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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 12 (June 15), 1998:
pp. 4632-4644
CD43 Interacts With Moesin and Ezrin and Regulates Its
Redistribution to the Uropods of T Lymphocytes at the Cell-Cell
Contacts
By
Juan M. Serrador,
Marta Nieto,
José L. Alonso-Lebrero,
Miguel A. del Pozo,
Javier Calvo,
Heinz Furthmayr,
Reinhard Schwartz-Albiez,
Francisco Lozano,
Roberto González-Amaro,
Paloma Sánchez-Mateos, and
Francisco Sánchez-Madrid
From the Servicio de Inmunología, Hospital de la Princesa,
Universidad Autónoma de Madrid, Madrid, Spain; the Department of
Pathology, Stanford University, Stanford, CA; the School of Medicine,
University of San Luis Potosí, San Luis Potosí, Mexico;
the Servicio de Inmunología, Hospital General Universitario
Gregorio Marañón, Madrid, Spain; the Servei d'Immunologia,
Hospital Clinic, Barcelona, Spain; and the Tumor Immunology Program,
German Cancer Research Center, Heidelberg, Germany.
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ABSTRACT |
Chemokines as well as the signaling through the adhesion molecules
intercellular adhesion molecule (ICAM)-3 and CD43 are
able to induce in T lymphocytes their switching from a spherical to a
polarized motile morphology, with the formation of a uropod at the rear
of the cell. We investigated here the role of CD43 in the regulation of
T-cell polarity, CD43-cytoskeletal interactions, and lymphocyte
aggregation. Pro-activatory anti-CD43 monoclonal antibody (MoAb)
induced polarization of T lymphocytes with redistribution of CD43 to
the uropod and the CCR2 chemokine receptor to the leading edge of the
cell. Immunofluorescence analysis showed that all three
ezrin-radixin-moesin (ERM) actin-binding proteins
localized in the uropod of both human T lymphoblasts stimulated with
anti-CD43 MoAb and tumor-infiltrating T lymphocytes. Radixin localized
at the uropod neck, whereas ezrin and moesin colocalized with CD43 in
the uropod. Biochemical analyses showed that ezrin and moesin coimmunoprecipitated with CD43 in T lymphoblasts. Furthermore, in these
cells, the CD43-associated moesin increased after stimulation through
CD43. The interaction of moesin and ezrin with CD43 was specifically
mediated by the cytoplasmic domain of CD43, as shown by precipitation
of both ERM proteins with a GST-fusion protein containing the CD43
cytoplasmic tail. Videomicroscopy analysis of homotypic cell
aggregation induced through CD43 showed that cellular uropods mediate
cell-cell contacts and lymphocyte recruitment. Immunofluorescence
microscopy performed in parallel showed that uropods enriched in CD43
and moesin localized at the cell-cell contact areas of cell aggregates.
The polarization and homotypic cell aggregation induced through CD43
was prevented by butanedione monoxime, indicating the involvement of
myosin cytoskeleton in these phenomena. Altogether, these data indicate
that CD43 plays an important regulatory role in remodeling T-cell
morphology, likely through its interaction with actin-binding proteins
ezrin and moesin. In addition, the redistribution of CD43 to the uropod region of migrating lymphocytes and during the formation of cell aggregates together with the enhancing effect of anti-CD43 antibodies on lymphocyte cell recruitment suggest that CD43 plays a key role in
the regulation of cell-cell interactions during lymphocyte traffic.
