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-Induced Lymphocyte Polarization and Chemotaxis1
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Servicio de Inmunología, Hospital de la Princesa, Universidad Autónoma de Madrid, Madrid, Spain; and
Department of Immunology and Oncology, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Universidad Autónoma de Madrid, Campus de Cantoblanco, Madrid, Spain
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(SDF-1). This
chemokine was able to directly activate p85/p110 PI3-kinase in whole
human PBL and to induce the association of PI3-kinase to the SDF-1
receptor, CXCR4, in a pertussis toxin-sensitive manner.
Two unrelated chemical inhibitors of PI3-kinase, wortmannin and
Ly294002, prevented ICAM-3 and ERM protein moesin polarization as well
as the chemotaxis of PBL in response to SDF-1. However, they did not
interfere with the reorganization of either tubulin or the actin
cytoskeleton. Moreover, the transient expression of a dominant negative
form of the PI3-kinase 85-kDa regulatory subunit in the constitutively
polarized Peer T cell line inhibited ICAM-3 polarization and markedly
reduced SDF-1-induced chemotaxis. Conversely, overexpression of a
constitutively activated mutant of the PI3-kinase 110-kDa catalytic
subunit in the round-shaped PM-1 T cell line induced ICAM-3
polarization. These results underline the role of PI3-kinase in the
regulation of lymphocyte polarization and motility and indicate that
PI3-kinase plays a selective role in the regulation of adhesion and ERM
proteins redistribution in the plasma membrane of
lymphocytes. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(SDF-1)4 is a
member of the CXC chemokine subfamily that attracts T as well as B
lymphocytes (4, 5, 6). Its role in T cell-HIV-1 infection has
been well characterized (7). Studies performed in CXCR4-
and SDF-1-deficient mice confer special significance to the role of
SDF-1 in lymphopoiesis as well as in cardiac and neural tissue
development (8, 9, 10, 11). Leukocyte chemotaxis is a crucial phenomenon both in the immune and inflammatory response (12). During chemotaxis, chemoattractant molecules, such as chemokines, induce lymphocyte polarization with generation of specialized cell compartments (13). Chemoattractant receptors concentrate at the cell leading edge, whereas the adhesion molecules ICAM-1, ICAM-3, and CD44 redistribute to the uropod, which is involved in the recruitment of bystander leukocytes (6, 14, 15, 16). Several cytoskeletal elements including the ERM protein moesin and the microtubule organizing center (MTOC) also concentrate at the uropod of polarized migrating lymphocytes (17, 18). Moreover, the actin-binding protein moesin interacts with ICAM-3 and its association increases during the polarization process of lymphocytes (17).
Signals regulating lymphocyte polarization and chemotaxis are largely
unknown. However, different signaling molecules have been proposed as
potential candidates in the regulation of such processes. One of these
molecules is the type I phosphatidylinositol 3-kinase (PI3-kinase),
which consists of an 85-kDa regulatory subunit responsible for
protein-protein interactions via Src-homology (SH), SH2, and SH3
domains, and a catalytic 110-kDa subunit (19). PI3-kinase
has been implicated in insulin-stimulated glucose uptake
(20), membrane ruffling induced by platelet-derived growth
factor (PDGF) or IL-2 (21, 22), and activation of
additional signaling molecules, such as p70 S6 kinase (23)
and Akt/protein kinase B (24). A G-protein-coupled
PI3-kinase, the PI3-kinase 
, which is not regulated by p85, has been
recently identified (25, 26). In addition, there is
evidence implicating seven transmembrane domain G-protein-coupled
receptors in the activation of p85/p110 PI3-kinase (27).
Furthermore, signaling by chemokines through their seven transmembrane
domain receptors has been reported to activate PI3-kinase
(28, 29, 30). However, controversial data exists regarding the
negative effect of PI3-kinase in chemokine-induced chemotaxis by using
chemical inhibitors (31).
