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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 95 No. 8 (April 15), 2000:
pp. 2462-2470
FOCUS ON HEMATOLOGY
Neutrophil polarity and locomotion are associated with surface
redistribution of leukosialin (CD43), an antiadhesive membrane
molecule
Stéphanie Seveau,
Hansuli Keller,
Frederick R. Maxfield,
Friedrich Piller, and
Lise Halbwachs-Mecarelli
From INSERM U507, Hôpital Necker, Paris, France; Institute of
Pathology, University of Bern, Switzerland; Department of Biochemistry,
Weill Medical College of Cornell University, New York, New York; Centre
de Biophysique Moléculaire, CNRS UPR 4301, Orléans, France.
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Abstract |
This study analyzed the behavior of an antiadhesive membrane
molecule, CD43, in neutrophil polarization and locomotion. CD43 cross-linking by antibodies induced neutrophil locomotion, with CD43
molecules clustered at the uropod of polarized neutrophils. In
contrast, CD11b/CD18 cross-linking by antibodies did not affect either
cell polarization or locomotion. Stimulation of suspended or adherent
neutrophils with chemotactic peptide results in cell polarization and
locomotion and a concomitant redistribution of CD43 to the uropod. This
process is entirely reversible. The study also investigated which
actin-binding protein could be involved in CD43 lateral redistribution.
 -Actinin and moesin are preferentially adsorbed on Sepharose beads
bearing a recombinant CD43 intracellular domain. Analysis by
immunofluorescence confocal microscopy shows a codistribution of moesin
during CD43 lateral redistribution. By contrast, -actinin is located
at the leading edge, an area devoid of CD43. These results shed new
light on the role of CD43 membrane redistribution, which appears to be
directly related to neutrophil polarity and locomotion.
(Blood. 2000;95:2462-2470)
© 2000 by The American Society of Hematology.
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Introduction |
Neutrophils circulate in the blood as spherical resting
cells. In response to inflammatory stimuli, neutrophils leave the blood
vessel by diapedesis and locomote across the extracellular matrix to
the inflamed area.1,2 Migrating cells acquire a polarized
morphology in which the leading edge, where lamellipodia are generated,
becomes differentiated from the rear, or uropod.3 Cell
locomotion is a complex, still poorly understood,
process.4-6 During locomotion, the leading edge extends in
the direction of migration; this membrane extension is associated with
F-actin polymerization.7-9 Contraction of the uropod,
probably by a myosin II-dependent process, may also pull the cell
forward toward the leading edge.10,11
Locomotion requires successive attachment and detachment of the cell
from the substratum and is controlled by cell adhesiveness. Both
adhesion at the front and release of the rear part of the cell regulate
cell morphology and locomotion.12-15 The behavior of
adhesion molecules, which mediate cellular attachment to the substratum, during cell locomotion, has been studied by various laboratories. Integrins on the adherent side of the cell connect the
cytoskeleton with the extracellular matrix to produce the forces
required for locomotion.16-18 Integrins remain attached to
the substratum while the cell moves forward and end up at the rear of
the cell.13,19 This rear attachment must then be released and new adhesion sites be created at the leading edge to ensure the
continuation of cell movement. It has been proposed that integrins recycling and shedding17,19,20 and the proteolytic
degradation of extracellular matrix21 are involved in these processes.
The possible function of uropod-localized membrane proteins in
particular antiadhesive moleculesin rear release has not been investigated. The main antiadhesive membrane molecule of leukocytes is
CD43 (leukosialin, sialophorin). CD43 is a heavily sialylated O-glycosylated surface protein, which is a major component of the
negatively charged repulsive barrier formed by the cell
glycocalyx.22-25
The antiadhesive function of CD43 has been established by in vitro
experiments in which CD43 transfection into CD43
cell lines diminishes cell adhesion,26 whereas, conversely, targeted disruption of the CD43 gene in CD43+ cells
increases cell adhesion.27 In vivo rolling and adhesion of
leukocytes to endothelial cells is enhanced in CD43-deficient mice
compared to wild-type mice.28 Leukocyte emigration into tissues is, however, impaired in these mice, suggesting a positive role
for CD43 in leukocyte adhesion and motility processes. Indeed, CD43 is
also potentially an adhesion molecule; it bears the sialyl-Lewis x
epitope and belongs to the mucin family of selectin counterreceptors. Various molecules, such as ICAM-1, E-selectin, or MHC-1, have been
proposed as putative ligands. This dual antiadhesive versus adhesive
function of CD43 has been recently reviewed.29
As far as neutrophil polarization and locomotion are concerned, we have
previously shown that CD43 cross-linking by antibodies induces CD43
redistribution in a cap structure by a cytoskeleton-driven process.
CD43 cross-linking induces the polarization of neutrophils in
suspension, with an F-actin-rich lamellipodium and a myosin-rich uropod, similar to what is observed on adherent locomoting
cells.30 CD43 has also been shown to be redistributed to
the uropod of lymphocytes crawling on a monolayer of endothelial cells
or on a protein-coated surface.31 While this work was in
progress, the CD43 intracellular domain was shown to interact with the
actin-binding protein members of the ezrin/radixin/moesin family
(ERM).32,33
This work offers evidence for a role of CD43 clustering in neutrophil
motility. Specifically, we show that CD43 cross-linking by antibodies
induces neutrophil polarity and locomotion, concomitant with CD43
relocation to the uropod. We also describe CD43 redistribution to the
uropod of either suspended or adherent neutrophils, following stimulation by chemotactic peptides. Kinetic analysis shows that CD43
redistribution parallels morphologic changes (cell
polarization) and locomotion by a reversible process.
