100 nM FMLP to
whole blood for 10 min resulted in marked CD11b/CD18 up-regulation.
Wortmannin had no effect on FMLP-stimulated CD11b/CD18 up-regulation
(Fig. 5A). Wortmannin also had no effect on FMLP
stimulated up-regulation of CD35 (CR1), a receptor localized
exclusively in the plasma membrane and secretory vesicles (41) (Fig. 5B). Levels of cell surface binding of a nonspecific
control mAb were small and were unaffected by wortmannin or FMLP (not
shown). Because our whole blood preparations contained plasma proteins,
including albumin, that might bind to and reduce the bioavailability of
wortmannin, we also tested the effect of wortmannin on FMLP-stimulated,
partially purified (buffy coat) neutrophils that had been washed free
of plasma (Fig. 5C). Although, as expected,
background levels of CD11b/CD18 expression on granulocytes were higher
in these preparations, the results in these assays were similar to
those observed in whole blood. Moreover, addition of 3% BSA to
preparations of purified neutrophils did not impair the ability of
wortmannin to inhibit FMLP-stimulated homotypic aggregation, indicating
that the drug remained bioavailable in the presence of albumin (not
shown).
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). As expected, FMLP-stimulated lysozyme
release was reduced by wortmannin at concentrations consistent with PI
3-K inhibition (IC50 30 nM). This discrepancy may
likely be explained by the recent observation by Sengeløv et al. that
CD11b/CD18 up-regulation in response to FMLP stimulation of neutrophils
is largely a marker of secretory vesicle rather than specific granule
release (see Discussion) (47).
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The failure of wortmannin to inhibit total CD11b/CD18 expression
led us to postulate that PI 3-K activation mediates changes in the
activation state of CD11b/CD18 already on the neutrophil surface.
Diamond and Springer have recently described CBRM1/5, a mAb that
recognizes an activation-specific neoepitope of CD11b/CD18 and is
capable of inhibiting stimulated adhesion of neutrophils to purified
ICAM-1 (9). Incubation of neutrophils with CBRM1/5 (8 µg/ml) resulted
in 25 ± 4% inhibition of FMLP-stimulated homotypic aggregation,
confirming that the CBRM1/5 epitope participates in neutrophil
aggregation (Fig. 7A).
Higher concentrations of CBRM1/5 (40 µg/ml) resulted in greater
inhibition of aggregation (47% inhibition, data not shown).
Surprisingly, although FMLP stimulated the expression of the CBRM1/5
epitope on neutrophils in buffy coat preparations, wortmannin had no
effect on CBRM1/5 epitope expression (Fig. 7B).
Similar results were obtained in a single experiment performed on
isolated neutrophils (not shown). We have previously observed that,
like wortmannin, NaS (3 mM) inhibits neutrophil agreggation but not
CD11b/CD18 up-regulation in response to FMLP (7). We now report that
NaS also fails to inhibit FMLP-stimulated expression of the
CBRM1/5-specific epitope (Fig. 7 A and B).
Taken together, these data indicate that expression of the
CBRM1/5-specific epitope is necessary but not sufficient for neutrophil
aggregation and that the effect of wortmannin on aggregation is
independent of changes in expression of the CBRM1/5 epitope.
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FMLP and other chemoattractants stimulate Erk activity in human
neutrophils in a manner (1) at least partially dependent on the small
GTP-binding protein Ras (11, 34, 48, 49), and (2) consistent with a
role for Erk in neutrophil adhesive function (11). As recent studies
have suggested that PI 3-K mediates G protein-stimulated activation of
Ras and Erk in both neutrophils (34, 50) and mitotic cells (51), we
tested whether PI 3-K mediates neutrophil adhesion via stimulation of
Erk activity. Whereas both FMLP and C5a stimulated Erk activity in
neutrophils, preincubation with wortmannin at concentrations sufficient
to inhibit PI 3-K inhibited Erk activity only in cells stimulated with
C5a (Fig. 8A). Like wortmannin, LY294002 at
concentrations that maximally inhibit PI 3-K activity inhibited C5a-,
but had little or no effect on FMLP-stimulated Erk activity (Fig. 8B). These data suggest that FMLP stimulates Erk via
a PI 3-K-independent pathway and that the role of PI 3-K in
FMLP-stimulated neutrophil adhesion is independent of Erk.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our data indicate that wortmannin, at concentrations consistent with PI
3-K inhibition, inhibits FMLP-stimulated homotypic aggregation. This
observation is similar to a recent report by Wise that wortmannin
inhibited FMLP-stimulated aggregation of rat neutrophils (53).
