From the Division of Infectious Diseases, Washington
University School of Medicine, St. Louis, Missouri 63110 and the
Departments of ¶ Immunology and
Cell Biology, Research
Institute of Scripps Clinic, La Jolla, California 92037-1092
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ABSTRACT |
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Circulating polymorphonuclear neutrophils (PMN)
are quiescent, nonadherent cells that rapidly activate at sites of
inflammation, where they develop the capacity to perform a repertoire
of functions that are essential for host defense. Induction of
integrin-mediated adhesion, which requires an increase in integrin
avidity, is critical for the development of these effector functions.
Although a variety of stimuli can activate integrins in PMN, the
signaling cascades involved are unclear. Phosphatidylinositol (PI)
3-kinase has been implicated in integrin activation in a variety of
cells, including PMN. In this work, we have examined activation of the
PMN integrin M
2, assessing both
adhesion and generation of the epitope recognized by the
activation-specific antibody CBRM1/5. We have found that PI 3-kinase
has a role in activation of
M
2 by immune
complexes, but we have found no role for it in
M
2 activation by ligands for trimeric G
protein-coupled receptors, including formylmethionylleucylphenylalanine (fMLP), interleukin-8, and C5a. Cytochalasin D inhibition suggests a
role for the actin cytoskeleton in immune complex activation of
M
2, but cytochalasin has no effect on
fMLP-induced activation. Similarly, immune complex activation of the
Rac/Cdc42-dependent serine/threonine kinase Pak1 is blocked
by PI 3-kinase inhibitors, but fMLP-induced activation is not. These
results demonstrate that two signaling pathways exist in PMN for
activation of
M
2. One, induced by Fc
R
ligation, is PI 3-kinase-dependent and requires the actin
cytoskeleton. The second, initiated by G protein-linked receptors, is
PI 3-kinase-independent and cytochalasin-insensitive. Pak1 may be in a
final common pathway leading to activation of
M
2.
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INTRODUCTION |
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Phagocytes are essential cells in host defense of metazoan
organisms because they prevent the systemic spread of invading pathogens. Phagocytic cells such as monocytes and polymorphonuclear leukocytes (PMN)1 circulate
throughout tissues to be able to initiate a rapid response to injury
and infection. At sites of inflammation and infection, these cells
perform many functions, including ingestion and killing of invading
organisms, generation of inflammatory mediators, and initiation of an
immune response. The acquisition of these effector functions required
for successful host defense is called phagocyte activation. Adhesion is
required to develop the full effector phenotype in phagocytes and,
indeed, in other leukocytes as well (reviewed in Refs. 1 and 2). We
have used human PMN as a model cell to study how adhesion regulates
this phenotypic change and the critical role of leukocyte integrins in
this process. PMN express 1,
2, and
3 integrins, but integrins other than the
2 family (also known as LeuCAM or CD18 integrins) are
present in low number. In particular, the CD18 integrin
M
2 plays a central role in PMN activation
at sites of inflammation (3-9). PMN integrins including
M
2 bind poorly to their ligands unless
the cells are exposed to inflammatory stimuli such as chemokines,
bacterial products, cytokines, complement fragments, or immune
complexes (10-15). These stimuli cause an increase in integrin avidity
through a process called "inside-out" signaling. The molecular
pathways of inside-out signaling are uncertain, but increases in
receptor affinity (16-19), receptor clustering (20), cytoskeletal
reorganization (19, 21, 22), and association with guanine nucleotide
exchange factors (23) may all be involved in the enhancement of
2 integrin avidity.