Because CD43 redistribution induced by cross-linking antibodies is
driven by an actomyosin-dependent contractile process,30 we
have investigated which actin-binding protein would link CD43 to the
F-actin network. We have first analyzed in vitro the possible interactions of actin-binding proteins with CD43 by performing affinity
chromatography of neutrophil cytosol on Sepharose beads bearing a
recombinant CD43 intracellular domain. Among the actin-binding proteins
tested, we found a preferential adsorption of  -actinin and moesin.
The physiologic relevance of these interactions has been assessed by
immunofluorescent analysis of adherent motile neutrophils. Moesin, but
not -actinin, is colocalized with CD43 at the rear of neutrophils,
confirming the possible function of moesin as the cytoskeletal linker
of CD43 to the F-actin network.
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Materials and methods |
Buffers and reagents
Hanks' balanced salt solution (HBSS) with or without
Ca++/Mg++ (GIBCO, Paisley, Scotland),
For-NLe-Leu-Phe-Nle-Tyr-Lys-OH 2.25 H2O (fNLPNTL)
and
N-Formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) were from Bachem (Bubendorf, Switzerland) and Sigma
(St. Louis, MO), respectively. The 102 mol/L
(fNLPNTL) or 103 mol/L (fMLP) stock
solutions in DMSO were stored at  20°C and diluted to 109 mol/L (fNLPNTL) or
108 mol/L (fMLP) for cell stimulation. Corresponding
dilutions of DMSO alone had no effect on cellular morphology and
surface molecule distribution. Fluorescein isothiocyanate
(FITC)-conjugated anti-CD11b mAb (clone Bear 1) and FITC-control mouse
IgG1 were from Immunotech (Marseille, France). Anti-CD43 mAbs: clone
MEM59 was from Biogenesis (Poole, UK) and clone L60 from Becton
Dickinson (Bedford, MA). F(ab')2 and Fab fragments of
clone MEM59 anti-CD43 were obtained by pepsin or ficin digestion using
Pierce (Rockford, IL) preparation kits according to the manufacturer's
instructions. Fc fragments were removed by absorption on protein
A-sepharose and purity of F(ab')2 and Fab fragments
assessed by polyacrylamide gel electrophoresis (PAGE) and silver
staining. A rabbit polyclonal antiserum was raised by injecting the
recombinant CD43 intracellular domain (see below) and antibodies
purified by affinity on the recombinant molecule. Antimoesin mAb (clone
38) was from Transduction Laboratories (Lexington, KY), rabbit
polyclonal antimoesin was a gift from Antony Bretscher (Cornell
University, Ithaca, NY). Rabbit anti--actinin was from Sigma
Immunochemicals and antivinculin mAb (clone V284) from Cymbus
Bioscience (Southampton, UK). Polyclonal antifimbrin and antiezrin
antisera were generous gifts from Monique Arpin (Curie Institute,
Paris, France).
FITC- or tetrarhodamine isothiocyanate (TRITC)-conjugated
F(ab')2 fragments of goat antimouse
F(ab')2, FITC- or TRITC-conjugated Fab fragments of
goat antimouse IgG (H+L) or TRITC-conjugated goat antirabbit IgG
(minimal cross-reactivity with human, mouse, and rat serum proteins)
were from Jackson Laboratories (West Grove, PA). Alexa 488-conjugated
goat antimouse IgGs were from Molecular Probes (Eugene, OR). All
antibodies were centrifuged before use, for 20 minutes at
11,600g, to remove immunoglobulin aggregates.
Normal goat serum (NGS) was from Sigma, and fetal calf serum (FCS) was
from GIBCO. Protease inhibitors phenylmethanesulfonylfluoride (PMSF),
diisopropyl-fluorophosphate (DFP), leupeptin, and chymostatin were
from Sigma.
Neutrophil isolation and labeling
Neutrophils were prepared at room temperature from
EDTA-anticoagulated blood from healthy adult volunteers. Neutrophils
were isolated by a 1-step density gradient centrifugation, on
Polymorphprep (Nycomed, Oslo, Norway), according to the manufacturer's
instructions. Residual erythrocytes were lysed in 0.2% NaCl for 1 minute and the osmolarity of the medium then equilibrated by the
addition of an equal volume of 1.6% NaCl. Cells were washed and
resuspended in the culture medium.
When mentioned, cells were labeled sequentially with primary and
secondary antibodies, for 30 minutes on ice, then washed twice with
cold medium. Primary antibodies: anti-CD43 mAbs (clone MEM59,
50µg/mL), anti-CD11b (clone Bear1, 10 µg/mL). Secondary antibodies:
TRITC-conjugated F(ab')2 or Fab fragments of goat antimouse IgG.