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INTRODUCTION |
THE POLARIZATION OF cell surface and
intracellular structures is important during differentiation for immune
response induction and cognate interactions between immune and other
cells.1 T lymphocytes migrate through lymphocyte organs and
extravasate into inflammatory sites to accomplish effector functions by
sequential engagement of adhesion and signaling
receptors.2-4 Effector activity of these cells involves
plasma membrane clustering of LFA-1 and CD4 and cytoskeletal
reorganization of microfilaments, microtubules, and
talin.5-7 Migrating cells also display other polarized
features, such as the localization of chemokine receptors towards the
leading edge of the cell,8,9 an area of the cell that
undergoes dynamic changes in the organization of the actin
cytoskeleton,10 and the uropod at the opposite pole where
intercellular adhesion molecules (ICAMs), CD44, and
CD43 molecules are clustered.11
CD43 (leukosialin, sialophorin) is a transmembrane glycoprotein that
bears a heavily O-glycosylated extracellular N-terminal region and
that is expressed by hematopoietic cells.12-15 In humans, low and high molecular weight (113 to 120 and 125 to 135 kD,
respectively) glycoforms of CD43 are generated by differential
O-glycosylation.16 Although its high negative charge may
provide a repulsive barrier that interferes with cellular adhesion
phenomena, this molecule was reported to be a counter-receptor for
ICAM-1 and major histocompatibility complex (MHC) class
I molecules.17,18 In this regard, CD43 has been described
as promoting leukocyte aggregation.19,20 However, the
functional analysis of T cells from CD43-deficient mice showed an in
vitro increase in their proliferative response to mitogens and an
enhanced homotypic cell aggregation capability, suggesting that CD43
could act as an antiadhesive molecule.21 In accordance,
CD43 expression in target cells diminishes its susceptibility to
cytolysis by T cytotoxic and natural killer cells.22,23
Thus, the role of CD43 as an adhesion receptor still remains
controversial. However, it is now clear that CD43 plays a role in the
regulation of T-lymphocyte adhesiveness and cellular
activation.24,25 In this regard, we previously described the regulatory role of CD43 on integrin-mediated T-cell adhesion to
endothelium and proteins of extracellular matrix.26 On the other hand, the existence of a putative endothelial cell ligand for
CD43 has recently been proposed, suggesting a role for this molecule in
the selective traffic of T cells through lymphoid tissues.27
Interaction of cell adhesion receptors with cytoskeletal proteins may
propagate signals for cellular reorganization.1 Previous studies have demonstrated that CD43 colocalized with the
ezrin-radixin-moesin (ERM) family of plasma membrane-cytoskeleton
cross-linkers implicated in the formation of membrane dynamic
structures such as filopodia, microvilli, and
microspicules.28-30 During cytokinesis of cell division,
these proteins interact directly or indirectly with the cytoplasmic
domain of CD43 in the cleavage furrow.31 In this report, we
explored the cellular localization of ERM proteins and CD43 in
polarized motile lymphocytes as well as in cell aggregates induced by
anti-CD43 antibodies. In addition, we studied the possible participation of CD43 in the formation of cell-cell contacts. Evidence
is presented for the association of moesin and ezrin with CD43 and its
coredistribution to the cellular uropods during T-cell aggregation and
cell recruitment induced through CD43.
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MATERIALS AND METHODS |
Antibodies and reagents.
The anti-ICAM-3 HP2/19 (IgG2a), anti-CD43 HP2/21 (IgM) and TP1/36
(IgG1), anti-CD44 HP2/9 (IgG1), anti-CD45 RP2/21 (IgG1), anti-CCR2
MCP-1R03 (IgG1), antimoesin/radixin 38/87 (IgG1), and anti-HLA-A,B
W6/32 (IgG1) monoclonal antibodies (MoAbs) have been described.9,32-36 The fluorescein isothiocyanate
(FITC)-conjugated 2D1 anti-CD45 (IgG1) MoAb was purchased from Becton
Dickinson (San Jose, CA). The moesin-specific polyclonal antiserum
(pAb) 95/2 was raised in rabbits by immunization with recombinant human moesin and purified by affinity chromatography.37 The
ezrin-specific pAb 90/3 was also raised in rabbits by immunization with
purified human ezrin.38 The affinity-purified polyclonal
antibodies 464 and 457, raised against unique peptides from murine
ezrin and radixin, respectively,39 were kindly provided by
Dr F. Solomon (Department of Biology and Center for Cancer Research,
MIT, Cambridge, MA). Recombinant human (rh) moesin was obtained as
described.38 Butanedione monoxime was purchased from Sigma
Chemical Co (St Louis, MO). The 80-kD fibronectin fragment (FN80) was a
generous gift of Dr A. García-Pardo (Centro de Investigaciones
Biológicas, Madrid, Spain). The chimeric ICAM-1-Fc, consisting of
the extracellular domains of ICAM-1 and the Fc region of
IgG1, was obtained as described.11
Cells.