Phosphatidylinositol-3 kinase (PI 3-kinase) has been implicated in the
inside-out signaling for integrin activation (24). PI 3-kinase
phosphorylates phosphatidylinositols (PI) at the D3 position, producing
PI 3-phosphate, PI (3,4)-bisphosphate, and PI (3,4,5)-trisphosphate
(PIP3) (25). Five isoforms of mammalian PI 3-kinase have
been discovered which appear to be products of distinct genes but have
overlapping patterns of expression (26-29). All known isoforms of
mammalian PI 3-kinase share sensitivity to the pharmacologic agent
wortmannin (26, 30, 31), which inhibits PI 3-kinase activity by binding
to the lipid-binding domain of the catalytic subunit (32, 33). Whereas
wortmannin specifically inhibits PI 3-kinase activity at concentrations
in the low nanomolar range (26, 30, 31), higher concentrations inhibit
PI 4-kinase and myosin light chain kinase activity (34, 35). A second
agent called LY294002 inhibits PI 3-kinase activity of the p85/p110
isoforms by binding the ATP-binding site of p110, but it has no effect
on PI 4-kinase activity at doses up to 100 µM (36). These
pharmacologic agents have been extremely useful in delineating cellular
activities in which PI 3-kinase has a role, including regulation of
adhesion. Wortmannin has been shown to inhibit 1
integrin-mediated adhesion to fibronectin of stem cell factor-treated
mast cells (37) and CD-2 transfected HL-60 cells (38); thrombin- and
Fc
RII-induced,
3 integrin-mediated aggregation of
platelets (39-41); and
2 integrin-dependent
homotypic adhesion of IL-2-treated lymphocytes (42). Use of PDGF and
CD28 receptor mutants that no longer bind PI 3-kinase has provided strong evidence that PI 3-kinase is important for regulating PDGF- and
CD28-induced adhesion in mast cells and HL-60 cells, respectively (43,
44). Furthermore, expression of dominant negative mutants of the p85
subunit of PI 3-kinase blocks CD7-induced activation of
1 integrins in human T cells (45). Although these data
strongly suggest that PI 3-kinase activity is an important early event in inside-out signaling regulating integrin-mediated adhesion, the
mechanism by which PI 3-kinase regulates adhesion is not clear nor is
the generality of the requirement for PI 3-kinase in integrin activation. PDGF receptors, for example, can activate
1
integrins by PI 3-kinase-dependent and -independent
mechanisms (44).
Whether PI 3-kinase has a role in M
2
activation in PMN is not known. Fc
R-induced phagocytosis (46),
fMLP-induced respiratory burst activity (32, 33, 47), and PDGF-induced
chemotaxis (48) in PMN are inhibited by wortmannin. Since each of these events depends on activated integrins, these data suggest the hypothesis that PI 3-kinase is a component of the inside-out signaling pathway regulating integrin activation in PMN. However, PMN contain an
intracellular pool of
M
2 which is rapidly
expressed at the plasma membrane upon activation (15, 49). While this
intracellular pool is not required for PMN binding to endothelia or for
aggregation (50-52), it is necessary for optimal adhesion (53). The
role of PI 3-kinase in regulating the expression of this intracellular pool at the plasma membrane is unknown.
We tested the importance of PI 3-kinase in regulating integrin
activation in PMN in two well characterized experimental systems for
activating 2 integrin-dependent adhesion.
Fc
R ligation activates
M
2 through
initiation of a tyrosine kinase cascade, whereas fMLP requires a
heterotrimeric G-protein to initiate signaling in PMN. Our results
suggest that two pathways exist for activating
2
integrin-dependent adhesion in PMN. The Fc
R-initiated
pathway is dependent on PI 3-kinase activity and is inhibited by
cytochalasin D, whereas the fMLP-induced increase in
M
2 avidity is independent of PI 3-kinase
and unaffected by cytochalasin D. Fc
R-mediated enhancement of
M
2 expression is inhibited by wortmannin,
but increased expression is not required for adhesion. Importantly, both pathways activate Pak1, a recently described serine/threonine kinase implicated in membrane ruffling and focal adhesion formation (54). Fc
R-induced activation of Pak1 is PI
3-kinase-dependent, whereas fMLP-induced activation of Pak1
is independent of PI 3-kinase, potentially placing Pak1 in a common
pathway leading to activation of
M
2
avidity. These data demonstrate that there is more than one molecular
pathway for inside-out signaling and suggest that the effects of
tyrosine kinase cascades, and G protein-dependent signaling
on integrin function may be mediated by distinct mechanisms that
converge on a common pathway involving Pak1.
