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PHAGOCYTES
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.
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 ± 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 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 "inside-out" 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 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 Cell culture
Isolation of human PMNs
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-leucyl-phenylalanine (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, 5µm pore size) were precoated for 30 minutes with 50µL 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 3-integrin21
(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 µg/mL aprotinin, 100µg/mL phenylmethylsulfonyl fluoride [PMSF], and 10µg/mL chymostatin) and debris removed by centrifugation. Lysates were precleared with 50 µL pre-equilibrated protein-G Sepharose (Pharmacia, Uppsala, Sweden). Immunoprecipitation of3-integrin was performed by addition of mAb AP3
(5µg/mL) followed by addition of 50 µL 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 encompassing3-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 µM 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 1µg/mL)24 prepared in 1% normal goat serum. After washing, the sections were then incubated with fluorescein isothiocyanate (FITC)-conjugated secondary antibody (Cappel, 1µg/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
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.
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 less potent PI3K inhibitor LY294002 (EC50 ~2 µM 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 chemoattractant-stimulated PMN transmatrix migration.
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-2-integrin 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-
PI3K inhibition blocks 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 by SDS-PAGE
and Western blots were probed with antiphosphotyrosine. As shown in
Figure 4, PI3K inhibition blocked
fMLP-stimulated 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 3-integrin tyrosine
phosphorylation.
Role for 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 µM. HIV-tat-linked peptide
corresponding to tyrosine 747 also increased transmigration (maximal
44% ± 0.5% at 50 µM, P < .025), but to a lesser
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.
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
Role of p72syk in PMN transmatrix migration Based on the observations that PI3K inhibition blocks surface3-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 selective
p72syk inhibitor piceatannol32 should block
surface
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
Recent studies have clearly defined PI3K as an important molecule in
acute inflammatory processes. Transgenic knockout mice lacking the
p110 Our studies provide unique insight into a prominent role for
The present studies provide direct evidence that cytoplasmic tail
tyrosine residue 759, and to a lesser extent residue 747, mediate PMN
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 In conclusion, as PMNs respond to chemoattractants and surface molecule
engage extracellular matrix ligands, phosphorylation of
We authors wish to thank Ms Kristin Synnestvedt for technical assistance.
Submitted August 3, 2000; accepted January 12, 2001.
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.
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.
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: colgan{at}zeus.bwh.harvard.edu.
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3.
Calderwood DA, Shattil SJ, Ginsberg MH.
Integrins and actin filaments: reciprocal Regulation of cell adhesion and signaling.
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2000;275:22607-22610 4. Schoenwaelder SM, Burridge K. Bidirectional signaling between the cytoskeleton and integrins. Curr Opin Cell Biol. 1999;11:274-286[CrossRef][Medline] [Order article via Infotrieve].
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Servant G, Weiner OD, Herzmark P, Balla T, Sedat JW, Bourne HR.
Polarization of chemoattractant receptor signaling during neutrophil chemotaxis.
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3-integrin
represents an endogenous "braking" mechanism during neutrophil
transmatrix migration
Top
Abstract
Introduction
, 
, and 
, and extracellular ligand coupling through
membrane-localized G proteins generates the signaling molecule
phosphatidylinositol 3,4,5-triphosphate.
) or wortmannin (
, 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 (
, C) or epithelia (
,
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 .
) followed by examination of
fMLP-stimulated (10
) or
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