Herein we have characterized the role of PI3-kinase in SDF-1-induced
polarization and chemotaxis of PBL. We found that SDF-1 induced
activation of PI3-kinase and association of p85 to CXCR4. Furthermore,
inhibition of PI3-kinase activity by either chemical compounds or
overexpression of its dominant negative form prevented the polarization
of adhesion molecules and ERM components as well as the chemotactic
response of lymphocytes to SDF-1. However, it caused no effect on
cytoskeletal rearrangements induced by the chemokine. On the other
hand, activation of PI3-kinase induced adhesion molecules polarization.
All together, these data underline the pivotal role of PI3-kinase in
lymphocyte polarization and chemotaxis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The anti-ICAM-3 TP1/24 (IgG2a), anti-LFA-1 LIA3/2
(IgG1), anti-CD45 D3/9 (IgG1), and RP2/21 (IgG1) mAb have been
previously described (32, 33). Rabbit anti-p85
polyclonal Ab (pAb) was purchased from Upstate Biotechnology (Lake
Placid, NY). Rabbit anti-Gs pAb (clone
K-20) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The
anti-moesin 38/87 mAb was a kind gift from Dr. R. Schwartz-Albiez
and has been previously described (34). The
anti--tubulin mAb was purchased from Sigma (St. Louis, MO). The
mouse anti-CXCR4 12G5 (IgG1) and 44708.111 (IgG2a) mAbs were
purchased from PharMingen (San Diego, CA) and R&D Systems (Minneapolis,
MN), respectively. The mouse anti-CCR5 CCR5.01 (IgM) mAb has been
previously described (16). The mouse anti-CXCR4
CXCR4.01 (IgM) mAb will be described elsewhere. Phalloidin-Texas Red
was obtained from Amersham Pharmacia Biotech (Molecular Probes, Eugene,
OR). Recombinant human SDF-1 was purchased from Peprotech (London,
U.K.). Bordetella pertussis toxin, the PI3-kinase inhibitors
wortmannin (WMN) and Ly294002, the mitogen-activated
protein/extracellular signal-related kinase kinase-1 (MEK1) antagonist
PD98059, and the protein kinase A blocker H-89 were obtained from
Calbiochem (La Jolla, CA). ATP and phosphatidylinositol (PI) were
obtained from Sigma.
Cells and cell lines
Peer 
CD3+ human T cell line was
grown in RPMI 1640 (Flow Lab., Irvine, U.K.) containing 10% FCS. PM-1
CD3+, CD4+ human T
lymphocyte line was kindly provided by Dr. S. Chen (Comprehensive
Cancer Center, Winston-Salem, NC). PBL were obtained as described
(14). Briefly, mononuclear cells were isolated from
freshly prepared buffy coats using a Ficoll-Hypaque density gradient,
followed by two rounds of adherence to plastic to deplete monocytes. A
representative cell population comprised 75%
CD3+ cells, 20% CD16+
cells, 4% CD19+ cells, and <2%
CD14+ cells.
p85 and CXCR4 immunoprecipitation and Western blot
Human normal lymphocytes were pretreated with or without 0.1
µM WMN, 20 µM Ly294002, 20 µM PD98059, 0.75 µg/ml
pertussis toxin or 2 µg/ml blocking anti-LFA-1 LIA3/2
for 30 min at 37°C. Thereafter, they were stimulated with 10 nM
SDF-1 for the indicated times under continuous stirring before being
washed twice in cold PBS. For Western blotting assays,
2x107 cells were lysed in a detergent buffer (20
mM triethanolamine, pH 8.0, 300 mM NaCl, 2 mM EDTA, 20% glycerol, 1%
digitonin, 10 mM sodium orthovanadate, 1 mM leupeptin, and 1 mM
aprotinin) for 30 min at 4°C under continuous stirring and then
centrifuged (15,000 x g, 15 min). The protein content
in the cell lysates was measured before the immunoprecipitation using a
protein detection kit (Pierce, Rockford, IL). Immunoprecipitations were
performed essentially as described (35). Briefly, protein
extracts precleared by incubation with 20 µg of anti-mouse IgM-