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MATERIALS AND METHODS |
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Reagents--
Cytochalasin D, PMA, fMLP, dimethyl sulfoxide,
rabbit polyclonal anti-BSA antiserum, C5a, BSA,
poly-L-lysine, glutaraldehyde, fluorescein
isothiocyanate-conjugated F(ab')2 sheep anti-mouse IgG
antibody, MBP, and EGTA were from Sigma. [-32P]ATP was
from ICN (Irvine, CA). Phosphate-buffered saline and 10× stock HBSS
were from Life Technologies, Inc. Protein A-conjugated Sepharose,
Ficoll-Paque, and Dextran T500 were obtained from Amersham Pharmacia
Biotech (Uppsala Sweden). IL-8 was purchased from Calbiochem (San
Diego, CA), and pertussis toxin was obtained from List Biologicals (Campbell, CA). 1 M stock Hepes and 7.5% sodium
bicarbonate were from BioWhittaker (Walkersville, MD). Wortmannin and
LY294002 were obtained from LC Laboratories (Woburn, MA). Fetal calf
serum (FCS) was from Hyclone (Logan, UT). Calcein and Celltracker Green CMFDA were from Molecular Probes (Eugene OR). Tissue culture plates and
96-well Immulon 2 plates were from Becton-Dickinson (Franklin Lakes,
NJ) and Dynatech (Chantilly, VA), respectively. Monoclonal Abs 3G8
(anti-Fc
RIII) (55), IV.3 (anti-Fc
RII) (56), IB4 (anti-
2, CD18) (57), W6/32 (anti-class I HLA) (58), and
B6H12 (anti-IAP, CD47) (59) were purified, and F(ab')2 was
prepared as described (60). CBRM1/5 (61) concentrated tissue culture supernatant and anti-Pak1 polyclonal antiserum (62) were prepared as described.
Preparation of PMN Suspensions-- Human PMN were isolated from whole blood exactly as described (63) except hypotonic lysis was not performed. PMN were greater than 98% viable as indicated by the exclusion of trypan blue dye. Cells were suspended in HBSS (Hanks' buffered salts solution with 20 mM Hepes and 8.9 mM sodium bicarbonate) with 1.0 mM Mg2+ and 1 mM Ca2+ (HBSS2+) or HBSS with 0.5 mM Mn2+ for adhesion assays and flow cytometry.
Adhesion Assay-- Purified human PMN (1 × 107/ml) were incubated with 2 µg/ml calcein in HBSS for 30 min at RT. The cells were washed once and resuspended in HBSS2+ at 2 × 106/ml. For adhesion experiments in the presence of Mn2+, the cells were washed in HBSS with 2 mM EGTA once, HBSS2+ or HBSS + 0.5 mM Mn2+ once, and resuspended in HBSS2+ or HBSS + 0.5 mM Mn2+. Cells were treated with wortmannin or LY294002 at the indicated concentration or Me2SO as a control for 15 min at 37 °C or with pertussis toxin (2 µg/ml) or control buffer in HBSS + 1% human serum albumin for 2 h at 37 °C. For antibody inhibition experiments, cells were incubated with 10 or 25 µg/ml of the appropriate antibody for 15 min at RT. 1 × 105 cells were added per well to Immulon 2 plates coated with BSA and a 1:25 dilution of rabbit anti-BSA to form IC or 5% FCS as described (64). For PMA or fMLP-stimulated adhesion, PMA (50 µg/ml final), fMLP (100 nM final), or Me2SO control was added to the cells after allowing them to settle onto FCS-coated wells for 6 min at RT. The cells were incubated at 37 °C for the indicated time. The fluorescence (485 nm excitation and 530 nm emission wavelengths) was measured using a fMax fluorescence plate reader (Molecular Devices, Sunnyvale, CA) before and after washing twice with 150 µl of phosphate-buffered saline. Percent adhesion was calculated by dividing the fluorescence after washing by the fluorescence before washing. In preliminary experiments, fluorescence was shown to be linearly related to cell number (data not shown).
