From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
PHAGOCYTES
Phosphoinositide 3-kinase modulation of ␤3-integrin represents an endogenous
“braking” mechanism during neutrophil transmatrix migration
Walter J. Bruyninckx, Katrina M. Comerford, Donald W. Lawrence, and Sean P. Colgan
During episodes of inflammation, neutrophils (polymorphonuclear leukocytes
[PMNs]) encounter subendothelial matrix
substrates that may require additional
signaling pathways as directives for
movement through the extracellular
space. Using an in vitro endothelial and
epithelial model, inhibitors of phosphoinositide 3-kinase (PI3K) were observed to
promote chemoattractant-stimulated migration by as much as 8 ⴞ 0.3-fold. Subsequent studies indicated that PMNs respond
in a similar manner to RGD-containing matrix substrates and that PMN-matrix interactions are potently inhibited by antibodies
directed against ␤3- but not ␤1-integrin antibodies, and that PI3K inhibitors block ␤3integrin dependence. Biochemical analysis
of intracellular ␤3-integrin uncoupling by
PI3K inhibitors revealed diminished ␤3integrin tyrosine phosphorylation and
decreased association with p72syk. Similarly, the p72syk inhibitor piceatannol promoted PMN transmatrix migration,
whereas HIV-tat peptide-facilitated loading of peptides corresponding to the ␤3integrin cytoplasmic tail identified the
functional tyrosine residues for this activity. These data indicate that PI3K-regulated ␤3-integrin represents a natural
“braking” mechanism for PMNs during
transit through the extracellular matrix.
(Blood. 2001;97:3251-3258)
© 2001 by The American Society of Hematology
Introduction
Migration of neutrophils (polymorphonuclear leukocytes [PMNs ])
to sites of inflammation requires the coordinated interplay of
soluble mediators, extracellular matrix ligands, and cell surface
adhesion molecules. To subserve this function, PMNs must traverse
endothelial cells lining the inner lumen of blood vessels. This
process of transendothelial migration requires engagement and
disengagement of a number of surface molecules and has been
extensively studied.1 After successful transendothelial migration,
PMNs encounter subendothelial extracellular matrices in transit to
inflammatory sites. Anchorage of cells to the extracellular matrix is
mediated in part by integrins, a large family of heterodimeric cell
surface proteins that mediate numerous cell functions, including
motility, differentiation, and proliferation.2 Integrin engagement of
matrix ligand results in highly regulated signal transduction
processes that cooperatively involve both “outside-in” and “insideout” pathways.3 Importantly, integrin signaling can vary depending
on the stimulus and on the cell type,4 the molecular details of which
are not fully understood at the present time.
Recent studies have identified an important role for phosphoinositide-3-OH kinase (PI3K) in leukocyte migration.5-7 The 4 known
isoforms of PI3K are ␣, ␤, ␥, and ␦, and extracellular ligand
coupling through membrane-localized G proteins generates the
signaling molecule phosphatidylinositol 3,4,5-triphosphate.8 PI3K
coordinates a number of leukocyte effector functions, including
leukocyte migration.5-7,9 Although not fully understood at present,
it appears that PI3K is central to stimulated chemotaxis. For
instance, mice lacking the catalytic domain of leukocyte-specific
PI3K␥ (p110␥) manifest chemotactic defects in vitro and in vivo6,7
and PI3K activation results in membrane localization of downstream protein kinases.5
In the present study, we examined the role of PI3K in PMN
interactions with endothelia, epithelia, and matrix substrates.
Detailed analysis revealed that PI3K activation by PMN chemoattractants results in functional regulation of surface ␤3-integrin
coupling. Further examination identified a candidate kinase (p72syk)
in such coupling of functional surface ␤3-integrin. Taken together,
these results identify PI3K-modulated, ␤3-integrin coupling as an
endogenous pathway for regulated PMN-matrix interactions.
From the Center for Experimental Therapeutics and Reperfusion Injury,
Brigham and Women’s Hospital, Harvard Medical School, Boston,
Massachusetts, and the Department of Biology, Hanover College, Indiana.
Reprints: Sean P. Colgan, Center for Experimental Therapeutics and
Reperfusion Injury, Brigham and Women’s Hospital, Thorn Bldg 704, 75
Francis St, Boston, MA 02115; e-mail: [email protected].
Submitted August 3, 2000; accepted January 12, 2001.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
Supported by National Institutes of Health grants DK50189, HL60569, project
3 of PO-1 DE13499, and by a grant from the Crohn’s and Colitis Foundation
of America.
BLOOD, 15 MAY 2001 䡠 VOLUME 97, NUMBER 10
Materials and methods
Cell culture
Human microvascular endothelial cells (HMVECs), a microvascular endothelial primary culture isolated from adult dermis, were obtained from
Cascade Biologics (Portland, OR) and cultured as previously described.10
Briefly, culture medium was supplemented with heat-inactivated bovine
calf serum, penicillin, streptomycin, Hepes, heparin, L-glutamine, and
endothelial mitogen factor. For preparation of experimental HMVEC
monolayers, confluent endothelial cells (passages 1, 2, or 3) were seeded at
about 2 ⫻ 105 cells/cm2 onto either permeable polycarbonate inserts or
6-well plates precoated with 0.1% gelatin. Endothelial cell purity was
assessed by phase microscopic “cobblestone” appearance and uptake of
fluorescent acetylated low-density lipoprotein. Oral epithelial cells (KB
cells) were obtained from the American Type Tissue Collection (ATTC;
Rockville, MD) and cultured as described previously.11
© 2001 by The American Society of Hematology
3251
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
3252
BLOOD, 15 MAY 2001 䡠 VOLUME 97, NUMBER 10
BRUYNINCKX et al
Isolation of human PMNs
The PMNs were freshly isolated from whole blood obtained by venipuncture from human volunteers and anticoagulated with acid-citrate-dextrose.12
Plasma and mononuclear cells were removed by aspiration from the buffy
coat following centrifugation (400g for 20 minutes) at 25°C. Erythrocytes
were removed using a 2% gelatin sedimentation technique. Residual
erythrocytes were removed by lysis in cold NH4Cl buffer. Remaining cells
were more than 90% PMNs as assessed by microscopic evaluation. PMNs
were studied within 2 hour of isolation.
Chemotaxis assay under agarose
Chemotaxis under agarose was used to assess the impact of PI3K inhibition
on PMN migration in isolation, as described previously,13 using human
PMNs and indicated concentrations of N-formyl-methionyl-leucylphenylalanine (fMLP) as a chemoattractant. PMN migration was measured
microscopically by the leading front method14 using an ocular micrometer
as described previously.15 From each plate, directed migration (migration
toward indicated concentrations of fMLP) and spontaneous migration
(adjacent wells without chemoattractant) were measured.