or IgG-agarose (Sigma) were centrifuged (15,000 x g, 1
min) and immunoprecipitated with the CXCR4.01, CCR5.01, or D3/9 mAb (5
µg/sample, 120 min, 4°C), followed by the anti-mouse IgM- or
IgG-agarose (20 µg/sample) for 60 min. Samples were centrifuged
(15,000 x g, 15 min, 4°C), and the agarose pellet
was washed twice with lysis buffer and three times with 50 mM Tris-HCl,
pH 7 (15,000 x g, 1 min, 4°C) and resuspended in
Laemmli buffer. For in vitro kinase assay, cells were lysed in lysis
buffer containing 50 mM Tris, pH 8.0, 1% Nonidet P-40, 150 mM NaCl,
0.5 mM EDTA, 1 mM NaF, 1 mM sodium pyrophosphate, 1 mM PMSF, 1 mM
aprotinin, 1 mM leupeptin, and 1 mM
Na3VO4. Cell lysates were
clarified by centrifugation at 15,000 x g at 4°C,
and equal amounts of protein were immunoprecipitated for 2 h at
4°C with 2 µg/ml of anti-p85 pAb or 5 µg/ml of anti-CXCR4
mAb, followed by incubation with 50 µl protein A-Sepharose (Amersham
Pharmacia Biotech) or 20 µg of anti-mouse IgM-agarose (Sigma) for
2 h at 4°C. Western blotting analysis was performed as described
(35). Immunoprecipitates were separated in SDS-PAGE and
transferred to nitrocellulose membranes. Protein loading was carefully
controlled using the protein detection kit and by reprobing the
membrane with the immunoprecipitating Ab.
PI3-kinase assay
The p85 or CXCR4 immunoprecipitates were washed twice with lysis
buffer, with 0.5 M LiCl, and with 50 mM Tris-HCl, pH 7.4/100 µM EDTA
before incubating with PI in reaction buffer (25 mM
MgCl2, 20 µM ATP, 50 mM Tris-HCl, pH 7.4, 10
µCi [-32P]ATP) (Amersham Pharmacia
Biotech) for 10 min at room temperature. The phosphorylation reaction
was stopped by the addition of 1 M HCl, and lipids were extracted with
CHCl3/methanol. The radiolabeled lipids were
resolved by thin-layer chromatography as described (36),
and radioactivity was analyzed with a Bio-Rad GS-525 Molecular Imager
System (Hercules, CA) with Molecular Analyst v. 2.1 software.
Calcium determination assay and flow cytometry analysis
Changes in intracellular Ca2+
concentration were monitored using the fluorescent probe Fluo-3/AM
(Molecular Probes, Eugene, OR). PBL (3 x
106 cells/ml) were suspended in HBSS containing
10 mM HEPES and loaded with 25 µl Fluo-3/AM (250 mM in DMSO) for 20
min at 37°C. Cells were then washed, resuspended in HBSS, and
incubated with SDF-1 at 10 nM in RPMI 1640. When indicated, the
cells were preincubated with 0.75 µg/ml pertussis toxin
for 30 min at 37°C. The intracellular calcium concentration was then
determined in a FACScan flow cytometer (Becton Dickinson, Mountain
View, CA) using CellQuest software. CXCR4 membrane expression on Peer T
cell line was measured by staining the cells with the 12G5 mAb plus a
1:50 dilution of an FITC-labeled goat F(ab')2
anti-mouse Ig (Dako, Carpinteria, CA) and analyzed by flow
cytometry.
Immunofluorescence microscopy and polarization assay
Immunofluorescence experiments were performed essentially as
described (37). Briefly, 1–1.5 x
106 PBL and 5 x105 Peer or
PM-1 T cells were incubated in flat-bottom 24-well plates (Costar,
Cambridge, MA) in a finalvolume of 500 µl complete medium on
coverslips coated with human fibronectin at 50 µg/ml. When indicated,
cells were pretreated with inhibitors for 30 min at 37°C under
continuous agitation. SDF-1 at the indicated concentration was
added, and cells were allowed to adhere for 30 min at 37°C in an
atmosphere containing 5% CO2. Cells were then
fixed in 3.7% formaldehyde in PBS for 10 min at room temperature
before being rinsed in TBS (50 mM Tris-HCl, 150 mM NaCl, pH 7.6).