Flow Cytometry-- Purified PMN (4 × 106/ml in HBSS2+) were treated with wortmannin (100 nM) or Me2SO for 15 min at 37 °C. For experiments with pertussis toxin, 1 × 107 cells/ml were incubated with pertussis toxin (2 µg/ml) or control buffer for 2 h at 37 °C and then washed. 2 × 106 cells were then treated with 30 µl insoluble IC (IIC) prepared exactly as described (65), fMLP (100 nM), C5a (50 nM), IL-8 (100 nM), or PMA (50 µg/ml) at 37 °C for 10 min, placed on ice, washed once with ice-cold wash buffer (phosphate-buffered saline, 1% FCS, 0.1% sodium azide), and resuspended in 100 µl of wash buffer plus primary antibody (25 µg/ml). Cells were incubated with primary antibody for 40 min on ice and then washed twice. After incubation with fluorescein isothiocyanate-conjugated F(ab')2 sheep anti-mouse IgG secondary antibody at a 1:50 dilution in 200 µl of wash buffer for 20 min on ice, cells were washed twice, and the relative fluorescence of gated PMN was measured using a EPICS XL (Coulter, Miami, FL) flow cytometer. For Mn2+ experiments, cells were treated with wortmannin, washed, resuspended in HBSS2+ or HBSS + 0.5 mM Mn2+, and incubated for 10 min at 37 °C and then placed on ice. Primary antibody was added directly to the cells (25 µg/ml) for 40 min on ice, washed twice, and incubated with secondary antibody as above. All washes were done with HBSS containing appropriate divalent cations.
Pak1 Kinase Assays--
Purified PMN were suspended at 1 × 107 cells/ml in HBSS2+. After pretreatment with
wortmannin, LY294002, pertussis toxin, or control buffer as described
above, 7.5 × 106 cells were added to 6-well plates
coated with IC or FCS as described (66) or stimulated in suspension
with fMLP (100 nM). After incubating at 37 °C, the cells
were lysed by adding cold 2× lysis buffer (1% Nonidet P-40, 150 mM NaCl, 5 mM EGTA, 50 mM NaF, 5 mM sodium pyrophosphate, 1 mM
NaVO4, 5 mg/ml leupeptin and aprotinin, and 1 mM diisopropyl fluorophosphate, final concentration) for 30 min on ice. Pak1 was immunoprecipitated from the lysates with 5 µl of
rabbit anti-Pak1 antiserum and 40 µl of a 1:1 slurry of Protein
A-Sepharose for 2 h at 4 °C. The immunoprecipitates were washed
four times with lysis buffer and two times with reaction buffer (25 mM Tris-HCl, pH 7.4, 10 mM MgCl2).
Kinase reactions were performed with the Pak1 immunoprecipitates by
adding 30 µl of reaction buffer with 2.5 µg of MBP to the beads,
incubating for 10 min at RT, followed by 10 µl of reaction buffer
containing 100 µM cold ATP and 0.5 µCi of
[-32P]ATP (4500 Ci/mmol) for a final ATP concentration
of 25 µM. The reactions were incubated for 20 min at
30 °C, after which the reaction was stopped with 50 µl of
SDS-polyacrylamide gel electrophoresis sample buffer containing 10%
SDS. Phosphorylation of MBP was detected by SDS-polyacrylamide gel
electrophoresis, transfer to polyvinylidene difluoride membranes, and
autoradiography. For each experiment, Pak1 protein was immunoblotted
using anti-Pak1 antiserum (1:1000) primary antibody, horseradish
peroxidase-conjugated goat anti-rabbit antiserum (20 µg/ml) secondary
antibody, and enhanced chemiluminescence substrate (ECL, Pierce) to
ensure that equivalent amounts of kinase protein were added to each
in vitro kinase reaction.
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RESULTS |
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PI 3-Kinase Activity Is Required for FcR-induced Activation of
M
2-dependent
Adhesion--
PI 3-kinase activity is required for adhesion of a
variety of cell types to fibronectin (37, 38), agonist-induced
aggregation and up-regulation of activated
IIb
3 expression in platelets (39-41),
and PDGF-induced chemotaxis in PMN (48), suggesting that PI 3-kinase
activity is important for integrin activation. Fc
R-induced
phagocytosis in PMN is blocked by wortmannin (46), suggesting that PI
3-kinase activity may be required for Fc
R-induced signal
transduction and effector functions in PMN. We used two pharmacologic
inhibitors of PI 3-kinase, wortmannin and LY294002, to test the
hypothesis that PI 3-kinase activity is required for Fc
R-induced,
2 integrin-dependent adhesion to IC in
PMN.