Video analysis of cell locomotion and CD43 redistribution after
antibody cross-linking
Prelabeled neutrophils (106/mL) were incubated at
37°C for 15 minutes in HBSS, then pelleted by centrifugation for 5 minutes. Cell suspension (5 µL), containing 106 cells,
was placed between a slide and a round coverslip (25 mm diameter), both
coated with FCS (for 2 hours at 37°C). The slide-coverslip preparation was sealed with paraffin and placed on the heated stage of
a Wild-Leitz Diavert microscope with a 40 × objective. Cells
were recorded on videotape for 10 minutes with a Sony CCD-IRIS, black
and white camera. The outline of the cell at the initial and final
position and the path traveled during 10 minutes was drawn on a
transparency. Cells remaining totally or partially within the outline
of the initial position are defined as stationary cells, and cells
found outside after 10 minutes are defined as locomoting cells. The
proportion of locomoting cells and their speed were determined by morphometry.
Analysis of CD43 distribution on fNLPNTL stimulation of
suspended neutrophils
Prelabeled neutrophils were suspended at 2 × 106
cells/mL and incubated at 37°C for 5 minutes in HBSS with
Ca++/Mg++, then fNLPNTL (109
mol/L) was added or not, for various times at 37°C. The incubation was stopped by addition of 3.7% paraformaldehyde (PFA) in
phosphate-buffered saline (PBS) for 15 minutes at room temperature.
After 2 washes in PBS, cells were cytocentrifuged on a slide, mounted
in fluoprep (BioMerieux, Marcy/Etoile, France) and analyzed by
differential interference contrast (DIC) and fluorescence
microscopy with a Wild-Leitz Diavert microscope.
Analysis of CD43 distribution on fMLP stimulation of adherent
neutrophils
As previously described,34 neutrophils, prelabeled or
not, were allowed to settle on coverslip bottom dishes precoated with 100 µg/mL human fibronectin (Collaborative Biomedical Products, Becton Dickinson), at 37°C for 5 minutes in the
incubation medium (150 mM NaCl, 5 mM KCl, 1 mM
MgCl2, 1 mM CaCl2, 20 mM HEPES, pH 7.4). Cells
were then stimulated with a bath application of 10 nM fMLP at 37°C.
After various times of incubation, cells were fixed in PBS containing
6.6% PFA and lysophosphatidyl-choline palmitoyl (LPCP) 0.1 mg/mL.
Where indicated, cells were labeled after the fixation step for CD43
(postlabeling). Fixed cells were incubated at room temperature for 2 hours in a blocking solution (PBS, 10% FCS). CD43 labeling was
performed at room temperature with an antibody solution at 20 µg/mL
(in PBS, 10% FCS). After washing in PBS, cells were incubated with
TRITC-F(ab')2 fragments of goat antimouse IgG (1/300,
in PBS, 10% FCS) for 30 minutes, then washed in PBS.
DIC and fluorescence microscopy were performed on a Leica DMIRB
microscope (Leica Microscopie and System, GmbH), equipped with a cooled
CCD camera (Frame Transfer Pentamax camera with a 512 × 512
back-thinned EEV chip, No. 512 EFTB (Princeton Instruments) driven by
Image-1/MetaMorph Imaging System software (Universal Imaging, West
Chester, PA). Images were acquired using a 63 × oil immersion
objective (1.4 NA).
Immunofluorescence analysis of CD43 extracellular and intracellular
domain localization.
Nonlabeled neutrophils were activated by fMLP to locomote on
fibronectin for 5 minutes at 37°C. Cells were fixed and
permeabilized (PFA 6.6%, LPCP 0.1 mg/mL in PBS), incubated in the
blocking solution, then labeled by successive 30-minute incubations,
with the antibodies: anti-CD43 extracellular domain (clone L60), Alexa
488-conjugated goat antimouse, rabbit anti-CD43 intracellular domain,
rhodamine-conjugated goat antirabbit antibodies. Fluorescence
microscopy was performed as described above. There was no
cross-reactivity between the different antibodies. The absence of
cross-talk between fluorophores was assessed by the following method.
We defined the acquisition parameters for imaging CD43 "extra"
(Alexa 488 labeling) and CD43 "intra" (rhodamine labeling) using
standard fluorescein and rhodamine filters. We acquired an image of
Alexa 488 single-labeled cells using the rhodamine filter and
reciprocally we acquired an image of rhodamine single-labeled cells
using the fluorescein filter. In both cases there was no observable
fluorescence cross-talk.
Time series acquisition.
Neutrophils were prelabeled with Fab fragments of anti CD43 antibodies
(clone MEM59) and with TRITC-conjugated Fab fragments of goat antimouse
antibodies. Cells were allowed to settle on fibronectin at 25°C for
5 minutes. Cells were then stimulated with a bath application of 10 nM
fMLP. Time series acquisition was performed on the microscope stage at
25°C. Every 10 seconds over a period of 400 seconds, fluorescent
images were recorded as previously described using a
40 × objective.
Adsorption of actin-binding proteins with
rCD43intra-Sepharose beads
Sepharose beads bearing the recombinant intracellular part of CD43
(CD43intra).
A plasmid encoded for the whole intracytoplasmic CD43 sequence
(PCD43intra, sequence shown in Figure 5) and expressed in
Escherichia coli. The region coding for the intracellular
domain of CD43 (from codon 283 to the end) was amplified from the full
length CD43 complementary DNA (cDNA).35 Primers contained
the appropriate restriction sites for in-frame insertion into the
bacterial expression vector QE-12 (Qiagen, Courtaboeuf, France), which
adds an N-terminal 6 His tag to the proteins coded by the
insert. The 6 His-tagged CD43intra was expressed in E coli
strain M15 (Qiagen) and purified as described on a nickel-charged
chelating-agarose column (ProBond from Invitrogen, Leek, the
Netherlands).36 The purified protein appeared on sodium
dodecyl sulfate (SDS)-PAGE as a single 18 kd band, as expected from the
sequence of the insert. This CD43intra preparation (1.5 mg), or 1.5 mg
bovine serum albumin (BSA, Sigma) as a control, was coupled to 1 mL
CNBr-Sepharose (Pharmacia, Upsala, Sweden) according to the
manufacturer's instructions.