ICAM-3 was visualized by staining the cells with the anti-ICAM-3
TP1/24 mAb plus a 1:50 dilution of an FITC-labeled goat
F(ab')2 anti-mouse Ig (Dako) or a 1:2500
dilution of Cy3-labeled goat F(ab')2
anti-mouse Ig (Amersham Pharmacia Biotech), when required. For
staining of cytoplasmic proteins, fixed cells were permeabilized
with 0.1% Triton X-100 for 5 min at room temperature before staining
with the appropriate mAbs or a 1:50 dilution of Texas red-labeled
phalloidin for actin visualization. Cells were observed using a Nikon
Labophot-2 photomicroscope with 40, 60, and 100x oil immersion
objectives. The proportion of uropod-bearing cells was calculated by
random choice of ten different fields (60x objective) of each
condition and by counting 400–500 cells. Images were acquired with a
Cohu high-performance charge-coupled device camera (Cohu, Tokyo,
Japan) coupled to the microscope and connected to a Leica Q550CW
workstation (Leica Imaging Systems, Cambridge, U.K.). Images were
visualized, processed, and stored by using the Leica QFISH software
v.V1.01 (Leica). Finally, images were printed with a Tektronix Phaser
440 color printer (Tektronix, Wilsonville, OR).
Expression constructs
The cDNAs for wild-type and dominant negative forms of p85
(p85wt and 
p85) and the activated mutant of p110 (p110CAAX) in
the pSG5 plasmid (Stratagene, La Jolla, CA) were kindly provided by Dr.
J. Downward (Imperial Cancer Research Fund, London, U.K.) and have been
previously described (38). The p85 and p110CAAX cDNAs were
subcloned into pCDNA3 through the EcoRI and BamHI
sites, respectively. The pEGFP-C1 (green fluorescent protein (GFP)
expression vector) was obtained from Clontech (Palo Alto, CA).
Transient transfection assay
Peer or PM-1 T cells (2 x 107) were washed twice with cold HBSS and resuspended in 500 µl cold Optimem medium (Life Technologies, Paisley, U.K.) before being placed in a 0.4-cm cuvette from Bio-Rad Laboratories (Hercules, CA). pCDNA3 plasmids containing the p85/p110 cDNAs of PI3-kinase (20 µg) and GFP (5 µg) were added, and transfections were conducted at 1200 µF/280 V using the Gene Pulser II electroporation system from Bio-Rad plus the capacitance extender plus device. After the electroporation burst, the cells were transferred to 25-cm2 flasks and cultured with RPMI 1640 containing 10% FCS and antibiotics (Flow Lab). The transfection efficiency was estimated by flow cytometry, excluding dead cells by staining with propidium iodide (Sigma).
Chemotaxis assay
Assays for lymphocyte chemotaxis were performed in polycarbonate
membranes, 6.5 mm diameter, 10 µm thickness, 5-µm-diameter pore
size Transwell cell culture chambers (Costar). Human normal lymphocytes
(100 µl at 10 x 106/ml) or Peer T cells
(100 µl at 5 x 106/ml) suspended in
RPMI1640/0.1% human serum albumin were added to the upper chamber and
SDF-1 was added to the lower well. When indicated, cells were
pretreated with or without 0.75 µg/ml pertussis toxin, 0.1
µM WMN, 20 µM Ly294002, 20 µM PD98059, 30 µM H-89, or 20
µg/ml of the 44708.111 anti-CXCR4 or TP1/24 anti-ICAM-3 mAb
for 30 min at 37°C. Thereafter, the cells were allowed to migrate for
90 min at 37°C in 5% CO2 atmosphere, after
which migrated cells were recovered from the lower part of the
chemotaxis chamber. For transient transfectants expressing mutant forms
of PI3-kinase, only cells coexpressing the GFP were quantified,
excluding nontransfected cells by flow cytometry. Counting of migrated
cells was performed by flow cytometry. Briefly, cells were stained with
propidium iodide and were counted during 1.5 min, calibrating the flow
rate of the FACScan with Trucount tubes (Becton Dickinson). Cell
chemotaxis was expressed as the migration index, which was calculated
with the following formula: number of cells in the lower well/[number
of cells in the lower well + (number of cells in the upper chamber
x100)].