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fMLP- and PMA-induced Adhesion Is PI 3-Kinase-independent--
PI
3-kinase could be required for integrin-dependent adhesion
because it is involved in an FcR-initiated signaling pathway which
results in an alteration in
M
2 avidity or
because it is involved in the cytoskeletal rearrangements which are
required for increased adhesion. To distinguish these possibilities, we assessed the role of PI 3-kinase in PMA and fMLP-induced PMN adhesion. PMN do not adhere to surfaces coated with FCS in the absence of
M
2 activation but adhere strongly when
stimulated with agonists (Fig. 3, A and B). In
contrast to its effect on sustained PMN adhesion to IC, wortmannin had
no effect on either PMA or fMLP-induced adhesion. fMLP (100 nM)-stimulated adhesion to FCS was maximal by 3 min,
decreased by 10 min, but remained above base-line adhesion for at least
30 min (Fig. 3A). Wortmannin treatment had no significant effect on fMLP-induced adhesion at any time point. In confirmation of
earlier reports (32, 33, 47), 10 nM wortmannin completely inhibited fMLP-induced respiratory burst activity in PMN (data not
shown). Furthermore, wortmannin pretreatment inhibited PI 3-kinase
activity in anti-p85 and anti-p110
immunoprecipitates with
IC50 of 5 and 20 nM, respectively,
demonstrating the efficacy of wortmannin treatment (data not shown).
Likewise, PMA (50 ng/ml) induced significant adhesion by 3 min which
continued to increase until 30 min and was unaffected by wortmannin
(Fig. 3B). Identical results were obtained for fMLP- and
PMA-induced adhesion to the
M
2 ligand fibrinogen (data not
shown). Like wortmannin, LY294002 had no effect on fMLP- or PMA-induced
adhesion (data not shown). The PKC inhibitor Gö6976 inhibited
fMLP, PMA, and IC-induced adhesion (data not shown).
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PI 3-Kinase-dependent and -independent Up-regulation of
M
2 Integrin--
When PMN are activated
two processes occur that can potentially affect adhesion. Cell surface
2 integrin expression increases as integrins stored in
intracellular granules are released to the cell membrane by
degranulation. Integrins already present on the cell surface also are
activated, causing an increase in avidity for ligand which is
independent of any increase in receptor expression. To determine
whether one or both of these steps was affected by the distinct fMLP
and IC signaling pathways and to determine whether one or both was
required for sustained adhesion, we examined these processes
independently. We first tested whether Fc
R-induced up-regulation of
2 integrin expression is affected by wortmannin using
flow cytometry to detect surface expression of
2 on PMN
stained with anti-
2 integrin antibody
F(ab')2.
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PI 3-Kinase-dependent and -independent Pathways for
M
2 Activation--
We next examined the
role of PI 3-kinase in activation of surface-expressed
M
2. We used a monoclonal antibody
(CBRM1/5) that specifically recognizes a neoepitope on activated
M
2 to detect activated
M
2 on the surface of stimulated PMN (61). IC, fMLP, and PMA all increased expression of the CBRM1/5 epitope (Fig.
6A and data not shown).
However, wortmannin and cytochalasin D inhibited the increased
expression of activated
M
2 only on PMN
stimulated with IC and not on fMLP- or PMA-stimulated PMN (Fig.
6A and data not shown). Thus, both Fc
R-induced
up-regulation of
M
2 expression and
activation are PI 3-kinase-dependent and also require the
actin cytoskeleton. In contrast, fMLP and PMA-induced up-regulation of
M
2 expression and activation are PI
3-kinase-independent and cytochalasin-insensitive.
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Activation of M
2 Avidity Is Necessary
and Sufficient for Sustained Adhesion to IC--
Whereas both
Fc
R-induced increases in
2 expression and activation
of the
M
2 activation epitope require PI
3-kinase activity, it is well established that receptor activation is
required, but increased receptor expression is not necessary for
M
2 adhesion in other systems (50-52,
61). To determine the requirements for sustained adhesion to IC, we
tested the effect of CBRM1/5 (61) on adhesion. Like wortmannin and the
anti-
2 antibody, CBRM1/5 inhibited sustained adhesion to
IC but had no effect on the initial
2-independent
adhesion (Fig. 7) Thus, activated
M
2 is necessary for sustained
adhesion.