Cytosol adsorption.
Cytosol was obtained by sequential centrifugation after neutrophil
lysis by nitrogen cavitation, as described.37 The cytosol was concentrated on amicon PM10 and frozen at
80°C. One milliliter of the concentrated
cytosol (corresponding to 2.5 × 108 neutrophils)
was first precleared for 1 hour with uncoupled Sepharose, then
incubated with 150 µL of CD43intra- or BSA-Sepharose. After 1 hour at
4°C on roller and 5 washes in relaxation buffer (5 × 1 mL),
specific elution was performed with 200 µL of recombinant CD43 (0.8 mg) followed by 2 times 200 µL of relaxation buffer. Elution was
completed with 0.5 mol/L NaCl. All eluted fractions were boiled in
unreduced sample buffer and analyzed by SDS-PAGE and Western blotting,
using various antibodies directed to actin-binding proteins.
Immunofluorescence analysis of -actinin and moesin
localization in relation to CD43 distribution
Neutrophils were allowed to locomote on fibronectin as described
above. After different times of stimulation by fMLP at 37°C, cells
were fixed and permeabilized by a 5-minute incubation (in 6.6% PFA and
0.1 mg/mL LPCP in PBS). Cells were then washed in 0.1 mol/L glycine in
PBS and incubated for 2 hours in a blocking solution (PBS, 10% FCS).
The following antibodies were then added sequentially (in PBS, 10%
FCS): anti-CD43 (clone L60, 20 µg/mL), Alexa 488-conjugated goat
antimouse (1/600), polyclonal rabbit antimoesin (1/300),
TRITC-conjugated goat antirabbit IgG (1/300). Each labeling step was
separated by 3 washes of 5 minutes in PBS. -Actinin labeling was
performed after cellular fixation for 5 minutes in 6.6% PFA,
glutaraldehyde 0.7%, and saponin 250 µg/mL in PBS. Cells were then
washed and incubated in the blocking solution. Cells were labeled with
anti--actinin antibodies (1:50 in PBS, 10% FCS) and with
TRITC-conjugated goat antirabbit IgG (1:300). Cellular fixation by
glutaraldehyde preserves the structure of -actinin but destroys CD43
epitopes, making double labeling impossible.
DIC and fluorescence microscopy were performed as described above. No
cross-reactivity between the different antibodies and no cross-talk
between the fluorophores were noted. Double-labeled cells were analyzed
by confocal microscopy using an Axiovert 100 mol/L inverted microscope
equipped with an LSM 510 laser scanning unit and a 63 × 1.4NA
plan Apochromat objective (Carl Zeiss). CD43 fluorescence (Alexa 488)
was excited with a 25 mW argon laser emitting at 488 nm, and the
emission was collected by 650 long pass filter. Moesin
fluorescence (rhodamine) was excited with a 1.0 mW helium/neon laser
emitting at 543 nm and was collected by a 530 to 560 long
pass filter. The 2 channels were scanned alternatively, having only 1 laser and 1 detector channel on at each time. No cross-talk between the
fluorophores has been detected, using the method described above for
CD43 "extra" and "intra" double labeling.
| |
Results |
CD43 cross-linking by antibodies induces neutrophil locomotion
We have previously shown that antibody cross-linking of CD43 at
37°C on suspended neutrophils induces the redistribution of CD43
into a cap structure and a front-to-tail polarization in 15% to 25%
of the cells.30 To assess if CD43 cross-linking results in
neutrophil locomotion, video microscopic analysis was performed on
neutrophils treated with anti-CD43 antibodies, in the absence of
stimulating agent. Cells were either unlabeled or treated with a mouse
monoclonal anti-CD43 antibody (clone MEM59) followed by a bivalent
F(ab')2 secondary antibody (to cross-link CD43), or a
monovalent Fab secondary antibody (to avoid cross-linking). Both
F(ab')2 and Fab secondary antibodies were TRITC
conjugated. After 15 minutes of incubation at 37°C in HBSS
Ca++/Mg++, to allow cross-linking and
redistribution of membrane molecules, cells were mounted between
FCS-coated coverslip and slide and video-analyzed, as described in
"Materials and methods".
The results of 3 independent experiments are shown in Table
1. CD43 cross-linking resulted in
locomotion of 73% neutrophils, as compared to the 22% of
spontaneously motile cells in the absence of antibody. Induction of
locomotion required CD43 cross-linking because it was not induced if
cross-linking was avoided by using monovalent Fab secondary antibodies
(22% of locomotion). Similar results were obtained with
F(ab')2 fragments of the anti-CD43 antibody, showing
that this induction of locomotion is independent of Fc receptors.