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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induces PI3-kinase activity and promotes its association
to CXCR4
The involvement of PI3-kinase in the different functional events
triggered by the binding of SDF-1 to its receptor was assessed
through an in vitro PI3-kinase assay. We found a 3-fold induction of
PI3-kinase activity in p85-immunoprecipitated cell lysates from
SDF-1-treated cells compared with unstimulated cells (Fig. 1
a) at the time point of
maximal lymphocyte polarization (see below, Fig. 4b). This
activation was abrogated by pretreatment of the cells with 0.1 µM WMN
or 20 µM Ly294002, two chemically distinct inhibitors of PI3-kinase
activity (19). In contrast, pretreatment with 20 µM
PD98059, a specific inhibitor of MEK1, did not affect PI3-kinase
activation (Fig. 1a).
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b). When PI3-kinase activity was
determined during the first 5 min after 10 nM SDF-1 stimulation, no
increases were observed in comparison to control cell treatments (data
not shown). To rule out integrin-dependent delayed activation of
PI3-kinase through homotypic contacts during incubation, cells were
treated with the chemokine under continuous stirring in the presence of
an anti-LFA-1 ß2 blocking mAb. The Ab neither activated
PI3-kinase by itself nor prevented SDF-1-induced PI3-kinase
activation (Fig. 1a), thus demonstrating the direct role of
chemokines in activating PI3-kinase in long-term stimulation.
To ascertain the possible mechanism of PI3-kinase activation by
SDF-1, the SDF-1 receptor from stimulated lymphocyte lysates was
immunoprecipitated with an anti-CXCR4 mAb, and an in vitro kinase
assay was performed. We found that SDF-1 increased PI3-kinase
activity associated with CXCR4. This activity showed a 4-fold induction
upon SDF-1 stimulation and was specifically inhibited by WMN and
Ly294002 (Fig. 2a), which
indicates the association of CXCR4 with a WMN-sensitive PI3-kinase
isoform. Pretreatment of the cells with a blocking mAb against CXCR4
abrogated SDF-1-induced PI3-kinase activity, demonstrating the
specificity of SDF-1 in activating a CXCR4-associated PI3-kinase
activity (data not shown). The kinetics of PI3-kinase activity
association to CXCR4 were coincident with total PI3-kinase activation,
as it peaked at 20 min, decreasing thereafter (Fig. 2b).
Further demonstration of the physical association of CXCR4 with
PI3-kinase was obtained by coprecipitation experiments with
anti-CXCR4 mAb followed by Western blotting with an anti-p85
pAb. PBL stimulation with SDF-1 for 20 min induced p85 association
to CXCR4, thus correlating with PI3-kinase activation (Fig. 2c). This association was specific because it was
neither observed with an isotype-matched anti-CCR5 nor with an
anti-CD45 mAb (Fig. 2c). To further demonstrate the
specificity of the interaction between p85 and CXCR4, p85 from
SDF-1-stimulated PBLs was immunoprecipitated followed by
immunodetection with the anti-CXCR4 mAb. Association of CXCR4 to
p85 was found only in SDF-1-stimulated cells, whereas no
association was observed in unstimulated cells (Fig. 2d). An
irrelevant pAb against the s subunit of heterotrimeric
Gs proteins was used as a control.
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in PBLs was
pertussis toxin-sensitive, cells were pretreated with 0.75
µg/ml of pertussis toxin and chemotaxis, calcium
mobilization, and PI3-kinase activation experiments were performed
(Fig. 3). As expected, both calcium
mobilization (Fig. 3a) and chemotaxis (Fig. 3b)
were blocked by pretreatment with pertussis toxin.
Interestingly, pertussis toxin also blocked SDF-1-induced
PI3-kinase activation both in CXCR4 (Fig. 3c) and in p85
immunoprecipitates (not shown). To further demonstrate the
role of Gi protein activation in SDF-1-induced
p85 association to CXCR4, coprecipitation experiments were
performed in pertussis toxin-pretreated cells.
Pertussis toxin also blocked p85 association to CXCR4 upon
SDF-1 stimulation (Fig. 3d).