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Pak1 Is Activated by IC and fMLP--
The small GTPases Rac and
Cdc42 have been found to be important for regulation of the actin
cytoskeleton and formation of integrin complexes in several cell types
(73). We investigated the possibility that Rac and/or Cdc42 regulated
integrin activation in PMN by examining the activation of the
Rac1/Cdc42 effector Pak1 which has been implicated in the regulation of
the actin cytoskeleton and formation of focal adhesions (54). Both fMLP and adhesion to IC activated Pak1 in PMN (Fig.
9, A and B). The kinetics of Pak1 activation in response to fMLP and IC was identical to
the kinetics of adhesion induced by these stimuli (Fig. 9, C
and D). Pak1 activation induced by fMLP or adhesion to IC
was unaffected by pretreatment with anti-2
F(ab')2, indicating that Fc
R ligation activates Pak1
independently of
2 integrins, suggesting that Pak1
activation may be a component of inside-out signaling (Fig.
9E). Importantly, we found that IC-induced Pak1 activation was dependent on PI 3-kinase activity and independent of a PT-sensitive G protein (Fig. 9, A and B). In contrast,
fMLP-induced Pak1 activation was inhibited completely by PT but was
unaffected by wortmannin (Fig. 9, A and B). Both
wortmannin and LY294002 inhibited IC-induced Pak1 activation with
IC50 of 5 nM and 5 µM (data not
shown), identical to the IC50 for inhibition of sustained
adhesion to IC. These data demonstrate that while Fc
R and the fMLP
receptor induce initially distinct signals, the signaling pathways
initiated by these receptors converge on the Rac/Cdc42-activated kinase
Pak1.
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DISCUSSION |
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A hallmark of neutrophil activation is the requirement for cell
adhesion to achieve full functional capacity. The 2
integrins, particularly
M
2, have a
central role in this adhesion-dependent activation, as
shown by the multiple, profound functional defects of PMN from patients
with leukocyte adhesion deficiency (3-8). Many lines of investigation
have demonstrated that unactivated PMN are poorly adherent, and
integrin-mediated adhesion is rapidly and reversibly induced by
activation stimuli (11, 12, 14). This regulation, which has been called
inside-out signaling, is a key event in PMN transendothelial migration,
in motility through extracellular matrix, and in induction of effector
functions at the site in inflammation. Whereas
and
chain
domains required for inside-out signaling have been studied in some
detail for several integrins (74-77), and integrin clustering and
alterations in association with cytoskeleton have been described as
consequences of inside-out signaling (19-22), the molecular details
of the signal transduction cascade involved in integrin activation
remain unclear.
We have identified two inside-out signaling pathways for activation of
M
2 integrin-dependent
adhesion, an Fc
R-induced, PI 3-kinase-dependent pathway
and an fMLP-induced PI 3-kinase-independent pathway. PMA-induced
adhesion is also PI 3-kinase-independent, either because PMA-activated
PKC is downstream of PI 3-kinase or because it is in the PI
3-kinase-independent pathway. Gö6976, an inhibitor of the
classical calcium-dependent PKC, inhibits adhesion to IC as
well as fMLP- and PMA-induced adhesion (data not shown), suggesting
that PKC is in a common pathway. Although we cannot definitively rule
out the possibility that the effect of wortmannin and LY294002 in our
experiments is a result of inhibition of other enzymes, the fact that
two inhibitors with distinct mechanisms of action cause the same
biologic effect argues strongly for specificity. Furthermore, the
wortmannin and LY294002 IC50 for inhibition of adhesion and
Pak1 activity induced by IC were 5 nM and 5 µM, respectively. These values agree with our own and
published values for inhibition of PI 3-kinase activity in PMN and
other cells (32, 33, 36, 40, 41, 45, 47, 78-80) and are 30- and
10-fold less than values reported for myosin light chain kinase
and PI 4-kinase, respectively (34, 35).