By contrast, cross-linking of CD11b/CD18 integrins by anti-CD11b
antibodies (clone Bear 1) and F(ab')2 secondary
antibodies resulted neither in neutrophil polarization nor in
locomotion (data not shown). Cross-linked CD11b molecules became
clustered in patches on round cells (Figure
1). The anti-CD11b were nonblocking antibodies and their cross-linking did not prevent cells from adhering
and locomoting on fibronectin when stimulated by fMLP (our unpublished
data).
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| Fig 1.
Combined DIC and fluorescence image (A), DIC image (B),
and fluorescence images (A', B') of living neutrophils.
Cells were labeled at 4°C with anti-CD43 mAbs (A, A') or
anti-CD11b mAbs (B, B') and TRITC-conjugated
F(ab')2 fragments of secondary antibodies. They were
analyzed at 37°C as described in "Materials and methods". (A,
A') Polarized, locomoting neutrophils with CD43 caps located at
the uropods. (B, B') Round, stationary neutrophils with uniformly
patched CD11b. Scale bar = 10 µm. (Panel A, the top left corner
cell is round, nonpolarized, and nonmotile.)
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CD43 redistribution is related to neutrophil
polarization and locomotion
Cell locomotion and cell morphology are functionally related because
polarization is a prerequisite for locomotion. The relationship between
CD43 redistribution and the cell morphology changes and locomotion was
explored. From movies obtained during the locomotion assay (from the
previous paragraph), cells were examined for the different patterns of
CD43 distribution (cap, patch, uniform), for the corresponding cell
morphologies (distinct polarization or not), and for their locomotor activity.
As shown in Table 2, during CD43
cross-linking experiments, CD43 molecules were capped on 94%
(± 4) of the total cell population; 78% (± 13) of these
capped cells were polarized and motile, with CD43 caps always located
at the uropod. Only 6% (± 4) of the total cell population had
redistributed CD43 in a patch structure; 17% (± 29) of
this patched cell population was motile (which represents only 1% of
the total cell population), regardless of whether cells were polarized
or not. In summary, cell polarity and locomotion appear to be related
to CD43 relocation at the uropod. The same correlation was
observed for spontaneously migrating cells (in the case of CD43
labeling with monovalent antibodies).
The proportion of polarized cells in this system was higher than the
previously reported 15% to 25% of front-tail polarized cells,
observed during CD43 cross-linking on cells in
suspension.30 Indeed, we observed that, after CD43
cross-linking, the contact of neutrophils with serum- or gelatin-coated
(data not shown) slide and coverslip increased the number of polarized cells.
Chemotactic stimulation of neutrophils induces CD43
redistribution to the cellular uropod, concomitant with cell
polarization and locomotion
Chemotactic stimulation of suspended neutrophils.
To assess if chemotactic peptide-induced cell polarization results in
surface redistribution of CD43, we analyzed the effect of fNLPNTL on
suspended cells, which had been fluorescently labeled with anti-CD43
mAb (clone MEM59) and TRITC-Fab fragments of secondary antibodies.
After various times of incubation with or without chemotactic peptide
(fNLPNTL 109 mol/L) at 37°C, cells were PFA
fixed and analyzed by microscopy. Three distinct patterns of CD43
distribution (uniform, patched, or capped) were observed that each
corresponded to a distinct cellular morphology (round smooth, nonpolar
with membrane projection, or polarized, respectively). Figure
2 summarizes 3 independent experiments.
Initially (time 0), more than 80% of cells were round without
protrusions and showed a uniform CD43 distribution. After 1 minute of
chemotactic stimulation, 84% of the cells became ruffled and showed a
patched CD43 distribution. Patching of CD43 clearly preceded cell
polarization, which was observed in a majority of cells after 15 minutes of stimulation. After 30 minutes of incubation at 37°C with
the chemotactic peptide, 75% of the cells were polarized with CD43
caps located at the uropod, whereas this phenotype was observed in only
8% of unstimulated cells. A similar redistribution of CD43 to the
uropod was observed when the secondary antibody was omitted, or when
cells labeled with Fab or F(ab')2 fragments of the
primary anti-CD43 monoclonal antibody were stimulated by fNLPNTL at
37°C (data not shown). By contrast, in the absence of fNLPNTL, CD43
labeling with anti-CD43 mAb and monovalent secondary antibody did not
induce either CD43 redistribution or morphologic changes when
neutrophils were incubated at 37°C for 0 to 30 minutes.
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| Fig 2.
Patterns of cell morphology.
Cells were labeled with anti-CD43 mAb and TRITC-Fab fragments of
secondary antibodies, then incubated in suspension at 37°C with or
without fNLPNTL (109 mol/L). After 1, 3, 5, 15, and 30 minutes, cells were PFA fixed and the different
patterns of cell morphology associated with CD43 distribution were
recorded by DIC and fluorescence microscopy. The results are expressed
in percent (mean ± SD) of the different patterns observed in 3 independent experiments.
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Chemotactic stimulation of adherent neutrophils.
We then investigated if CD43 was also redistributed to the uropod when
neutrophils were crawling on an adherent surface. Unlabeled neutrophils, adherent to human fibronectin, were stimulated with 10 nM
fMLP for various times. Cells were then fixed and postlabeled with anti-CD43 as described in "Materials and methods". As
shown in Figure 3A, 30 seconds after the
chemotactic peptide stimulation, neutrophils presented a ruffled
morphology and CD43 was partially redistributed. After 60 seconds,
neutrophils started to spread on the substratum and CD43 redistribution
progressed. After 2 minutes of stimulation, locomoting neutrophils were
polarized and had redistributed most of their CD43 to their uropods,
leaving a faint CD43 labeling of the cell body.