Wortmannin also inhibited aggregation stimulated by the
chemoattractants C5a and LTB4, suggesting a common
pathway for stimulation of aggregation by this group of G
protein-linked receptor agonists. Wortmannin inhibition of
chemoattractant-stimulated aggregation was partial rather than total
(maximum inhibition was 
50%, even at concentrations of wortmannin
(100 nM) and LY294002 (100 µM) sufficient to completely inhibit
FMLP-stimulated PI 3-K activation (20, 25, 28, 29, 32, 36)), suggesting
that other, PI 3-K-independent pathways for adhesion may also be
present in the neutrophil. In contrast to its effects on
chemoattractant signaling, wortmannin had no effect on aggregation
stimulated by the direct PKC activator PMA, confirming that
chemoattractants and PMA stimulate aggregation by distinct mechanisms
(54) and consistent with the failure of wortmannin to inhibit other
PMA-stimulated neutrophil functions such as
O2
generation (20, 29, 36). Whereas several groups have observed that
wortmannin and LY294002 do not inhibit chemoattractant-stimulated
calcium influx in neutrophils (36, 50), our data indicate that
wortmannin partially inhibits neutrophil aggregation in response to
calcium influx. However, this effect of wortmannin was small compared
with its effect on neutrophils stimulated with chemoattractants.
Cross et al. have recently suggested that wortmannin may not be a specific inhibitor of PI 3-K because, in Swiss 3T3 cells lacking PI 3-K activity, wortmannin at nanomolar concentrations inhibits bombesin-stimulated cytosolic phospholipase A2 (cPLA2) activity (55). This observation is unlikely to be applicable to FMLP-stimulated neutrophil aggregation, because (1) in the absence of manipulation of extracellular calcium concentrations, FMLP stimulates little or no cPLA2 activity in neutrophils (56), and (2) we observed no effect of bombesin on neutrophil aggregation (data not shown). Nonetheless, we confirmed the role of PI 3-K in neutrophil aggregation by demonstrating that LY294002, a specific inhibitor of PI 3-K that acts by a mechanism distinct from wortmannin, also inhibited FMLP-stimulated aggregation at appropriate concentrations. LY303511, a close analogue of LY294002 that fails to inhibit PI 3-K (32), had no effect on FMLP-stimulated aggregation, confirming the specificity of these reagents. Whereas wortmannin irreversibly inhibits PI 3-K via nucleophilic addition of the kinase onto the C-21 position of the D ring of wortmannin (24), LY294002 reversibly inhibits PI 3-K via interactions with the PI 3-K ATP-binding site (32). Consistent with these disparate mechanisms of action, our data indicate that wortmannin and LY294002 inhibition of FMLP-stimulated aggregation are irreversible and reversible, respectively.
FMLP stimulation of neutrophil heterotypic adhesion to endothelial cells is similar in some respects to stimulation for homotypic aggregation. In each case, neutrophil adhesiveness appears to depend upon changes in the affinity state of CD11b/CD18 rather than upon changes in integrin surface expression (7, 8). Moreover, although heterotypic adhesion has traditionally been measured after 10 to 30 min of FMLP stimulation, the kinetics of aggregation and heterotypic adhesion are similar, each peaking at approximately 2 min (57). Heterotypic adhesion differs from homotypic aggregation in that the major cognate ligand for CD11b/CD18 on endothelial cells is ICAM-1, whereas the neutrophil ligand for homotypic aggregation is not ICAM-1 but has yet to be identified. Whether differences exist in the intrinsic regulation of neutrophil CD11b/CD18 for homotypic vs heterotypic adhesion is not known. We observed that wortmannin inhibited FMLP-stimulated adhesion to ECV304 cells at concentrations similar to those affecting homotypic aggregation, consistent with a role for PI 3-K in stimulated neutrophil adhesiveness in general. We took advantage of the irreversibility of wortmannin to wash neutrophils after incubation with wortmannin but before their exposure ECV304 monolayers. Wortmannin inhibition of heterotypic adhesion was thus due to effects on neutrophils and not endothelium. Moreover, FMLP does not stimulate endothelial cells for neutrophil adherence (57). Nonetheless, our data do not exclude the possibility that PI 3-K may also mediate increases in endothelial adhesiveness for neutrophils in response to stimuli such as IL-1.
Our observation that wortmannin reduces chemoattractant-stimulated aggregation of cytoplasts as well as intact neutrophils confirms that PI 3-K mediation of neutrophil aggregation does not occur via effects on receptor number. We were nonetheless surprised to observe that up-regulation of CD11b/CD18, a marker for neutrophil activation, was unaffected by wortmannin or LY294002. CD11b/CD18 up-regulation occurs via fusion of granular and plasma membranes, resulting in the movement of stored, preformed CD11b/CD18 to the neutrophil surface. The CD11b/CD18-containing compartment has been shown to co-sediment with neutrophil-specific granules, and the release of this compartment to the plasma membrane in response to FMLP is PI 3-K dependent (33). However, Borregaard et al. have recently demonstrated that CD11b/CD18 is contained in both neutrophil-specific granules and a class of smaller granules designated secretory vesicles (58, 59). Although the majority of CD11b/CD18 is contained in specific granules, stimulation of neutrophils with FMLP results in a much larger mobilization of secretory vesicles to the plasma membrane (47). Up-regulation of CD11b/CD18 in response to FMLP is thus due mainly to secretory vesicle release. Our data thus demonstrate a discrepancy between specific granule release and CD11b/CD18 up-regulation and suggest that, in contrast to its effects on specific and azurophilic granules, PI 3-K does not mediate the FMLP-stimulated degranulation of secretory vesicles. Indeed, we observed that wortmannin had no effect on FMLP-stimulated up-regulation of CD35, a marker for secretory vesicles but not specific or azurophilic granules (41).