Recently, several groups have suggested an important role for PI
3-kinase in inside-out signaling by lymphocyte receptors including the
IL-2 receptor, CD2, CD7, and CD28 (38, 42, 43, 45), by FcRIIA or
thrombin receptors on platelets (39-41), and by stem cell factor
receptors on mast cells (37). These studies generally have had as their
integrin targets
1 and
3 integrins, which
may differ significantly from leukocyte
2 integrins in their mechanisms for regulation (23, 67). The finding that a PI
3-kinase-independent pathway exists for activation of
1-dependent adhesion by the PDGF receptor
(44) and that PMA and
1 integrin-activating antibodies
induce adhesion in wortmannin-treated HL60 cells (38) suggest that PI
3-kinase is an element in the signal transduction cascade for
1 integrin activation. Our demonstration that
2 integrin activation by the physiologic agonist fMLP as
well as PMA can occur in PMN in which PI 3-kinase has been inhibited
further establishes that PI 3-kinase is a component of inside-out
signal transduction and is not required for adhesion itself.
G protein-coupled receptors can activate PI 3-kinase and use it to
stimulate PIP3 production, respiratory burst activity, and
activation of Raf-1 and ERK1/2 (33, 47, 81). In other cells, trimeric G
protein-linked receptors activate the p110 isoform of PI 3-kinase by
both the
and
subunits of the heterotrimeric G protein (31).
The wortmannin sensitivity of this isoform may be less than the
classical p85/p110 heterodimeric kinase, but the IC50 for
wortmannin is still less than 50 nM (31). Indeed, in our
system, wortmannin pretreatment inhibited PI 3-kinase activity in
anti-p110
immunoprecipitates with an IC50 of 20 nM (data not shown). Wortmannin has been shown to inhibit
completely fMLP-induced PIP3 production in PMN with an
IC50 of 5 nM (32, 33). Wortmannin inhibited
fMLP-induced respiratory burst activity in our system with an
IC50 of 2 nM (data not shown). Thus, activation
of PI 3-kinase by fMLP is entirely wortmannin-sensitive in PMN; hence, wortmannin insensitivity demonstrates that PI 3-kinase activation is
not required for the activation of
M
2.
This second, PI 3-kinase-independent, pathway for activating
M
2 can be initiated by several ligands for seven transmembrane receptors, including fMLP, C5a, and IL-8. This
result is consistent with the finding that wortmannin does not inhibit
fMLP- or IL-8-induced chemotaxis (48). Interestingly, wortmannin does
inhibit PDGF-induced chemotaxis in PMN (48). These data suggest that
Fc
R and PDGF receptor, which activate tyrosine kinase cascades,
utilize PI 3-kinase for integrin activation, whereas G protein-linked
receptors do not. Thus, whether PI 3-kinase is involved in integrin
activation may depend on whether the initial stimulus initiates a
tyrosine kinase cascade. However, PI 3-kinase is not absolutely
required for all tyrosine kinase-initiated integrin activation, because
PDGF receptor mutants that cannot bind PI 3-kinase but are able to bind
phospholipase C
are perfectly capable of activating
1
integrins in mast cells (44). At physiologic concentrations of PDGF,
PDGF receptor-initiated activation of Erk1 and Erk2 is inhibited by
wortmannin, but at higher concentrations of PDGF, activation of Erk1/2
can occur by a PI 3-kinase-independent, phospholipase C
- and
PKC-dependent pathway, suggesting that PI 3-kinase activity
is critical at low but not high signal strength (82).
Many PMN responses to immune complexes require
M
2, including sustained adhesion (8, 83).