Human T lymphoblasts were prepared from peripheral blood mononuclear
cells by treatment with phytohemagglutinin (PHA) 0.5% (Pharmacia,
Uppsala, Sweden) for 48 hours. Cells were then washed and cultured in RPMI 1640 (Flow Laboratories, Irvine, Scotland) containing 10% fetal calf serum (FCS; Flow
Laboratories) and 50 U/mL interleukin-2 (IL-2) kindly provided by
Eurocetus (Madrid, Spain). T lymphoblasts cultured by
10 to 15 days were used in all experiments. These cells were analyzed
by flow cytometry and their phenotype was 98% CD3 and 99% CD45RO;
also, they show a heterogeneous expression of chemokine receptors CCR2
and CCR5.9,40 Melanoma cells and CD8+
tumor-infiltrating lymphocytes (TIL) were isolated from melanoma specimens from patients with metastatic melanoma (Department of Pathology, Hospital General Universitario Gregorio Marañón, Madrid, Spain). These cells were cultured in AIM-V medium (Flow Laboratories) containing 10% HY-ultroser serum (Pharmacia) and 5,000 U/mL IL-2.40 Jurkat cells were grown in complete medium without the addition of IL-2.
Immunofluorescence microscopy.
Immunofluorescence experiments were performed as
described.11 Briefly, 1 × 106 T
lymphoblasts were incubated in flat-bottommed, 24-well plates (Costar
Corp, Cambridge, MA) in a final volume of 400 µL complete medium on
coverslips coated with FN80 at 20 µg/mL. The polarization-inducing anti-CD43 HP2/21 (supernatant) and TP1/36 at 4 µg/mL were added and
cells were allowed to remain in a cell incubator at 37°C and 5%
CO2 atmosphere. After 30 minutes, cells were fixed with
3.7% (wt/vol) paraformaldehyde in phosphate-buffered saline (PBS), pH
7.4, at room temperature and then rinsed in TBS. Fixed cells were
incubated with specific MoAb or pAb. After washing, cells were stained
with an FITC-labeled rabbit F(ab) 2 antimouse IgG, donkey antirabbit IgG (Pierce, Rockford, IL), or
Cy3-tagged goat antimouse IgG (Amersham, Pittsburgh, PA). For double
immunofluorescence analysis, cells were saturated with 10% normal
mouse serum in TBS. Coverslips were then incubated with biotinylated
MoAb, followed by washing and labeling with either FITC-extrAvidin
(Sigma) or Cy3-streptavidin.
Cell aggregation assays.
Homotypic cell aggregation was performed as previously
described.33 Briefly, T lymphoblasts were incubated in
flat-bottommed, 24-well microtiter plates (Costar Corp) at 4 × 106/mL in a final volume of 0.5 mL of complete medium.
Then, 1:20 dilution of anti-CD43 HP2/21 supernatant was added and cells
were incubated at 37°C and 5% CO2 atmosphere.
Aggregation was then determined at different periods of time by direct
visualization of the plate with an inverted microscope and counting the
free cells of at least five randomly chosen areas of 0.025 mm2. Results were expressed as a percentage of aggregated
cells.
For immunofluorescence staining, after either 10 minutes (small cell
aggregates) or 30 minutes (large cell aggregates) of incubation, cells
were fixed with 3.7% paraformaldehyde in PBS for 10 minutes at room
temperature and rinsed in TBS. To directly visualize CD43, cells were
stained with a 1:50 dilution of an FITC-labeled rabbit
F(ab)2 antimouse IgG (Pierce). To visualize moesin,
after cell permeabilization with 0.1% Triton X-100, cell aggregates
were incubated with the biotinylated antimoesin 38/87 MoAb followed by
washing and labeling with 1:1,000 dilution of Cy3-streptavidin. Cells
were observed using a Nikon Eclipse-800 photomicroscope with 60×
and 100× oil immersion objectives. Preparations were photographed
with Ektachrome 400 film (Eastman Kodak Co, Rochester,
NY).