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| Fig 3.
CD43 distribution in neutrophils developing locomotory
activity on fibronectin.
(A) Neutrophils were allowed to settle on fibronectin for 5 minutes at
37°C and were stimulated by fMLP for 30, 60, and 120 seconds. Cells
were then fixed and labeled for CD43. (B) After 5 minutes of locomotion
on fibronectin, at 37°C, cells were fixed and double labeled for
CD43 extracellular domain (Alexa 488) and for CD43 intracellular domain
(rhodamine). Scale bars = 10 µm.
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To demonstrate that CD43 uropod localization was not the result of a
specific shedding of CD43 at the leading edge, we analyzed the
respective distribution of CD43 intracellular and extracellular domains
on neutrophils crawling on fibronectin. We performed double labeling of
CD43 extracellular domain (clone L60, which does not label the residual
cell-associated CD43 after CD43 shedding23) and of CD43
intracellular domain (with polyclonal antibodies recognizing shedding-independent intracellular epitopes). As shown in Figure 3B, both fluorescent markers were very similarly distributed
at the uropod. This result confirms the hypothesis of the
redistribution of CD43 toward the uropod.
To follow CD43 redistribution by video microscopy in real time,
neutrophils were prelabeled for CD43 (with Fab fragments of primary and
secondary antibodies) before the locomotion assay. Neutrophils were
induced to locomote on the microscope stage at 25°C to slow down
the process, and a fluorescent image was recorded every 10 seconds for
400 seconds. Figure 4A represents the
fluorescent images (at 60-second intervals) of a representative time
series acquisition of migrating neutrophils. Here again CD43 clearly redistributed to the uropod. Until 120 seconds after fMLP stimulation, neutrophils were stationary but displayed a ruffled morphology with
CD43 partially redistributed. At 140 seconds after fMLP stimulation, neutrophils started to spread, then to locomote. This induction of
locomotion paralleled a clear redistribution of CD43 to the uropod.
We obtained the same result when the coverslips were coated with human
vitronectin (data not shown).
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| Fig 4.
Time series of neutrophils developing or losing polarity
and locomotor activity on a fibronectin-coated surface.
Cells were labeled for CD43 (with Fab fragments of primary and
secondary antibodies) at 4°C, then allowed to settle on the
fibronectin-coated surface for 5 minutes at 25°C. fMLP (10 nM) was
applied and fluorescence images were acquired every 10 seconds. (A)
Images have been selected at 60-second intervals. The lines in the last
panel represent the tracks of the cell centroids during the 320 seconds
of stimulation. (B) The first image represents the uropod of a
neutrophil after 5 minutes of locomotion. The cells were then washed in
a medium without fMLP. In B, images have been selected at the indicated
times (0, 60, 140, and 190 seconds). Scale bar = 10 µm.
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Figure 4B shows that CD43 redistribution is reversible. Cells were
labeled for CD43 (with Fab fragments of primary and secondary antibodies), then allowed to locomote on fibronectin as described above. After 5 minutes of locomotion, cells were washed and incubated in the medium without fMLP to allow the cells to revert to a spherical shape. The first panel of Figure 4B represents the fluorescent uropod
of a cell before the removal of fMLP. After the washes, pictures were
taken at the indicated times (0, 60, 100, 140, and 190 seconds) over a
period of 190 seconds. As shown in Figure 4B, CD43 molecules originally
clustered at the uropod progressively returned to a uniform
distribution all around the cell when fMLP was washed off.
Actin-binding proteins interact with the intracytoplasmic tail
of CD43
Neutrophil cytosol was applied to a Sepharose column bearing either
the recombinant intracellular portion of CD43 (Figure 5) or BSA, which was chosen as a control
because of its similar charge (BSA isoelectric point of 5.4-5.8, as
compared to 5.81 for rCD43intra). Among proteins specifically eluted
with purified rCD43intra from CD43intra-Sepharose (Figure
6, lane C), but not the BSA-Sepharose
column (Figure 6, lane B), we identified -actinin and moesin by
Western blotting. An unidentified band, slightly below the moesin band,
reacted nonspecifically with mouse IgGs. Faint bands were also observed
in the CD43intra eluate but not the BSA eluate with antiezrin and
antiradixin antibodies (data not shown). Because of the high level of
homology (70-80%) between ERM proteins, we cannot exclude a
cross-reaction of these antibodies with moesin. Western blot analysis
of neutrophil cytosol showed that it mainly contains moesin, a very low
amount of ezrin, and no detectable radixin (data not shown).
Antivinculin revealed a similar eluted band in samples eluted either
from CD43intra or the BSA eluate, showing nonspecific binding of
vinculin to Sepharose columns. Fimbrin was not detected in either
eluate, although clearly detected in the whole cytosol with the
antifimbrin polyclonal antibody (data not shown).
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| Fig 5.
Sequence of recombinant CD43intra (137aa).
The sequence of CD43 intracytoplasmic tail begins at threonine n22. It
was translated from DNA sequence CD43 (base 943-1293).
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| Fig 6.
Analysis of actin-binding proteins binding to
rCD43intra-Sepharose.