This study demonstrates that the recruitment of
M
2 function requires PI 3-kinase activity from Fc
R ligation. In our experiments,
M
2 activation was measured by
quantitating binding of the mAb CBRM1/5. CBRM1/5 recognizes a
neoepitope induced in a subset of
M
2 upon
activation by agonists that induce adhesion (61). The characteristics
of this subset remain unknown; however, it is clear that this subset of
receptors is required for agonist-induced adhesion to
M
2 ligand (61). Although adhesion to IC
induced both increased surface expression of the integrin and the
conformational change associated with binding of CBRM1/5, increased
surface expression was not required for sustained adhesion. In
contrast, the conformational change in
M
2
recognized by CBRM1/5 was required for sustained adhesion to IC. This
is similar to the conclusions about the role of
M
2 in PMN-endothelial adhesion (52) and
in PMN aggregation (50, 51) and makes
M
2
activation similar to activation of
L
2, another
2 integrin which exhibits regulated adhesion
without changes in surface expression (84). Thus, while PI 3-kinase is
involved in activating regulated secretion in PMN which results in
increased plasma membrane expression of
M
2, its role in sustaining adhesion to
immune complexes requires only induction of integrin activation.
Sustained PMN adhesion to IC leads in turn to enhanced generation of
LTB4 (8), superoxide (83), and mediators of inflammation.
Our data demonstrate that FcR and seven transmembrane receptor
ligation induce distinct pathways that converge into a common pathway
for activation of
M
2 avidity. A potential
effector of this common pathway is the Rac/Cdc42-activated kinase Pak1.
Fc
R-induced Pak1 activation is dependent on PI 3-kinase activity,
whereas fMLP activation of Pak1 is independent of PI 3-kinase,
consistent with their effects on
M
2
activation. For both IC and fMLP, Pak1 activation is independent of
M
2 ligation as assessed by antibody inhibition, suggesting it is upstream of integrin activation. Cdc42 and
Rac regulate focal complex formation and adhesion in fibroblasts and
the macrophage cell line Bac1.2F5 (85, 86), consistent with the
possibility that they regulate integrin avidity. Since there are
several effector pathways initiated by both Rac and Cdc42, it is
possible our data reflect a requirement for these small GTPases rather
than for Pak1 itself. Our suggestion that Pak1 regulates integrin
activation is supported by the finding that expression of an activated
form of Pak1 in Swiss 3T3 cells causes large focal adhesions to form
and actin accumulation in lamellipodia (54). Interestingly, PDGF and
insulin receptor-induced actin cytoskeletal re-organization mediated by
Rac are inhibited by wortmannin, while LPA and bombesin responses,
which signal via G protein-linked receptors, are not (87), again
suggesting that tyrosine-kinase pathways generally activate Rac and
Pak1 by a PI 3-kinase-dependent mechanism while the G
protein-dependent receptor-initiated pathway does not. It
is intriguing as well that cytohesin-1, a cytosolic regulator of
2-mediated adhesion which binds to the
2
cytoplasmic tail, is a guanine nucleotide exchange factor for Arf-1
(85, 88). We suggest that PI 3-kinase is a necessary effector of
tyrosine kinase-mediated but not seven transmembrane receptor-mediated
integrin regulation in PMN. These cascades converge at activation of
Pak1 through the small GTPases Rac and Cdc42. This model predicts that
the rapid, reversible activation of integrin-mediated adhesion that is
necessary for chemotaxis and transendothelial migration induced by
chemoattractants is controlled by a distinct proximal pathway from
that which activates the sustained, integrin-mediated adhesion
necessary for PMN effector functions such as Fc
R-mediated
phagocytosis.
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FOOTNOTES |
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* This work was supported by grants from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by a Physician Scientist Award.
** To whom correspondence should be addressed: Division of Infectious Diseases, Washington University School of Medicine, Box 8051, 660 S. Euclid Ave., St. Louis, MO 63110. Tel.: 314-362-2125; Fax: 314-362-9230.
1 The abbreviations used are: PMN, polymorphonuclear neutrophil(s); IC, immune complexes; IIC, insoluble immune complexes; fMLP, formylmethionylleucylphenylalanine; IAP, integrin-associated protein (CD47); HLA, human leukocyte antigen; PI, phosphatidylinositol; PMA, phorbol 12-myristate 13-acetate; MBP, myelin basic protein; BSA, bovine serum albumin; FCS, fetal calf serum; IL, interleukin; mAb, monoclonal antibody; PT, pertussis toxin; HBSS, Hanks' buffered salts solution; PIP3, PI (3,4,5)-trisphosphate; PDGF, platelet-derived growth factor; RT, room temperature; PKC, protein kinase C.
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