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-induced cytoskeletal
rearrangements
Chemokines such as RANTES, MCP-1, macrophage inflammatory protein
(MIP)-1, and MIP-1ß induce cell polarization with redistribution
of adhesion molecules to the uropod in different lymphoid subsets
including T lymphocytes and NK cells (16, 37). We have
previously reported that SDF-1 is the most potent chemokine
in inducing cell polarization on tonsil and peripheral blood B
lymphocytes (6). Accordingly, we have found that SDF-1
induced a dose-dependent polarization of PBL, as assessed by ICAM-3
redistribution to the cellular uropod (Fig. 4a). Kinetic studies of
SDF-1-induced cell polarization showed a maximal response around
20–30 min, thereafter declining by 60 min (Fig. 4b).
To explore the role of PI3-kinase in SDF-1-induced lymphocyte
polarization, PBL were treated with different doses of WMN and Ly294002
and then immunofluorescence experiments were performed (Fig. 5). Both inhibitors blocked the
SDF-1-induced ICAM-3 redistribution to the uropod in a
dose-dependent fashion, whereas the MEK1 inhibitor PD98059 did not
inhibit adhesion molecules polarization (Fig. 5a). The PKA
inhibitor H-89, which prevents lymphocyte polarization
(37), was included as a control of inhibition (Fig. 5a). Dose-response curves of the inhibitory effect of
Ly294002 and WMN on ICAM-3 redistribution to the uropod are shown in
Fig. 5c, whereas quantification of the inhibitory effect of
the inhibitors tested is shown in Fig. 5d.
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b). Both Ly294002
and WMN were able to inhibit ICAM-3 and moesin clustering at the
uropod, whereas they did not affect the cell morphology, the
localization of the MTOC, nor the overall actin redistribution (Fig. 5b, and data not shown).
Chemotaxis assays showed that SDF-1-induced directional migration
was partially inhibited by WMN or Ly294002, which indicates the
involvement of PI3-kinase in this phenomenon and further reinforces the
linkage between cell polarization and migration (Fig. 6). Neither the MEK1 inhibitor PD98059
nor the protein kinase C blocker BIM-II affected PBL chemotaxis toward
SDF-1. As a control for specificity of the interaction between CXCR4
and SDF-1, PBLs were preincubated with 20 µg/ml of the 44708.111
(IgG2a) anti-CXCR4 mAb, which abrogated SDF-1-mediated
chemotaxis. The isotype-matched anti-ICAM-3 TP1/24 mAb was used as
a control.
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We have previously reported the constitutively polarized phenotype
displayed by several T cell lines such as Peer and HSB-2 with ICAM-3
localized in one pole of the cells (37). To further assess
the involvement of PI3-kinase in the regulation of lymphocyte polarity,
transient cotransfection assays with different forms of the PI3-kinase
subunits and a plasmid encoding GFP were performed. We found that the
overexpression of a dominant negative form of p85 (p85) induced a
marked decrease in the spontaneous polarization of the Peer T cell line
(Fig. 7a). Transfection of
p85wt partially inhibited cell polarization, whereas overexpression of
an activated mutant of p110 (p110CAAX) had no effect on polarization
(Fig. 7a). The quantitative estimation of the effect of
overexpression of PI3-kinase forms on Peer T cell polarization is shown
in Fig. 7b.
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in a dose-dependent,
pertussis toxin-sensitive fashion (Fig. 8, a and b, and
data not shown). We next tested the effect of overexpression of the
dominant negative form of p85 in SDF-1-induced chemotaxis. The
overexpression of p85 in Peer T cells reduced chemotaxis to levels
comparable to WMN- or Ly294002-treated cells (Fig. 8c, and
data not shown). p85wt diminished lymphocyte migration but to a lower
extent than p85, whereas p110CAAX had no effect on lymphocyte
chemotaxis.
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p85 had no effect on ICAM-3 redistribution
(Fig. 9, a and b).