Whole cytosol (A) and the 2 first fractions eluted with purified
rCD43intra from BSA-Sepharose (B) or from rCD43intra-Sepharose (C) were
submitted to 7.5% acrylamide-PAGE electrophoresis and analyzed by
Western blotting with control mouse IgG1, anti--actinin or
antimoesin.
|
|
Localization of actin-binding proteins during CD43
redistribution
Neutrophils were allowed to locomote for various time at 37°C on
fibronectin (30, 60, and 140 seconds). Cells were then fixed and
permeabilized and blocked with FCS, then double immunofluorescence of
CD43 and moesin was carried out. Figure 7A
represents the matching DIC, CD43 fluorescence (Alexa 488), and moesin
fluorescence (TRITC) images. We observed a colocalization between CD43
and moesin, both molecules following the same kinetics of
redistribution. After 30 seconds of stimulation they were both
partially redistributed and colocalized, before any morphologic
polarization. When cells were polarized and motile, moesin and CD43
colocalized at the uropod, whereas a small proportion of these
molecules remained in other cell areas. To more clearly delineate CD43
and moesin distribution, we performed confocal microscopic analysis of
double-labeled cells as shown in Figure 7B. Nineteen images were
acquired every 0.5 µm. Panels A and B represent a summation of the 19 planes of the stack, for CD43 and for moesin imaging, respectively.
Panels C and D represent a single plane from the middle of the stack for CD43 and for moesin imaging, respectively. Moesin labeling was
located very close to the plasma membrane and again perfectly colocalized with CD43.
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| Fig 7.
CD43 (Alexa 488) and moesin (TRITC) double labeling of
migrating neutrophils.
Cells, stimulated by fMLP, were allowed to locomote on fibronectin as
described in "Materials and methods". Cells were then fixed and
permeabilized after 30, 60, or 140 seconds of locomotion. After washes
and saturation steps, cells were double labeled for CD43 and moesin. A
represents DIC and fluorescent images as indicated. B represents
confocal images of CD43 (A, C) and of moesin (B, D). A and B represent
a summation of the 19 planes of the stack; C and D represent a single
plane from the middle of the stack. Scale bars = 10 µm.
|
|
Double labeling of CD43 and -actinin could not be performed because
the respective staining conditions were not compatible. However,
parallel staining of these molecules allowed us to exclude their
colocalization. Figure 8 shows that
-actinin is located at the leading edge of polarized neutrophils and
is not detectable in the uropod, where CD43 molecules are concentrated.
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| Fig 8.
DIC and fluorescent images of neutrophils labeled
for -actinin.
Cells, stimulated by fMLP, were allowed to locomote on fibronectin for
2 minutes. Cells were then fixed and permeabilized and labeled for
-actinin. Arrows indicate the position of the lamellipodium;
asterisk indicates the uropod position. Scale bar = 10µm.
|
|
| |
Discussion |
Antibody cross-linking of CD43 induces CD43 capping and triggers
neutrophil polarization and locomotion. This induction of migration is
associated with a polar redistribution of CD43 to the uropod. These
phenomenon are Fc independent because they were also observed when
F(ab')2 fragments of anti-CD43 antibodies were used
instead of whole IgGs. It is the first description of an induction of
neutrophil locomotion by antibody cross-linking of a defined membrane
molecule, namely CD43, in the absence of other stimulating agents.
Under the same conditions, antibody cross-linking of other receptors
such as the integrin CD11b/CD18 and HLA molecules (data not shown)
results, in our hands, in a patched distribution of the receptors and
does not induce either morphologic changes or cell motility.
This induction of cell locomotion by cross-linking of a surface
molecule is reminiscent of the motile behavior of B lymphocytes triggered by the capping of membrane immunoglobulins.38
Also, antibody cross-linking of L 2 and 41 integrins has
been reported to stimulate T-lymphocyte locomotion, but only on
surfaces coated with extracellular matrix proteins, suggesting that a
costimulation by adhesive substrates is required in that
process.39 Cross-linking by antibodies may mimic
cross-linking by substratum-bound molecules. More recently,
simultaneous cross-linking of CD3 and CD2 by antibodies was shown to
result in a clustering of both molecules at the T-lymphocyte uropod and
to stimulate cell migration within 3-dimensional collagen matrices,
presumably via specific T-cell activation.40 We did not
address here the question of a possible effect of adhesion on CD43
redistribution induced by antibody cross-linking. We found that the
proportion of polarized cells following anti-CD43 cross-linking increased from 15% to 25% in suspended neutrophils30 to
73% when cells were placed between 2 coated slides for locomotion assays. This suggests that the contact with a surface facilitates polarization and locomotion. A similar observation has been reported during the capping of surface Igs, where B-lymphocyte polarization was
decreased if cells were not allowed to settle onto a
surface.41
A second important point is that neutrophil stimulation by chemotactic
peptide, in the presence or in the absence of an anti-CD43 mAb, results
in CD43 membrane redistribution to the uropod and cell polarization,
CD43 molecules being always concentrated at the uropods. This
redistribution is observed for both suspended and adherent cells. A
proportion of CD43 molecules are known to be shed from neutrophil
surface during neutrophil activation and adhesion.23 The
observed CD43 asymmetric distribution could thus result from a
localized shedding of CD43 molecules at the leading edge. Our results
using a polyclonal antibody directed against the intracellular domain
of CD43 exclude this hypothesis and show that all CD43 molecules are
concentrated at the uropod (whether they had released their
extracellular portion or not). CD43 redistribution to uropods has also
been described on T lymphocytes polarized by chemokines or
spontaneously polarized when in contact with endothelial
cells.31 A similar clustering of another membrane sialomucin, PSGL-1, has recently been observed at the uropod of neutrophils activated by nanomolar concentrations of
fMLP.42 We conclude that the leukocyte polarization process
may include lateral redistribution of specific membrane glycoproteins
to the uropod, although the function of this phenomenon is still unknown.