Time-lapse videomicroscopy.
Video microscopy analysis was performed using a Nikon Diaphot 300 inverted microscopy equipped with a Sony SSC-M350CE CCD black and white
videocamara coupled to a Sony SVT-5000P time lapse videocassette
recorder and a Sony PVM-1453MD video monitor (Japan).
Cell recruitment assays.
T lymphoblasts were allowed to attach for 20 minutes at 37°C to
35-mm plastic petri dishes (Costar Corp) previously coated with
ICAM-1Fc (10 µg/mL) in the presence of anti-CD43 HP2/21. After the
addition of a second layer of T lymphoblasts, cell-cell interactions
were recorded for 1 hour under phase contrast using a 20×
objective. Images were acquired every 3.2 seconds, and sequential frames were digitalized by using Optimas software (Optimas Corp, Bothell, WA).
Immunoprecipitation and Western blot analysis.
T lymphoblasts either untreated or treated with the anti-CD43 HP2/21
MoAb adhered to FN80-coated dishes were lysed by incubation for 20 minutes in 1.5 mL RIPA buffer containing 0.1% sodium dodecyl sulfate
(SDS), 0.5% deoxycholate, 1% Nonidet P-40, 150 mmol/L NaCl, 50 mmol/L
Tris (pH 8), 1 mmol/L p-amidino phenylmethyl sulfonyl fluoride
(PMSF), and 15 µg/mL leupeptin. Cell lysate was
removed from the dish with a rubber policeman and then precleared by
centrifugation at 10,000g for 20 minutes. Immunoprecipitations
were performed with 45 µL of TP1/36 anti-CD43 or RP2/21 anti-CD45
(control) MoAb directly coupled to Sepharose 4B beads (Pharmacia) at 1 mg/mL. Proteins bound to Sepharose beads were eluted by boiling in
sample buffer, subjected to SDS 7.5% polyacrylamide gel
electrophoresis (PAGE) under reducing conditions, and transferred onto
a nitrocellulose membrane (Millipore, Bedford, MA) in
Tris-Glycine-Methanol buffer during 25 minutes at 17 V using a
Transfer-Blot SD Semi-Dry Transfer Cell (Bio-Rad Laboratories,
Hercules, CA). To detect moesin, membranes were soaked overnight in TBS
containing 3% bovine serum albumin (BSA) and washed three times with
TBS-0.1% Tween 20 during 15 minutes, followed by 1 hour of incubation
with a 1:1,000 dilution of rabbit antimoesin 95/2 pAb. After three
washes, blots were incubated with a peroxidase-conjugated goat
antirabbit IgG (Pierce) and developed using an enhanced
chemiluminiscence system (Amersham Corp). To detect the presence of
ezrin coprecipitated with CD43, the 95/2 and secondary antibodies were
removed from the membrane by treating it with a
stripping buffer (100 mmol/L 2-mercaptoetanol, 2% SDS, 62.5 mmol/L
Tris-HCl, pH 6.7) at 50°C for 30 minutes. Then, blots were reprobed
with the antiezrin 90/3 pAb.
Construction, expression, and purification of GST-CyD7CD43 fusion
protein.
The cytoplasmic region of CD43 without the nucleotides coding for the
first seven residues plasma membrane-proximal was amplified by
polymerase chain reaction (PCR) and cloned as Sal I-Not
I fragments into pGEX-4T (Pharmacia LKB Biotechnology, Uppsala,
Sweden). For amplification of the CD43 cytoplasmic
region (from residues T294 to P400), the primers SalI43T8
(5-CAATTTGTCGACAACTGGGGCCCTCGTGCTGAGC-3) and NotCD43
(5-ATAAGAATGCGGCCGCCGACACTTAAGGGGCAGCC-3) were used.
Expression of GST-fusion proteins in DH1OB cells and purification were
performed following the manufacturer's instructions. The proteins were
stored at 4°C on glutathione Sepharose 4B beads as a 50% slurry in
10 mmol/L Tris, pH 7.4, 140 mmol/L NaCl, 0.5% Triton X-100, 0.02%
azide, and protease inhibitors (Complete Protease Inhibitors;
Boehringer Mannheim Corp, Indianapolis, IN). The amount of fusion
protein was stimated by coomassie blue staining of SDS-PAGE.