Together, these results underline the key role of PI3-kinase in the
control of lymphocyte membrane polarity.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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is able to induce the
polarization of PBL in a dose-responsive manner, which is followed by a
chemotactic response. SDF-1 also induced the activation of
PI3-kinase type I and its association with the CXCR4 receptor. In
addition, we have found that PI3-kinase is involved in the polarization
and chemotactic responses of lymphocytes to
SDF-1.
RANTES, MCP-1, and SDF-1 have been described to be able to induce
activation of PI3-kinase in several cell types, including human
peripheral T lymphocytes, T cell lines, and mouse-transfected cells
(28, 29, 30). In addition, a previous work, which is in
agreement with our results (30), found that SDF-1 is a
potent inducer of PI3-kinase activity in a CXCR4-transfected mouse cell
line. It is of interest that the kinetics of SDF-1-induced
PI3-kinase activation parallels the induction of cell polarization
triggered by this chemokine. These results differ from another report,
in which MCP-1-induced PI3-kinase activation was observed as early as
30 s (29). It is feasible that chemokines activate
PI3-kinase in two waves, the earlier at 15 s to 2 min, which would
be comparable to chemokine-induced calcium mobilization, and the later
at 15–30 min, which could be involved in cell polarization. However,
we have been unable to detect PI3-kinase activation at early time
points. In contrast, our results demonstrate the association of
PI3-kinase activity to CXCR4 upon ligand triggering in lymphocytes in a
pertussis toxin-sensitive fashion. Although the association
of different signaling proteins has been previously described for
several chemokine receptors, including CCR2 (35), the
association of PI3-kinase to CXCR4 in human lymphocytes had
not so far been reported.
The involvement of PI3-kinase in SDF-1-mediated lymphocyte
polarization and ICAM-3 redistribution is demonstrated by the
inhibitory effect of two unrelated p85/p110 PI3-kinase antagonists,
Ly294002 and WMN. SDF-1-induced migration was also reduced, but not
completely inhibited, by WMN and Ly294002, which reflects the
involvement of PI3-kinase in chemokine-induced chemotaxis. These
results have been confirmed in other cell lines, such as T
lymphoblasts, which express both CCR5 and CXCR4. Whereas
RANTES-induced cell migration was completely inhibited,
SDF-1-induced chemotaxis was partially abolished in T
lymphoblasts (not shown), which could be due to the different
potency of both chemokines. Likewise, Ganju et al. have described
partial inhibition of SDF-1-induced chemotaxis in the L1.2 mouse
pre-B lymphoma cell line stably transfected with CXCR4
(30). In this regard, the effect of WMN on cellular
chemotaxis has not been uniform and is apparently cell lineage and
chemoattractant dependent. Hence, it has been described that PI3-kinase
inhibitors are ineffective in IL-8- and fMLP-induced neutrophil
chemotaxis (31), whereas they are able to block the
migration induced by RANTES and MCP-1 in peripheral blood T and
transfectant cells, respectively (28, 29).
Previous studies performed on different cellular systems such as T cell-APC conjugate formation and epithelial cells have pointed out the key regulatory role of small GTP-binding proteins on cell polarization (39, 40, 41). Active mutants of Cdc42 and Rac1 disrupt the constitutive polarization of mammary epithelial cells (41). In addition, T cells stably transfected with either the activated mutant or the dominant negative forms of Cdc42 display an abnormal positioning of the MTOC during Ag presentation (40). Moreover, a specific effect of Cdc42 is exerted on the migration of macrophages induced by GM-CSF (42). PI3-kinase is involved in signaling cascades in which small GTP-binding proteins participate. In this regard, PI3-kinase seems to activate Rac in the PDGF-induced membrane ruffling pathway but not in the c-Jun N-terminal kinase/mitogen-activated protein kinase pathway (43). PI3-kinase apparently does not activate Rac in T lymphocytes (44), but it has been shown that it acts upstream of Rac in the IL-2-induced membrane ruffling pathway in T cells (22).