The failure of wortmannin to inhibit CD11b/CD18 up-regulation is consistent with a model in which PI 3-K mediates changes in the affinity state, rather than total number, of CD11b/CD18 receptors. Accordingly, we tested the effects of wortmannin on the expression of an activation-specific CD11b/CD18 neoepitope identified by binding of mAb CBRM1/5 (9). Although CBRM1/5 inhibited FMLP-stimulated aggregation (demonstrating a role for its target epitope in aggregation), we observed that neither wortmannin nor NaS (which also inhibits aggregation) inhibited expression of the CBRM1/5 epitope in response to FMLP. These data suggest that the CBRM1/5 epitope may be necessary but not sufficient for aggregation, indicating that PI 3-K must mediate aggregation via mechanisms independent of the CBRM1/5 epitope. It is possible that PI 3-K may mediate changes in the activation state of CD11b/CD18 other than those detected by CBRM1/5. However, our data do not exclude the possibility that PI 3-K may mediate changes in the expression or activation state of an as yet unidentified accessory molecule required for aggregation. The ability of wortmannin to inhibit both homotypic aggregation (unknown counterligand) and heterotypic adhesion of neutrophils to endothelial cells (ICAM-1 counterligand) suggests that such an accessory molecule would be unlikely to represent the CD11b/CD18 counterligand for aggregation.
The effectors of PI 3-K (or its 3-phosphorylated phosphoinositol
products) in mediating neutrophil functions remain to be determined.
One possible candidate is Erk, which in mitotically competent cells has
been shown to play roles in signaling for cell growth and division
(60). In terminally differentiated, postmitotic neutrophils, Erk
undergoes activation upon stimulation of G protein-linked receptors by
chemoattractants (11, 61). Moreover, although recent evidence suggests
that Erk activation may play no role in neutrophil
O2
generation (62), we have recently observed an association between Erk
activation and neutrophil aggregation (11). In neutrophils as well as
in other cell types, a number of groups have recently reported a role
for PI 3-K in G protein-dependent activation of Erk (51), although one
of these studies reported that wortmannin inhibits guinea pig
neutrophil Erk activity in response to platelet-activating factor only
at concentrations far above those required to inhibit PI 3-K (50). In
contrast, we observed that neither wortmannin nor LY294002 inhibited
FMLP stimulation of neutrophil Erk at concentrations consistent with
their effects on PI 3-K or adhesion. Indeed, concentrations of
wortmannin and LY294002 sufficient to completely inhibit
FMLP-stimulated PI 3-K activation had little or no effect on
FMLP-stimulated Erk activity. Our data on FMLP-stimulated Erk would
appear to conflict with the previous observation by Knall et al. that
wortmannin inhibits C5a-stimulated Erk activation (34). However, we too
observed wortmannin inhibition of C5a-stimulated Erk in neutrophils.
These findings thus confirm our previous observation that signaling by
FMLP and other chemoattractants is likely to involve divergent as well
as convergent pathways (11). Moreover, the failure of wortmannin and
LY294002 to inhibit Erk in response to FMLP suggests that PI 3-K
signaling for aggregation is Erk independent and that a position for
Erk in neutrophil aggregation must be located upstream of, or parallel
to, PI 3-K signaling. These data resemble a recent report by Sue-A-Quan
et al. that wortmannin inhibits FMLP stimulation of neutrophil Erk only
at concentrations significantly higher than those required to inhibit
neutrophil
O2
generation, suggesting that Erk is unlikely to participate in the PI
3-K-dependent pathway for NADPH oxidase assembly (28). Although our
data do not rule out the formal possibility that neutrophil aggregation
stimulated by C5a depends upon the sequential activation of PI 3-K and
Erk, such a mechanism would seem unlikely. Klarlund et al. have
recently demonstrated in vitro that
phosphoinositide-3,4,5-trisphosphate (the predominant product of PI 3-K
in neutrophils) binds specifically to the plekstrin homology domain of
cytohesin-1 (63), a protein that can also bind to integrin
ß2 cytoplasmic domains and that has been implicated in
the enhancement of cell-cell interactions (64). If applicable, these
observations suggest a possible mechanism for Erk-indpendent, PI
3-K-dependent neutrophil adhesion.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Michael Pillinger, Department of Medicine, Room NB16N1, New York University Medical Center, 550 First Avenue, New York, NY 10016. E-mail address:
3 Abbreviations used in this paper: PI 3-K, phosphatidylinositol 3-kinase; MBPp, myelin basic protein peptide; LTB4, leukotriene B4; NaS, sodium salicylate; MOPC, mineral oil plasmacytoma; O2, superoxide anion; IC50, 50% inhibitory concentration.
4 C. Capodici, M. H. Pillinger, G. Han, M. R. Philips, and G. Weissmann. Integrin-dependent homotypic adhesion of neutrophils: arachidonic acid activates Raf-1/Mek/Erk via a 5-lipoxygenase-dependent pathway. Submitted for publication.
Received for publication May 16, 1997. Accepted for publication October 27, 1997.
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