PMN transmigration assay
The PMN transmigration assay has been previously detailed12 and was
modified to accommodate endothelia, epithelia, or matrix substrates. For
experimental treatment, PMNs were pre-exposed to wortmannin (Calbiochem, La Jolla, CA), LY294002 (Calbiochem), piceatannol (Biomol,
Plymouth Meeting, PA) or vehicle (concentration of carrier equivalent to
the highest concentration of reagent used) for 15 minutes at 25°C with
occasional mixing. Prior to addition of PMNs, confluent endothelial or
epithelial monolayers were washed free of media with Hanks balanced
saline solution (HBSS) containing Ca⫹⫹ and Mg⫹⫹. Transmigration assays
were performed by the addition of pretreated PMNs to the upper chambers
after chemoattractant (10 nM fMLP unless otherwise noted) was added to
the opposing (lower) chambers. At time zero, 1 ⫻ 106 PMNs were added
and transmigration was allowed to proceed for 30 minutes at 37°C.
Transmigration was quantified by assaying for the PMN azurophilic granule
marker myeloperoxidase (MPO) as described previously.16,17 In subsets of
experiments, we assessed PMN migration across matrix-coated substrates.
To circumvent issues of matrix-integrin ligand redundancies,18 a general
matrix was established. Acellular, matrix-coated permeable support substrates were generated as follows. Bare polycarbonate permeable supports
(Corning-Costar, Cambridge, MA, 5␮m pore size) were precoated for 30
minutes with 50␮L 0.1% gelatin (derived from porcine skin, 175 Bloom,
Attachment Factor, Cascade Biologics) followed by addition of media
(containing 10% newborn calf serum, Gibco, Grand Island, NY) for an
additional 30 minutes. Inserts were rinsed in HBBS and PMN transmigration was assessed using similar conditions as described above.
Antibodies
Functionally inhibitory monoclonal antibodies (mAbs) were as follows:
␤1-integrin19 (clone LM534, obtained from Chemicon International, Temecula, CA), ␤2-integrin20 (clone 44a, obtained from ATTC), and ␤3integrin21 (clone AP3, obtained from GTI, Brookfield WI). A control mAb
directed against MHC class I22 (clone W6/32, obtained from ATTC) was
used as a nonfunctional PMN binding control. All mAbs were diluted to
indicated concentrations in HBSS (containing Ca⫹⫹ and Mg⫹⫹, with 10
mM Hepes, pH 7.4, Sigma, St Louis, MO) during functional assays.
Immunoprecipitation/Western blotting
The PMNs (1 ⫻ 107 PMN/well) were attached to a matrix substrate
prepared on 6-well, tissue culture-treated plastic plates (Corning-Costar).
Plates were centrifuged at 400g to uniformly settle PMNs onto the matrix
substrate, followed by addition of fMLP (final concentration 10 nM) for
indicated periods. Cells were lysed (1% [vol/vol] Triton X-100, 25 mM
Tris/HCl, pH 8.1, 5 mM EDTA, 10 ␮g/mL aprotinin, 100␮g/mL phenylmethylsulfonyl fluoride [PMSF], and 10␮g/mL chymostatin) and debris re-
moved by centrifugation. Lysates were precleared with 50 ␮L preequilibrated protein-G Sepharose (Pharmacia, Uppsala, Sweden).
Immunoprecipitation of ␤3-integrin was performed by addition of mAb
AP3 (5␮g/mL) followed by addition of 50 ␮L pre-equilibrated protein-G
Sepharose and overnight incubation. Immunoprecipitates were washed 3
times in lysis buffer, boiled in nonreducing sample buffer (2.5% sodium
dodecyl sulfate [SDS], 0.38 M Tris pH 6.8, 20% glycerol, and 0.1%
bromophenol blue), separated by nonreducing SDS-polyacrylamide gel
electrophoresis (SDS-PAGE), transferred to nitrocellulose, and blocked
overnight in blocking buffer. Primary antibody to phosphotyrosine (clone
PY20 from Transduction Labs, Lexington, KY) or anti-p72syk (rabbit
polyclonal generated against amino terminal peptide, Santa Cruz Biotechnology, Santa Cruz, CA), as indicated, was added for 3 hours followed by
wash and addition of species-matched, peroxidase-conjugated secondary
antibody (Cappel, West Chester, PA). Labeled proteins were visualized by
enhanced chemiluminescence (ECL; Amersham, Arlington Heights, IL).
HIV-tat peptide treatment of PMNs
Peptides corresponding to amino acids encompassing ␤3-integrin cytoplasmic tail tyrosine at position 747 (sequence ANNPLYKEATS, where bold
indicates tyrosine 747; single-letter amino acid codes), tyrosine at position
759 (sequence FTNITYRGT, where bold indicates tyrosine 759) or a
scrambled peptide control (SPLAQAVRSSSR) were synthesized (Synpep,
Dublin, CA) with the HIV-tat peptide as an N-terminal leader sequence to
facilitate loading into intact cells.23 Peptides were solubilized in DMSO at
stock concentrations of 50 mM and diluted in HBBS. PMNs were
preincubated with indicated concentrations of peptide or vehicle control
(DMSO at 1:1000 dilution) for 20 minutes at 25°C prior to addition to
matrix substrates in a gradient of fMLP. Transmigration was assessed as
described above.
For localization of HIV-tat peptides, PMNs were attached to coverslips
(2.5 ⫻ 103 PMN/mL, 30 minutes, 37°C), loaded with HIV-tat peptides (50
␮M in HBSS, 30 minutes, 37°C), washed with HBSS, fixed with
paraformaldehyde (1% wt/vol for 10 minutes), and permeabilized or not, as
indicated, with Triton X-100 (0.2% vol/vol for 5 minutes). Monolayers
were then incubated for 1 hour with anti–HIV-tat mAb (a kind gift from Dr
Eric Vives, Institut de Genetique Moleculaire, Montpellier, France, used at
1␮g/mL)24 prepared in 1% normal goat serum. After washing, the sections
were then incubated with fluorescein isothiocyanate (FITC)-conjugated
secondary antibody (Cappel, 1␮g/mL) for 30 minutes. The sections were
then mounted in phosphate-buffered saline (PBS)–polyvinylalchohol and
viewed with a fluorescence microscope (Olympus BH2, Melville, NY). As
a control for background labeling, control sections were incubated with
secondary antibody only.
To investigate the phosphorylation of synthetic ␤3-integrin HIV-tat
peptides, high-performance liquid chromatography (HPLC) was used as
described before.25 Briefly, PMNs were loaded with peptide (50 ␮M in
HBSS) and brought to 37°C in the presence of 10 nM fMLP; the reaction
was allowed to proceed for 15 minutes (optimized from pilot experiments)
before termination by snap freezing in liquid nitrogen. Lysate/peptide mixes
were thawed and fractionated through molecular weight cutoff filters (⬍ 5
kd), and the profile of HIV-tat phosphopeptides was determined by HPLC.