Notably, our data indicate that PI3-kinase plays a selective role in the regulation of ICAM-3 and moesin redistribution on the plasma membrane rather than a general role in the regulation of cell morphology and tubulin cytoskeleton polarization. These data concur with studies performed by other authors, in which they demonstrate that this enzyme is not required in TCR-mediated reorientation of the MTOC in Jurkat T cells (45). PI3-kinase is likely to be acting on adhesion molecule redistribution through other signaling proteins, such as guanosine exchange factors of the Rho subfamily, which possess pleckstrin homology domains and can bind PI(3, 4, 5)-trisphosphate, directing these molecules to their activation sites within the plasma membrane (46). Thus, signaling through chemokine receptors will involve the triggering of many intracellular pathways in a PI3-kinase-dependent manner (47). It is tempting to speculate that the partial, but not complete, inhibition of chemotaxis achieved with PI3-kinase inhibitors could be due to aberrant positioning of adhesion molecules within the plasma membrane. In this regard, polarized cells would retain certain locomotor capabilities, but the inappropriate redistribution of adhesion molecules would reduce chemotaxis.
We have also addressed the role of PI3-kinase in lymphocyte polarization using transient transfection assays. The blockade of the constitutively polarized distribution of the adhesion molecule ICAM-3 of Peer T cells by a dominant negative mutant of PI3-kinase agrees with our results obtained with chemical inhibitors as well as the work by Stowers et al. (40), in which chemical inhibition of PI3-kinase impaired T cell polarization during Ag presentation. In contrast, partial inhibition of ICAM-3 clustering induced by p85wt could be due to the regulatory role of p85 in the activation of downstream effectors of PI3-kinase. Thus, overexpression of p85wt would compete with activated endogenous p85/p110 for downstream mediators, thus interfering with the putative signaling leading to ICAM-3 positioning within the cell membrane. In contrast, the overexpression of a constitutively activated mutant form of PI3-kinase, p110CAAX, induces ICAM-3 redistribution in the nonpolarized T cell line PM-1, thus indicating that PI3-kinase activation is not only necessary but sufficient to induce membrane receptor polarization.
Our data using either chemical inhibitors or the dominant negative
mutant of p85 indicate that PI3-kinase is also involved in the
lymphocyte migration induced by SDF-1. The prominent role of
PI3-kinase in cell motility has been previously found through the
overexpression of activated mutant forms of p110 in epithelial as well
as carcinoma cell lines. In all cases, overexpression of activated p110
led to an increased cell motility and invasive capability (41, 48). In contrast, the chemokine-induced directional motility of
T lymphocytes is impaired by PI3-kinase inhibitors (Refs.
28 and 30 and this report), which further
underlines the essential role of PI3-kinase in cell motility. The fact
that a complete abrogation of SDF-1-induced chemotaxis was not
achieved could be due to a bypass of the inhibitory signals delivered
by WMN, Ly294002, and p85 by other PI3-kinase isoforms, such as the
Gß-coupled PI3-kinase . In this regard, pertussis
toxin has been demonstrated to prevent PI(3, 4, 5)-trisphosphate
accumulation in MCP-1-stimulated T cell lines by inhibition of the
WMN-insensitive PI3-kinase C2 (29). Further studies are
required to establish the possible role of PI3-kinase C2 in
chemokine-induced lymphocyte polarization to acquire a more complete
view of the involvement of PI3-kinase activity in the migratory
behavior of lymphocytes.
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Vascular Biology, The Scripps Research Institute, CVN223/VB4, 10550 North Torrey Pines Road, La Jolla, CA 92037.
3 Address correspondence and reprint requests to Dr. Francisco Sánchez-Madrid, Servicio de Inmunología, Hospital de la Princesa, c/Diego de León, 62, E-28006 Madrid, Spain. E-mail address:
4 Abbreviations used in this paper: SDF-1, stromal cell-derived factor-1; GFP, green fluorescent protein; MCP-1, monocyte chemotactic protein-1; pAb, polyclonal Ab; MTOC, microtubule organizing center; PI3-kinase, phosphatidylinositol 3-kinase; SH, Src homology; PDGF, platelet-derived growth factor; WMN, wortmannin; MEK, mitogen-activated protein/extracellular signal-related kinase kinase-1; MIP, macrophage inflammatory protein; p85wt, wild-type p85; p85, dominant negative p85.
Received for publication April 13, 1999. Accepted for publication July 20, 1999.
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