In vitro binding of GST-CyD7CD43 fusion protein to cell extracts.
Jurkat cells (2 × 107 cells/mL) were labeled
overnight with a mixture of [35S]methionine/cysteine (500 µCi) in methionine/cysteine-free RPMI 1640 medium supplemented with
10% dialyzed fetal bovine serum. 35S-labeled Jurkat cells
were disrupted in 1 mL lysis buffer (10 mmol/L Tris/HCl, pH 7.6, 150 mmol/L NaCl, 1% Nonidet P-40, 5 mmol/L EDTA, 50 mmol/L sodium
fluoride, 0.4 mmol/L sodium orthovanadate, 10 mmol/L iodoacetamide, 5 mmol/L sodium pyrophosphate, 1 mmol/L penoxymethyl sulphofluoride, and
10 mg/mL aprotinin, leupeptin, pepstatin A, chymostatin, and
 1-antitrypsin) on ice for 10 minutes, followed by 10 minutes of
microcentrifugation. The supernatant was mixed with approximately 50 µg of GST-fusion proteins attached to glutathione Sepharose 4B beads
for 12 hours at 4°C. The beads were collected by centrifugation and
washed twice with lysis buffer alone, twice with lysis buffer plus
0.1% SDS, twice with lysis buffer plus 0.65 mol/L NaCl, and twice with
lysis buffer alone. The beads were then subjected to SDS-PAGE 8% under
reducing conditions.
| |
RESULTS |
Engagement of CD43 induces T-cell polarization with redistribution of
membrane and cytoskeletal proteins.
We have previously reported the regulatory role of CD43 in cell
morphology and its redistribution towards the uropods of
polarized T lymphocytes.26 Herein, we investigated
whether the engagement of CD43 is also capable to redistribute other
membrane receptors as well as cytoskeletal components to different
specialized compartments of the cell. Fluorescence microscopy analysis
of T lymphoblasts adhered to FN80 and stimulated with anti-CD43 MoAb
showed that, during cell polarization, the chemokine receptor CCR2
redistributed to the leading edge of the cell
(Fig 1i, A and B, and 1ii), whereas CD43
was concentrated at the cellular uropod (Fig 1i, A). In contrast, CD45
did not redistribute during CD43-induced cell polarization (Fig 1i, C).
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| Fig 1.
Polarized distribution of CD43 and CCR2 on migrating T
lymphoblasts. (i) T lymphoblasts adhered to FN80 were stimulated with the TP1/36 anti-CD43 MoAb for 30 minutes at 37°C and then fixed as
described under the Materials and Methods. In (A), cells were double-stained with anti-CD43 (orange) and biotinylated-anti-CCR2 chemotactic receptor MoAb (green), and both fluorescences were photographed on the same frame by double exposure. In (B) and (C),
cells were stained for CCR2 and CD45 (FITC-conjugated anti-CD45), respectively. Arrows point to the cellular uropods, whereas arrowheads point to the leading edge. Bar = 10 µm. (ii) T lymphoblasts were allowed to adhere to FN80-coated coverslips and then were stimulated with the TP1/36 anti-CD43, HP2/9 anti-CD44, or D3/9 anti-CD45 MoAb.
Cells were then stained for CCR2 and the percentage of cells on which
the chemokine receptor was redistributed calculated by random choice of
10 fields for each condition and direct cell counting (400 to 500). The
arithmetic mean ± SD of three independent experiments is shown.
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Immunofluorescence staining of ERM cytoskeletal proteins using specific
monoclonal and polyclonal antibodies showed that, in anti-CD43-treated
T lymphocytes, CD43, ezrin, and moesin (Fig 2i, A, B, and D, respectively) redistributed to the cellular uropods, whereas radixin showed a staining pattern at the uropod neck (Fig 2i,
C). Furthermore, double immunofluorescence staining showed that CD43
(Fig 2ii, A and C) colocalized with moesin (Fig 2ii, B) and ezrin (Fig
2ii, D) at the uropod tips. In addition, a weak signal of ezrin was
also detected at the leading edge (Fig 2ii, D).