Peptide levels were measured with a 10%:90% H2O/CH3CN gradient (30
minutes) mobile phase (1 mL/min) on a reverse-phase HPLC column (Luna
5␮ C18, 150X 4.6 mm, Phenomonex, Torrence, CA). Absorbance was
monitored at 220 nm. UV absorption spectra were collected and peptides
were identified by their chromatographic elements (retention time, UV
spectra, and coelution with standards).
Data presentation
Time-course data and concentration responses were compared by analysis
of variance (ANOVA) for significance and individual comparisons were
made by Student t test. All results are presented as the mean and SEM of
n experiments.
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
BLOOD, 15 MAY 2001 䡠 VOLUME 97, NUMBER 10
Results
PI3K inhibitors promote transmigration
As an initial series of experiments, we examined whether inhibition
of PI3K influences PMN migration. Pretreatment of PMNs with the
potent and irreversible PI3K inhibitor wortmannin (10 nM) significantly inhibited PMN chemotaxis (Figure 1A, P ⬍ .01 by ANOVA)
as well as spontaneous migration (Figure 1B, P ⬍ .01 by ANOVA)
under agarose.
We next addressed whether similar observations were apparent
in a more physiologically relevant setting (PMN transendothelial
and epithelial migration). As shown in Figure 1C, and in stark
contrast to results observed under agarose, wortmannin increased
fMLP-stimulated PMN transendothelial and transepithelial migration in a concentration-dependent manner (median effective concentration [EC50] ⬃10 nM, P ⬍ .01 by ANOVA), with concentrations
as low as 1 nM resulting in a significant increase (P ⬍ .05).
Subsequent experiments revealed that endothelial cells were not
necessary in this response and that PI3K inhibitors promote PMN
transmigration across matrix substrates to a similar extent as in the
presence of endothelial cells (14 ⫾ 0.5-fold increase over control,
Figure 1D). In addition, these observations were not specific for
fMLP, because PMN transmatrix migration in response to LTB4 (10
nM gradient) was similarly increased by inhibition of PI3K
(maximal 6 ⫾ 0.4-fold increase with 100 nM wortmannin over
untreated controls, P ⬍ .001). Importantly, the presence of a
chemotactic gradient was necessary, since no differences between
wortmannin-treated and untreated PMNs were evident in the
absence of fMLP (data not shown). Moreover, these observations
were not specific for wortmannin and were also evident using the
Figure 1. Inhibition of PMN PI3K promotes transendothelial, transepithelial,
and transmatrix migration. Isolated human PMNs were examined for chemotaxis
(A) or spontaneous migration (B) under agarose following pre-exposure of PMNs to
vehicle (f) or wortmannin (o, 10 nM, 15 minutes, 22°C) toward indicated concentrations of fMLP. Data are derived from 4 separate experiments with results expressed
as mean ⫾ SEM distance migrated. In panels C and D, PMNs were pre-exposed to
indicated concentrations of wortmannin and assessed for fMLP-stimulated (10⫺8 M)
transmigration across confluent endothelia (p, C) or epithelia (u, C) or across
matrix-coated substrates (D). Transmigrated PMNs were quantified by determination
of MPO content following termination of the assay. Data are derived from 8 to 12
monolayers in each condition from at least 3 separate experiments with results
expressed as mean ⫾ SEM number transmigrating PMNs .
PI3 KINASE REGULATION OF ␤3-INTEGRIN
3253
less potent PI3K inhibitor LY294002 (EC50 ⬃2 ␮M with maximal
7 ⫾ 0.3-fold increase over untreated PMNs , P ⬍ .01 by ANOVA).
Original studies with this transmigration model have indicated
that PMNs do not degranulate during transit through cell monolayers.16,17,26 Nonetheless, recent studies have suggested that integrin-mediated adhesion may potentate PMN degranulation.27
Because our transmigration assay uses MPO as a biochemical
marker, we ruled out that these findings with wortmannin could
result from differences in MPO content. To do this, we examined
PMN MPO content in the presence and absence of wortmannin
before and after transmigration. Following transmigration, PMNs
were collected and quantified by hemocytometer and MPO content
was compared to resting PMNs. This analysis revealed no differences between wortmannin treatment groups (mean OD absorbance units of 0.26 ⫾ 0.01 and 0.27 ⫾ 0.02/104 PMNs in the
presence and absence of 10 nM wortmannin, respectively, n ⫽ 4,
P ⫽ not significant), or between PMNs that had migrated across
endothelial (mean OD absorbance units of 0.25 ⫾ 0.03 and
0.27 ⫾ 0.01/104 PMNs before and after transmigration, respectively, n ⫽ 4, P ⫽ not significant) or epithelial monolayers (mean
OD absorbance units of 0.23 ⫾ 0.03 and 0.28 ⫾ 0.04/104 PMNs
before and after transmigration, respectively, n ⫽ 4, P ⫽ not
significant), suggesting that MPO is an appropriate biochemical
marker for analysis of PMN transmigration. Taken together, we
conclude that inhibition of PI3K promotes chemoattractantstimulated PMN transmatrix migration.
␤3-Integrin dependence
We next determined whether PI3K-dependent promotion of PMN
transmigration required surface integrin activity. Initial insight was
gained by the observation that matrix proteins were absolutely
required for PI3K-dependent increases in PMN transmigration.
Indeed, although wortmannin significantly increased PMN migration across a general matrix-coated substrate (Figure 1D), transmigrations of wortmannin-treated PMNs across bare (ie, no matrix
coating) polycarbonate substrates were either equivalent to control
or even decreased at high concentrations of wortmannin (eg,
31% ⫾ 7% decrease at 500 nM, P ⬍ .05); suggesting the necessity
for matrix ligands in wortmannin-mediated PMN responses. Based
on these data, and numerous previous observations that integrins
bind matrix components,2 we screened a panel of functionally
inhibitory mAbs directed against ␤1-, ␤2-, or ␤3-integrins (Figure
2). Compared to binding control mAb against major histocompatibility complex (MHC) class I, anti-␤1 mAb did not influence PMN
transmatrix migration in the presence or absence of any wortmannin concentration tested (P ⫽ not significant for all). Anti–␤2integrin mAb blocked PMN transmigration by as much as
88% ⫾ 6.0%, with approximately equal potency in the presence
and absence of wortmannin. These findings that PI3K inhibition
does not differentially influence ␤2-integrin function are consistent
with previous observations.28
In contrast to these findings with anti–␤1- or ␤2-integrin
antibodies, differential responses were evident in the presence of
functionally inhibitory anti–␤3-integrin antibodies. Although anti-␤3
mAb significantly blocked PMN transmatrix migration in untreated
PMNs (79% ⫾ 5.2%, compared to binding controls, P ⬍ .01), the
inhibitory influence of anti-␤3 mAb was lost under conditions of
inhibited PI3K activity (P ⫽ not significantly different from binding control in PMNs pretreated with either 10 or 100 nM
wortmannin). The anti–␤3-integrin dose-response curve is shown
in Figure 3 and demonstrates a nearly complete loss of anti-␤3
inhibition in PMNs pre-exposed to 100 nM wortmannin (P ⬍ .01
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
3254
BRUYNINCKX et al
BLOOD, 15 MAY 2001 䡠 VOLUME 97, NUMBER 10
by SDS-PAGE and Western blots were probed with antiphosphotyrosine. As shown in Figure 4, PI3K inhibition blocked fMLPstimulated tyrosine phosphorylation in a time-dependent (10 nM
fMLP) and concentration-dependent fashion. In the presence of
wortmannin, a slight increase in tyrosine phosphorylation was
consistently observed at 10 minutes, although analysis beyond this
time point did not reveal additional phosphorylation (data not
shown). These findings were not a result of decreased PMN
adhesion to matrix substrates, as determined morphologically and
by biochemical quantification of PMN adhesion (using the marker
MPO, data not shown). Moreover, these findings were not a result
of decreased PMN ␤3-integrin content (refer to control ␤3-integrin
Western blots adjacent to phosphotyrosine blots in Figure 4). These
data suggest a role for PI3K in chemoattractant-stimulated ␤3integrin tyrosine phosphorylation.