It is tempting to establish a connection between the 2 following
observations: (1) CD43 redistribution induced by cross-linking antibodies results in cell locomotion, with CD43 caps located at the
uropod, with CD43 redistribution preceding and appearing to trigger
cell locomotion; and (2) spontaneously locomoting cells or cells
activated by chemotactic peptides redistribute CD43 to the uropods. In
both cases, the induction of locomotion results in CD43 redistribution
to the uropod. Altogether, these sets of observations strongly suggest
that CD43 redistribution and motility are closely related processes.
CD43 clustering on the cell membrane is mediated by an
actomyosin-dependent contractile process during CD43 cross-linking by
antibodies.30 We investigated the interactions of CD43 with cytoskeletal proteins likely to be involved in CD43 redistribution during neutrophil motility. Among the various cytoskeleton molecules present in neutrophil cytosol, we found that moesin and -actinin, but not vinculin or fimbrin, were preferentially adsorbed on the recombinant intracellular portion of CD43 bound to Sepharose. Immunofluorescence analysis, however, precluded that an interaction of
CD43 with -actinin would mediate CD43 lateral redistribution at the
cell surface of neutrophils. Indeed -actinin was localized at the
leading edge of the polarized neutrophil and was not detected in the
CD43-containing uropod. On the other hand, a nearly perfect colocalization of moesin with CD43 was observed. We conclude that moesin bridges CD43 to the cytoskeleton and mediates CD43
redistribution to the uropod, or that CD43 and moesin both belong to a
membrane and submembrane structure that is organized during neutrophil motility. This conclusion is in accordance with several reported data
showing that ERM family members appear as F-actin linkers to the plasma
membrane of neutrophils,43 ERM colocalize with CD43 and
CD44 at the cleavage furrow of dividing lymphocytes,44 and
ERM molecules interact specifically with CD44 and CD43
intracellular domains.32,33
Our observation of a relationship between CD43 redistribution to the
tail and neutrophil motility is relevant to the general function of
uropods in cell locomotion. Two hypothesis based on CD43 and motility
properties can be put forward:
1. In the antiadhesive hypothesis, a repulsive uropod would facilitate
the release of adhesion at the rear of the cell. The concentration of a
negatively charged repulsive molecule such as CD43 in the uropod could
fulfill this function. Furthermore, CD43 redistribution may clear
negatively charged antiadhesive molecules from the leading edge of
polarized neutrophils.24,45
2. In the adhesive hypothesis, an adhesive uropod would allow the cell
to lean on the substratum when exerting forward traction forces.
Indeed, transient interactions of the uropod with collagen fibers have
been described on T cells migrating through 3-dimensional collagen
lattices.46 In this model, CD43 would interact positively with the substratum. This would allow, as previously
proposed,46 "the uropod to stabilize the cell's
position for the formation of new contacts at the leading edge, pushing
of the cell body forward." Putative ligands of CD43 could be the
positively charged heparin-binding domains present on most proteins of
the extracellular matrix, such as fibronectin, laminin, or vitronectin.
Each of these hypotheses may be coupled to the idea of an important
role of rear contractions in cell movement. CD43 has been shown to
concentrate in the cleavage furrow of dividing cells,44 where strong myosin II-dependent contractions occur. CD43 localization in the myosin II-rich uropod during cell locomotion suggests that the
anchoring of actomyosin fibers to transmembrane molecules such as CD43
could promote propulsive contractions involved in locomotion. Indeed,
whether CD43 was cross-linked by antibodies or not, cell migration was
associated with a striking localization of CD43 molecules at the rear
of the cell, a localization that never changed during locomotion.
| |
Acknowledgments |
We thank Dr Verena Niggli (Institute of Pathology, Bern, Switzerland),
Dr Monique Arpin (Curie Institute, Paris, France), and Dr Anthony
Bretscher (Cornell University, Ithaca, NY) for helpful discussion; Drs
Lynda Pierini and Robert Eddy for their critical reading of the
manuscript; Dr Minoru Fukuda for providing the CD43
cDNA; and Virginie Cuelhe and
Nicole Bureaud for expert technical assistance.
| |
Footnotes |
Submitted July 13, 1999; accepted December 9, 1999.
Supported by fellowships ASTF 8598 and ASTF 8713 to S.S. from
the European Molecular Biology Organization (EMBO), Heidelberg, Germany; and N° MLD/CM/ML-CN3/97 from the Association pour la Recherche contre le Cancer (ARC), Paris, France. This work was also
supported in part by National Institutes of Health grant GM34770
(F.R.M.).
Reprints: Stephanie Seveau, Department of Biochemistry, Weill
Medical College of Cornell University, 1300 York Avenue, New York, NY
10021; e-mail: sseveau{at}mail.med.cornell.edu.
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.
| |
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Abstract
Introduction