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| Fig 2.
Ezrin and moesin colocalize with CD43 at the uropods of T
lymphoblasts stimulated with anti-CD43 MoAb. T lymphoblasts were allowed to adhere to coverslips coated with FN-80 and then stimulated with the anti-CD43 HP2/21 MoAb. (i) Cells were fixed and stained for
CD43 (A), ezrin (90/3 pAb) (B), radixin (C), and moesin (38/87 MoAb)
(D). Note the redistribution of all three ERM proteins as well as CD43
to cellular uropods. (ii) Cells were double-stained for CD43 (HP2/21
MoAb) (A and C, orange) and for moesin (38/87 MoAb) (B, green) or ezrin
(90/3 pAb) (D, green), as described under the Materials and Methods.
Arrows point to cellular uropods.
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To further analyze the spatial codistribution of CD43 and moesin, we
performed immunofluorescence analysis on CD8+ TIL adhered
to FN80. These in vivo activated cells (CD45RO+,
CD69+) were isolated from patients with melanoma. In these
cells, CD43 and moesin were also colocalized at its pronounced uropods
(Fig 3i). When TIL were allowed to adhere
to melanoma cells, they displayed the polarized morphology typical of
motile cells with CD43 clustered in the cellular uropods (Fig 3ii).
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| Fig 3.
CD43 clusters at the uropod of polarized TILs and
colocalizes with moesin. (i) TILs were allowed to adhere to coverslips
coated with FN80. Then, single (A and B) or double (C and D)
immunostainings were performed as described under the Materials and
Methods. (A and C) Staining with the anti-CD43 HP2/21 MoAb. (B and D)
Staining with the antimoesin 38/87 MoAb. Arrows point to cellular
uropods. (ii) TILs were cocultured on a monolayer of autologous
melanoma cells for 1 hour at 37°C. Fixed cells were then stained
with the anti-CD43 TP1/36 MoAb (A). In (B), the same field was
photographed under bright field conditions. Note in (A) the typical
migratory morphology of the TIL on melanoma cell (MC), showing the
frontal leading edge (L) and the trailing uropod (U).
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Interaction of CD43 with ERM proteins in T lymphoblasts.
To determine whether moesin and ezrin physically interact with CD43,
cell lysates from T lymphoblasts either nonstimulated or stimulated
with the anti-CD43 MoAb HP2/21 were immunoprecipitated with the TP1/36
anti-CD43 MoAb or with the RP2/21 anti-CD45 MoAb as control,
transferred to nitrocellulose membranes, and blotted with pAb against
either moesin (Fig 4A) or ezrin (Fig 4B).
Coprecipitation of moesin and ezrin was observed in immunoprecipitates
from CD43 (Fig 4A and B, lanes 3 and 4, respectively), whereas neither
moesin nor ezrin were detected in the CD45 immunoprecipitates (Fig 4A and B, lanes 1 and 2). An increase in the amount of moesin associated to CD43 was detected in immunoprecipitates from cells pretreated with
the polarization-inducing HP2/21 anti-CD43 MoAb compared with untreated
cells (Fig 4A, lanes 3 and 4). In contrast, comparable amounts of ezrin
were found associated to CD43 in treated and untreated cells (Fig 4B,
lanes 3 and 4).
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| Fig 4.
Association of ezrin and moesin with CD43 in polarized T
lymphoblasts. T cells either unstimulated (lanes 1 and 3) or stimulated with anti-CD43 HP2/21 MoAb (lanes 2 and 4) were allowed to adhere to
FN80. Cells were then lysed and the soluble fraction was
immunoprecipitated with the anti-CD45 RP2/21 (lanes 1 and 2) and
anti-CD43 TP1/36 MoAb (lanes 3 and 4). A human recombinant moesin
standard was run in the right lanes. Each immunoprecipitate as well as
standards were immunoblotted with the antimoesin 95/2 pAb after SDS
7.5% PAGE (A) and reprobed with the anti-ezrin 90/3 pAb (B) as
described under the Materials and Methods.