Figure 2. Role of integrins in PI3K-mediated increases in PMN transmatrix
migration. Isolated human PMNs were pre-exposed to indicated concentrations of
wortmannin and/or antibodies (10 ␮g/mL for each) directed against MHC class I (f),
anti–␤1-integrin (p), anti–␤2-integrin (u), or anti–␤3-integrin (z) followed by examination of fMLP-stimulated (10⫺8 M) transmigration across matrix-coated substrates (A).
Transmigrated PMNs were quantified by determination of MPO. Panel B represents
data converted to percent inhibition relative to control binding antibody. Data are
derived from 9 to 12 monolayers in each condition from at least 3 separate
experiments with results expressed as mean ⫾ SEM number transmigrating PMNs .
by ANOVA). Important in this regard, wortmannin did not
influence ␤3-integrin expression (assessed by flow cytometry and
immunoprecipitation of surface biotinylated PMN ␤3, data not
shown). These data suggest that blockade of PI3K inhibits surface
␤3 function during stimulated PMN-matrix interactions.
PI3K inhibition blocks ␤3-integrin tyrosine phosphorylation
The above results indicate that PI3K inhibitors functionally block
␤3-integrin during PMN interactions with matrix. The short cytoplasmic tail of ␤3-integrin contains tyrosine residues at positions
747 and 759, which serve as functional kinase substrates.29 Thus,
we next addressed whether PI3K was important in ␤3-integrin
tyrosine phosphorylation. To do this, PMNs were attached to a
matrix substrate and activated for various periods of time with
fMLP (10 nM). The ␤3-integrin immunoprecipitates were resolved
Figure 3. PI3K-mediated uncoupling of ␤3-integrin–mediated inhibition of PMN
transmatrix migration; antibody dose response. Isolated human PMNs were
pre-exposed to wortmannin (100 nM, 䡺) or vehicle (f) and/or indicated concentrations of antibody directed against ␤3-integrin followed by examination of fMLPstimulated (10⫺8 M) transmigration across matrix-coated substrates (A). Transmigrated PMNs were quantified by determination of MPO content following termination
of the assay. Panel B represents data converted to percent inhibition relative to no
antibody controls. Data are derived from 8 to 10 monolayers in each condition from at
least 3 separate experiments with results expressed as mean ⫾ SEM number
transmigrating PMNs .
Role for ␤3-integrin cytoplasmic tail
Previous studies have indicated that both of the ␤3-integrin
cytoplasmic tail tyrosines (at positions 747 and 759) can be
functionally important, depending on the cell type.29 Thus, we
addressed whether functional inhibition of ␤3-integrin cytoplasmic
tail tyrosines would result in responses similar to PI3K inhibition
(ie, enhanced PMN transmatrix migration). To do this, intact PMNs
were loaded with peptides corresponding to amino acids spanning
the ␤3-integrin cytoplasmic tail tyrosine at position 747 (ANNPLYKEATS) or position 759 (FTNITYRGT). Loading of intact
PMNs was accomplished by synthesizing peptides with the 12
amino acid HIV-tat peptide motif (YGRKKRRQRRRG) at the
N-terminus, a maneuver that for unknown reasons significantly
facilitates peptide uptake across intact plasma membranes.23 Incubation of HIV-tat–linked peptides with PMNs followed by assessment of PMN transmatrix migration revealed a predominant role
for tyrosine 759 (Figure 5). Indeed, the HIV-tat-FTNITYRGT
peptide increased PMN migration in a concentration-dependent
manner with a 97% ⫾ 6.2% increase at 50 ␮M. HIV-tat–linked
peptide corresponding to tyrosine 747 also increased transmigration (maximal 44% ⫾ 0.5% at 50 ␮M, P ⬍ .025), but to a lesser
Figure 4. Inhibition of PI3K blocks fMLP-activated ␤3-integrin tyrosine phosphorylation. Isolated human PMNs were pre-exposed to vehicle or wortmannin (3 nM in
the top panel or indicated concentration in the bottom panel) followed by adhesion to
matrix substrates. Adherent PMNs were exposed to fMLP (10 nM) for indicated
periods of time (top panel) or for 10 minutes (bottom panel). Cells were lysed and
␤3-integrin was immunoprecipitated and resolved by SDS-PAGE. Tyrosine phosphorylation was assessed by immunoblot. Data are representative from 3 separate
experiments.
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
BLOOD, 15 MAY 2001 䡠 VOLUME 97, NUMBER 10
PI3 KINASE REGULATION OF ␤3-INTEGRIN
3255
Figure 5. HIV-tat peptide–mediated loading of peptides corresponding to ␤3-integrin cytoplasmic tail
promotes PMN transmatrix migration. In panel A,
isolated human PMNs were pre-exposed to indicated
concentrations of HIV-tat peptides motifs corresponding
to ␤3-integrin cytoplasmic tyrosine 747 (closed circles),
tyrosine 759 (closed squares), or scrambled peptide for
20 minutes at room temperature followed by examination
of fMLP-stimulated (10⫺8 M) transmigration across matrixcoated substrates. The immunofluorescence insets for
each peptide localize HIV-tat peptide within permeabilized (P) and nonpermeabilized (NP) PMNs following
peptide loading (see “Materials and methods”). Arrowheads demonstrate predominant localization of peptides
at the cytoplasmic face of PMNs. Transmigrated PMNs
were quantified by determination of MPO. Data are
derived from 9 monolayers in each condition from 4
separate experiments with results expressed as mean ⫾
SEM number transmigrating PMNs. (B) The ␤3-integrin
cytoplasmic sequence is a kinase substrate. Coincubation of HIV-tat peptides coupled to a scrambled peptide
(top panel), the FTNITYRGT peptide (middle panel), or
the ANNPLYKEATS peptide (bottom panel) with fMLPactivated PMNs were analyzed for phosphorylation by
HPLC (representative tracings of 2 experiments are
shown). Arrows indicate position of unphosphorylated or
phosphorylated standards.
extent than those targeting tyrosine 759 (P ⬍ .025 compared to
those targeting tyrosine 747). A scrambled peptide synthesized with
HIV-tat linker was used as a control for these experiments and did
not influence transmigration at any concentration tested (P ⫽ not
significant at all concentrations). As a control for these experiments, localization of HIV-tat peptide in adherent, permeabilized
PMNs revealed equivalent loading between different peptides
(Figure 5, inset), with an obvious increase in membrane localization of peptides targeting the ␤3-integrin cytoplasmic tail (ANNPLYKEATS and FTNITYRGT) but not in the scrambled peptide.
Such data suggest that these peptides localize to an appropriate
compartment within the cell (ie, membrane proximal) and that
differential loading does not explain the functional results shown in
Figure 5A.
Figure 6. Role of p72syk in PI3K-mediated uncoupling of ␤3-integrin–mediated
transmatrix migration. (A) Isolated human PMNs were pre-exposed to vehicle
(⫺Wort) or wortmannin (⫹Wort, 100 nM) and adhered to matrix substrates (top 2
blots) or left in suspension. PMNs were exposed to fMLP (10 nM) for indicated times
(0-30 minutes). Cells were lysed and ␤3-integrin was immunoprecipitated and
resolved by SDS-PAGE. Association with p72syk was assessed by immunoblot . Data
are representative from 3 separate experiments. (B) PMNs were pre-exposed to
vehicle (C), wortmannin (WT, 100 nM) or indicated concentrations of the p72syk
inhibitor piceatannol for 20 minutes at room temperature followed by examination of
fMLP-stimulated (10⫺8 M) transmigration across matrix-coated substrates. Transmigrated PMNs were quantified by determination of MPO content following termination
of the assay. Data are derived from 8 to 10 monolayers in each condition from 3
separate experiments with results expressed as mean ⫾ SEM number transmigrating PMNs .
As additional evidence that these HIV-tat peptides are functional, we examined the direct phosphorylation of synthetic
phosphopeptides by fMLP-stimulated PMNs using a recently
described HPLC assay. As shown in Figure 5B, this analysis
revealed significant phosphorylating activity of the ANNPLYKEATS
peptide (mean 49% conversion in 5 minutes, n ⫽ 2) as well as the
FTNITYRGT peptide (mean 68.6% conversion in 5 minutes,
n ⫽ 2). These data indicate that synthetic peptides targeting the
␤3-integrin cytoplasmic tail serve as substrates for tyrosine kinase(s). No detectable phosphorylation was evident in scrambled
peptide exposed to similar conditions. Taken together, these data
suggest that inhibition of ␤3-integrin cytoplasmic tail tyrosines
results in a similar functional response as that of PI3K inhibition.
Role of p72syk in PMN transmatrix migration
Based on the observations that PI3K inhibition blocks surface
␤3-integrin function, that inhibitors of PI3K block tyrosine phosphorylation, and that HIV-tat peptides targeting inhibition of
␤3-integrin cytoplasmic tyrosines promote PMN transmatrix migration, we sought to gain insight into kinases important in tyrosine
phosphorylation. Recent studies indicated that p72syk, a kinase
expressed predominantly in myeloid-derived cells,30 coordinates
integrin function in platelets.31 Thus, we examined the influence of
PI3K inhibition on p72syk interactions with ␤3-integrin in PMNs .
As shown in Figure 6 (left panel), activation of matrix-adherent
PMNs with fMLP (10 nM), and subsequent ␤3-integrin immunoprecipitation/p72syk Western blot resulted in a time-dependent association between p72syk and ␤3-integrin. This response was evident by 5
minutes of activation and dominant by 30 minutes. Similar analysis
from PMNs pretreated with wortmannin (100 nM) revealed a
nearly complete abolition in p72syk–␤3-integrin association (Figure
6). Importantly, as shown in Figure 6, p72syk–␤3-integrin association was not observed in nonadherent PMNs exposed to fMLP (10
nM), suggesting that PMN adherence to matrix substrates provides
an “outside-in” signal for such associations. Such findings were not
a result of decreased PMN ␤3-integrin content (see control
␤3-integrin Western blots adjacent to phosphotyrosine blots in
Figure 6).
Based on these data, we thus predicted that the relatively
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
3256
BRUYNINCKX et al
selective p72syk inhibitor piceatannol32 should block surface ␤3integrin function in a similar manner as PI3K inhibition and should
manifest as increased PMN transmatrix migration (Figure 1). As
shown in Figure 6 (right panel), pretreatment of PMNs with
piceatannol, but not vehicle control, resulted in a concentrationdependent increase (P ⬍ .025 by ANOVA) in PMN transmatrix
migration (5.2 ⫾ 0.3-fold increase at 30 ␮M piceatannol). We next
compared the influence of piceatannol on PMN transendothelial
and transepithelial migration with bare insert migration. This
analysis revealed that piceatannol (30 ␮M pretreatment of PMNs
for 30 minutes) significantly enhanced PMN migration across
endothelial (210% ⫾ 65% increase over untreated controls, n ⫽ 3,
P ⬍ .025) and epithelial monolayers (160% ⫾ 50% increase over
untreated controls, n ⫽ 3, P ⬍ .05) but did not influence migration
across bare inserts (10% ⫾ 2% increase over untreated controls,
n ⫽ 3, P ⫽ not significant), thus confirming our hypothesis that
matrix ligands are critical for this response. These data suggest that
the p72syk-selective inhibitor piceatannol, like the PI3K inhibitor
wortmannin, functionally blocks ␤3-integrin function during PMN
interactions with matrix. Taken together, such data provide evidence that PI3K modulation of surface ␤3-integrin activity functions as an endogenous “braking mechanism” during chemoattractant-stimulated PMN migration across matrix substrates.
Discussion
The process of PMN transmigration occurs through a series of steps
orchestrated by the interplay of surface adhesion molecules and
soluble mediators derived from the local milieu. Following transendothelial migration, PMNs encounter subendothelial matrix substrates, and at present, the molecular mechanism(s) used by PMNs
to traverse the extracellular matrix are not fully understood. In
these studies, we hypothesized a role for PMN PI3K in regulation
of PMN trafficking through vascular endothelial cells. We observed
a seemingly paradoxical enhancement in PMN migration under
conditions of inhibited PI3K activity, and a series of experiments
identified PI3K uncoupling of ␤3-integrin as a mechanism for this
response. These results unveil 2 novel concepts with regard to basic
mechanisms of leukocyte function. First, PI3K is central to
chemoattractant-stimulated PMN-matrix interactions via modulation of the cytoplasmic tail of ␤3-integrin, and second, these studies
define PMN ␤3-integrins as an endogenous regulatory mechanism
during leukocyte trafficking, the uncoupling of which results in
enhanced leukocyte movement though matrix substrates.
Recent studies have clearly defined PI3K as an important
molecule in acute inflammatory processes. Transgenic knockout
mice lacking the p110␥ subunit of PI3K manifest defects in some
PMN responses, including protein kinase Akt activation, formation
of 3⬘-phosphorylated lipids, superoxide anion generation, and
chemotaxis,6,7,33 and that the p110␥ subunit may be the major PI3K
activated during chemoattractant stimulation of PMNs.34 Although
it is difficult to compare our results to these in the mouse, consistent
with our studies using the under agarose assay and bare insert
experiments, decreased in vitro chemotaxis was observed in PI3K
knockout mice PMNs when migrating across polycarbonate membranes not imprinted with matrix proteins.6,7 It should also be noted
that some controversy exists regarding the immediate role of PI3K
in PMN migration on nonmatrix substrates. Although we (see
“Results”) and others35-38 have observed that inhibition of PI3K
decreases PMN motility on nonphysiologic substrates (ie, plastic),
other studies have suggested only partial or selective inhibition39-41
BLOOD, 15 MAY 2001 䡠 VOLUME 97, NUMBER 10
or no influence of PI3K inhibitors on PMN chemotaxis.42 Similar to
our experiments with PI3K inhibition (see “Results”), no differences were observed in matrix-mediated adhesion in PMNs derived
from PI3K-deficient mice.6,7 Thus, it is likely that significant
variability exists between these different methods and that mechanisms to explain similarities and differences are likely complicated
and difficult to resolve. Additionally, it is difficult to directly
compare our results to those in the p110␥ null mice, because human
PMNs express all 4 known PI3K subunits (␣, ␤, ␥, and ␦) and
wortmannin inhibits all forms of PI3K.43 Nonetheless, our present
observations using more physiologic substrates (endothelia, epithelia, or matrix) indicate the necessity for matrix ligands as a “second
signal” in PMN motility under conditions of inhibited PI3K, as
would be the case in vivo. Of note in this regard, in vivo defects
associated with PI3K deficiency included neutrophilia and decreased peritoneal elicitation of PMNs and macrophages.6,7 Although the role of ␤3-integrins in leukocyte extravasation has only
been indirectly addressed in vivo, the inflammatory phenotype is
clearly similar to that of PI3K deficiency. Indeed, Lindberg and
colleagues demonstrated that targeted disruption of integrinassociated protein (IAP) results in defective PMN accumulation
and PMNs that show impaired ␤3-integrin–dependent ligand binding.44 Thus, it is possible that PI3K-coupled ␤3-integrin could
contribute to the inflammatory defect associated with the PI3K-null
phenotype during inflammatory processes.
Our studies provide unique insight into a prominent role for
␤3-integrins in PMN interactions with postendothelial matrix
substrates. It is unlikely that PMN ␤3-integrins directly interact
with endothelial surface proteins because stripping endothelial
cells did not influence PMN migration (see “Results”). Rather, it
appears that PMNs use ␤3-integrins as a postendothelial, matrix
adhesive mechanism, likely via RGD-containing peptides.45 Previous studies addressing the role of ␤3-integrins in leukocyte
transmigration have focused primarily on monocytes,46 and revealed that the ␤3-chain of ␣v␤3 enhanced monocyte migration
through endothelia via a cooperative mechanism involving ␤2- and
␤3-integrins. These studies with monocytes did not address whether
such responses could be mediated by direct ␤3-integrin–matrix
interaction, but it is unlikely that a similar mechanism is important
in PMN ␤3-integrin function. For example, although anti–␤2integrin mAb potently blocks PMN transmatrix migration, no
differences were evident in ␤2-integrin function under conditions
of PI3K inhibition (ie, wortmannin did not block this response,
Figure 2). These findings are consistent with previous studies
demonstrating no impact of PI3K inhibitors on ␤2-integrin function
in PMNs.28 Similarly, we did not observe an appreciable influence
of functionally inhibitory anti–␤1-integrin antibodies in the presence or absence of PI3K inhibitors. Taken together, these data
identify ␤3-integrin as an important, and previously unappreciated,
molecule in the PMN transmigratory cascade. Of note in this
regard, we have not characterized the matrix in detail. The matrices
used here required precoating with both gelatin and newborn
calf serum (see “Materials and methods”), and because calf serum
contains relatively high levels of vitronectin and fibinogen,47
PMN ␤3-integrins presumably use these serum factors as ligands
for transmatrix migration. We have not, however, specifically
determined the immediate role of each serum component in
this response.
The present studies provide direct evidence that cytoplasmic tail
tyrosine residue 759, and to a lesser extent residue 747, mediate
PMN ␤3-integrin function and that the tyrosine kinase p72syk
contributes to these observations in this transmatrix migration
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
BLOOD, 15 MAY 2001 䡠 VOLUME 97, NUMBER 10
PI3 KINASE REGULATION OF ␤3-INTEGRIN
model. Because PMNs are not amenable to transfection or mutationbased experiments, HIV-tat peptide-facilitated loading of consensus peptides corresponding to the cytoplasmic domains were used.
These results revealed that peptides targeting tyrosine residue 759
were substantially more potent (10- to 50-fold) than those targeting
residue 747, which is within the well conserved NPXY motif found
in a number of integrins,48 including both ␤1- and ␤3-integrin
studied here (Figures 2 and 3). Important in this regard, the
␤2-integrin cytoplasmic tail does not express a membrane proximal
NPXY motif,29 and although anti–␤2-integrin antibodies significantly inhibit PMN transmatrix migration (Figure 2), no differences were observed with PI3K inhibition.
Consistent with previous mutational analysis,49 our data using
HIV-tat peptide-coupled consensus motifs suggest that tyrosine
759 may be relatively more important for cytoskeletal linkage than
tyrosine residue 747, and that phosphorylation of the membrane
proximal position 747 may mediate a generalized postligand
binding event common to a number of integrin ␤ chains.48 It is also
likely that the membrane distal cytoplasmic domain consensus
(NITY) is a better substrate for tyrosine kinase p72syk and functions
directly through PI3K signaling, because p72syk has a demonstrated
importance in the sustained phosphorylation of B-cell Akt following PI3K activation.50 Moreover, formation of the functional
p72syk–␤3-integrin complex likely requires integrin ligation because the association was not evident in nonadherent PMNs, and
the relatively specific p72syk inhibitor piceatannol induced a
functional response consistent with PI3K uncoupling of ␤3-integrin
3257
(ie, increased fMLP-stimulated transmigration). These findings
support those of Sarkar and coworkers31 and Gao and colleagues,51
which revealed that p72syk activation required ␤3-integrin occupancy. Thus, it is possible that loading PMNs with peptides
corresponding to tyrosine residue 759 indirectly regulates Akt
phosphorylation through p72syk-␤3 interactions. It is also possible
that p72syk is a substrate for Akt. The minimal sequence for efficient
phosphorylation by Akt is RXX(S/T)X* where R is arginine, X any
amino acid, S/T is serine/threonine, and X* is a hydrophobic
residue.43 In fact, our examination of the published p72syk amino
acid sequence52 revealed 3 previously unappreciated binding sites
for Akt (RVLTV at amino positions 253-257, RIKSY at positions
292-296, and RKSSP at positions 304-308), providing at least the
possibility that p72syk is an Akt substrate.
In conclusion, as PMNs respond to chemoattractants and
surface molecule engage extracellular matrix ligands, phosphorylation of ␤3-integrin in association with p72syk defines a PI3Kdependent modulation of PMN transmatrix migration. Future
studies are necessary to define the exact sequence of activation and
the coordination of “outside-in” and “inside-out” components for
this modulation.
Acknowledgment
We authors wish to thank Ms Kristin Synnestvedt for technical
assistance.
References
1. Gonzalez-Amaro R, Sanchez-Madrid F. Cell adhesion molecules: selectins and integrins. Crit
Rev Immunol. 1999;19:389-429.
12. Colgan, SP, Dzus AL, Parkos CA. Epithelial exposure to hypoxia modulates neutrophil transepithelial migration. J Exp Med. 1996;184:1003-1015.
2. Shimizu Y, Rose DM, Ginsberg MH. Integrins in
the immune system. Adv Immunol. 1999;72:325380.
13. Colgan SP, Gasper PW, Thrall MA, Boone TC,
Blancquaert A-MB, Bruyninckx WJ. Neutrophil
function in normal and Chediak-Higashi syndrome cats following administration of recombinant canine granulocyte colony-stimulating factor.
Exp Hematol. 1992;20:1229-1234.
3. Calderwood DA, Shattil SJ, Ginsberg MH. Integrins and actin filaments: reciprocal Regulation of
cell adhesion and signaling. J Biol Chem. 2000;
275:22607-22610.
4. Schoenwaelder SM, Burridge K. Bidirectional signaling between the cytoskeleton and integrins.
Curr Opin Cell Biol. 1999;11:274-286.
5. Servant G, Weiner OD, Herzmark P, Balla T, Sedat JW, Bourne HR. Polarization of chemoattractant receptor signaling during neutrophil chemotaxis. Science. 2000;287:1037-1040.
6. Sasaki T, Irie-Sasaki J, Jones RG, et al. Function
of PI3Kgamma in thymocyte development, T cell
activation, and neutrophil migration. Science.
2000;287:1040-1046.
7. Hirsch E, Katanaev VL, Garlanda C, et al. Central
role for G protein-coupled phosphoinositide 3-kinase gamma in inflammation [see comments].
Science. 2000;287:1049-1053.
8. Toker A, Cantley LC. Signalling through the lipid
products of phosphoinositide-3-OH kinase. Nature. 1997;387:673-676.
9. Li J, Fenton RA, Cutler BS, Dobson JG. Adenosine enhances nitric oxide production by vascular
endothelial cells. Am J Physiol. 1995;269:C519–
C523.
10. Lennon PF, Taylor CT, Stahl GL, Colgan SP. Neutrophil-derived 5⬘-adenosine monophosphate
promotes endothelial barrier function via CD73mediated conversion to adenosine and endothelial A2B receptor activation. J Exp Med. 1998;188:
1433-1443.
11. Madianos PN, Papapanou PN, Sandros J. Porphyrimonas gingivalis infection of oral epithelium
inhibits neutrophil transepithelial migration. Infect
Immun. 1997;65:3983-3990.
14. Nelson RD, Quie PG, Simmons RL. Chemotaxis
under agarose: a new and simple method for
measuring chemotaxis and spontaneous migration of human polymorphonuclear leukocytes and
monocytes. J Immunol. 1975;115:1650-1656.
15. Blancquaert, A-MB, Colgan SP, Bruyninckx
WJ. Chemotactic and chemokinetic activity of
Streptococcous faecalis culture supernatant for
equine neutrophils. Vet Immunol Immunopathol.
1988;19:285-297.
16. Parkos CA, Delp C, Arnaout MA, Madara JL.
Neutrophil migration across a cultured intestinal
epithelium: dependence on a CD11b/CD18-mediated event and enhanced efficiency in the physiologic direction. J Clin Invest. 1991;88:16051612.
17. Colgan SP, Parkos CA, Delp C, Arnaout MA,
Madara JL. Neutrophil migration across cultured
intestinal epithelial monolayers is modulated by
epithelial exposure to interferon-gamma in a
highly polarized fashion. J Cell Biol 1993;120:
785-795.
18. Lowell CA, Berton G. Integrin signal transduction
in myeloid leukocytes. J Leukoc Biol. 1999;65:
313-320.
19. Giancotti FG, Ruoslahti E. Elevated levels of the
alpha 5 beta 1 fibronectin receptor suppress the
transformed phenotype of Chinese hamster ovary
cells. Cell. 1990;60:849-859.
PJ. Amino acid 489 is encoded by a mutational
“hot spot” on the beta 3 integrin chain: the CA/TU
human platelet alloantigen system. Blood. 1993;
82:3386-3391.
22. Barnstable CJ, Bodmer WF, Brown G, et al. Production of monoclonal antibodies to group A
erythrocytes, HLA and other human cell surface
antigens-new tools for genetic analysis. Cell.
1978;14:9-20.
23. Schwarze SR, Dowdy SF. In vivo protein transduction: intracellular delivery of biologically active
proteins, compounds and DNA. Trends Pharmacol Sci. 2000;21:45-48.
24. Vives E, Brodin P, Lebleu B. A truncated HIV-1 Tat
protein basic domain rapidly translocates through
the plasma membrane and accumulates in the
cell nucleus. J Biol Chem. 1997;272:1601016017.
25. Taylor CT, Furuta GT, Synnestvedt K, Colgan SP.
Phosphorylation-dependent targeting of cAMP
response element binding protein to the ubiquitin/
proteasome pathway in hypoxia. Proc Natl Acad
Sci U S A. 2000;97:12091-12096.
26. Nash S, Stafford J, Madara JL. Effects of polymorphonuclear leukocyte transmigration on barrier function of cultured intestinal epithelial monolayers. J Clin Invest. 1987;80:1104-1113.
27. Rainger GE, Rowley AF, Nash GB. Adhesion-dependent release of elastase from human neutrophils in a novel, flow-based model: specificity of
different chemotactic agents. Blood. 1998;92:
4819-4827.
28. Capodici C, Hanft S, Feoktistov M, Pillinger MH.
Phosphatidylinositol 3-kinase mediates chemoattractant-stimulated, CD11b/CD18-dependent cellcell adhesion of human neutrophils: evidence for
an ERK-independent pathway. J Immunol. 1998;
160:1901-1909.
20. Todd RF, Nadler LM, Schlossman SF. Antigens
on human monocyte identified by monoclonal antibodies. J Immunol. 1981;126:1435-1442.
29. LaFlamme SE, Homan SM, Bodeau AL, Mastrangelo AM. Integrin cytoplasmic domains as
connectors to the cell’s signal transduction apparatus. Matrix Biol. 1997;16:153-163.
21. Wang R, McFarland JG, Kekomaki R, Newman
30. Turner M, Schweighoffer E, Colucci F, Di Santo
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
3258
BLOOD, 15 MAY 2001 䡠 VOLUME 97, NUMBER 10
BRUYNINCKX et al
JP, Tybulewicz VL. Tyrosine kinase SYK: essential functions for immunoreceptor signalling. Immunol Today. 2000;21:148-154.
31. Sarkar S, Rooney MM, Lord ST. Activation of integrin-beta3-associated syk in platelets. Biochem J.
1999;338:677-680.
32. Oliver JM, Burg DL, Wilson BS, McLaughlin JL,
Geahlen RL. Inhibition of mast cell Fc epsilon R1mediated signaling and effector function by the
Syk-selective inhibitor, piceatannol. J Biol Chem.
1994;269:29697-29703.
33. Li Z, Jiang H, Xie W, Zhang Z, Smrcka AV, Wu D.
Roles of PLC-beta2 and -beta3 and PI3Kgamma
in chemoattractant-mediated signal transduction.
Science. 2000;287:1046-1049.
34. Naccache PH, Levasseur S, Lachance G,
Chakravarti S, Bourgoin SG, McColl SR. Stimulation of human neutrophils by chemotactic factors
is associated with the activation of phosphatidylinositol 3-kinase gamma. J Biol Chem. 2000;275:
23636-23641.
35. Haribabu B, Zhelev DV, Pridgen BC, Richardson
RM, Ali H, Snyderman R. Chemoattractant receptors activate distinct pathways for chemotaxis and
secretion. Role of G-protein usage. J Biol Chem.
1999;274:37087-37092.
36. Knall C, Worthen GS, Johnson GL. Interleukin
8-stimulated phosphatidylinositol-3-kinase activity
regulates the migration of human neutrophils independent of extracellular signal-regulated kinase and p38 mitogen-activated protein kinases.
Proc Natl Acad Sci U S A. 1997;94:3052-3057.
37. Jing Q, Xin SM, Zhang WB, Wang P, Qin YW, Pei
G. Lysophosphatidylcholine activates p38 and
p42/44 mitogen-activated protein kinases in
monocytic THP-1 cells, but only p38 activation is
involved in its stimulated chemotaxis. Circ Res.
2000;87:52-59.
38. Siddiqui RA, English D. Phosphatidylinositol 3⬘kinase-mediated calcium mobilization regulates
chemotaxis in phosphatidic acid-stimulated human neutrophils. Biochim Biophys Acta. 2000;
1483:161-173.
39. Niggli V, Keller H. The phosphatidylinositol 3-kinase inhibitor wortmannin markedly reduces chemotactic peptide-induced locomotion and increases in cytoskeletal actin in human
neutrophils. Eur J Pharmacol. 1997;335:43-52.
40. Coffer PJ, Geijsen N, M’rabet L, et al. Comparison of the roles of mitogen-activated protein kinase kinase and phosphatidylinositol 3-kinase
signal transduction in neutrophil effector function.
Biochem J. 1998;329:121-130.
41. Xiao YQ, Minami K, Mue S, Ohuchi K. Pharmacological analysis of protein kinases responsible for
chemotaxis of rat peritoneal neutrophils. Eur
J Pharmacol. 1998;360:195-204.
42. Thelen M, Uguccioni M, Bosiger J. PI 3-kinasedependent and independent chemotaxis of human neutrophil leukocytes. Biochem Biophys Res
Commun. 1995;217:1255-1262.
45. Suehiro K, Plow EF. Ligand recognition by beta 3
integrins. Keio J Med. 1997;46:111-114.
46. Weerasinghe D, McHugh KP, Ross FP, Brown EJ,
Gisler RH, Imhof BA. A role for the alphavbeta3
integrin in the transmigration of monocytes. J Cell
Biol. 1998;142:595-607.
47. Grabham PW, Gallimore PH, Grand RJ. Vitronectin is the major serum protein essential for NGFmediated neurite outgrowth from PC12 cells. Exp
Cell Res. 1992;202:337-344.
48. Blystone SD, Williams MP, Slater SE, Brown EJ.
Requirement of integrin beta3 tyrosine 747 for
beta3 tyrosine phosphorylation and regulation of
alphavbeta3 avidity. J Biol Chem. 1997;272:
28757-28761.
49. Schaffner-Reckinger E, Gouon V, Melchior C,
Plancon S, Kieffer N. Distinct involvement of
beta3 integrin cytoplasmic domain tyrosine residues 747 and 759 in integrin-mediated cytoskeletal assembly and phosphotyrosine signaling.
J Biol Chem. 1998;273:12623-12632.
50. Gold MR, Scheid MP, Santos L, et al. The B cell
antigen receptor activates the Akt (protein kinase
B)/glycogen synthase kinase-3 signaling pathway
via phosphatidylinositol 3-kinase. J Immunol.
1999;163:1894-1905.
43. Vanhaesebroeck B, Alessi DR. The PI3K-PDK1
connection: more than just a road to PKB. Biochem J. 2000;3:561-576.
51. Gao J, Zoller KE, Ginsberg MH, Brugge JS, Shattil SJ. Regulation of the pp72syk protein tyrosine
kinase by platelet integrin alpha IIb beta 3. EMBO
J. 1997;16:6414-6425.
44. Lindberg FP, Bullard DC, Caver TE, Gresham
HD, Beaudet AL, Brown EJ. Decreased resistance to bacterial infection and granulocyte defects in IAP-deficient mice. Science. 1996;274:
795-798.
52. Law CL, Sidorenko SP, Chandran KA, et al. Molecular cloning of human Syk. A B cell proteintyrosine kinase associated with the surface immunoglobulin M-B cell receptor complex. J Biol
Chem. 1994;269:12310-12319.
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
2001 97: 3251-3258
doi:10.1182/blood.V97.10.3251
Phosphoinositide 3-kinase modulation of β3-integrin represents an
endogenous ''braking'' mechanism during neutrophil transmatrix migration
Walter J. Bruyninckx, Katrina M. Comerford, Donald W. Lawrence and Sean P. Colgan
Updated information and services can be found at:
http://www.bloodjournal.org/content/97/10/3251.full.html
Articles on similar topics can be found in the following Blood collections
Cell Adhesion and Motility (790 articles)
Hemostasis, Thrombosis, and Vascular Biology (2485 articles)
Phagocytes (969 articles)
Signal Transduction (1930 articles)
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of
Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.