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Interaction of the cytoplasmic tail of CD43 with moesin and ezrin.
To further confirm the interaction of CD43 with moesin and ezrin, we
performed precipitation studies with a GST fusion protein containing
the CD43 cytoplasmic region (Fig 5). All
attempts to induce the expression in DH1OB cells of a GST-fusion
protein containing the full-length cytoplasmic region of CD43 failed.
However, we were able to induce the expression of a GST-fusion protein
bearing the cytoplasmic region of CD43 lacking the first seven
N-terminal residues, designated as GST-CyD7CD43 (Fig 5A). We found that
the GST-CyD7CD43 fusion protein specifically precipitated two bands of
78 and 80 kD from metabolically labeled Jurkat T-cell lysates, whereas
no protein band was observed in precipitates from GST control protein
(Fig 5B). Western blot analysis performed in parallel showed that the
78- and 80-kD proteins bound to GST-CyD7CD43 corresponded to moesin and
ezrin, respectively (Fig 5C). These results further indicate that the
cytoplasmic tail of CD43 interacts with moesin and ezrin.
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| Fig 5.
Interaction of the cytoplasmic region of CD43 with moesin
and ezrin from Jurkat cell lysates. (A) Coomassie blue-stained SDS-PAGE of GST-CyD7CD43 fusion protein purified with glutathione Sepharose 4B
beads. (B) Specific association of two proteins of 78 and 80 kD with
GST-CyD7CD43. Jurkat cells were metabolically labeled and lysed. After
discarding cell debris and nuclei, supernatants were collected and
allowed to bind to equivalent amounts of GST and GST-CyD7CD43 proteins
bound to glutathione Sepharose 4B beads by overnight incubation at
4°C. Bound proteins were sequentially washed with lysis buffer
containing 0.1% SDS and 0.65 mol/L NaCl and then subjected to 8%
SDS-PAGE. Before drying, the gel was incubated for 30 minutes in
Amplify solution (Amersham Corp). (C) Precipitates from unlabeled
Jurkat cells were performed as in (B), SDS-PAGE separated, and
immunoblotted with the antimoesin 95/2 (lanes 1 and 2) or the antiezrin
90/3 (lanes 3 and 4) pAb. Molecular weights in kilodaltons are
indicated at the right.
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CD43 and moesin are redistributed to cell-cell contacts during
homotypic T-cell aggregation induced by anti-CD43 MoAb.
The induction of homotypic leukocyte aggregation by anti-CD43 MoAb has
been described.20,41,42 In this regard, we have previously
selected a number of anti-CD43 MoAb for their ability to promote rapid
and strong homotypic aggregation of normal T lymphoblasts and U937
myelomonocytic cells.26 However, little is known about the
dynamics of aggregation and the nature of cell-cell interactions
established during this phenomenon. In an attempt to understand this
issue, we performed time-lapse videomicroscopy studies of homotypic
T-lymphoblast aggregate formation induced by the anti-CD43 MoAb HP2/21
(Fig 6i).
Five minutes after addition of anti-CD43, T cells initiated locomotion
on the substratum, displayed a polarized morphology, and established
early cell-cell interactions with other polarized and spherical cells,
mainly through their uropods, forming motile small aggregates (Fig 6i). One hour later, motile small cell aggregates contacted with other small
aggregates through externally exposed cell uropods generating large
cellular aggregates (Fig 6i). Immunofluorescence studies performed in
parallel showed that CD43 as well as moesin were concentrated at the
uropods of small cell aggregates that were mediating cell-cell
interactions (Fig 6ii, A and B, and iii, A, respectively). On the other
hand, in large cell aggregates, CD43 and moesin were located in the
uropods projected towards the outer milieu (Fig 6iii, C, and iii, B,
respectively) as well as in cell-cell contacts. These results strongly
suggested that the clusters of CD43 projected by the prominent uropods
could facilitate the interaction of this molecule with a putative
ligand to allow formation of cell aggregates.
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| Fig 6.
Dynamics of CD43 and moesin during cell aggregation. (i)
T lymphoblasts adhered to FN80 were treated with the proaggregatory anti-CD43 HP2/21 MoAb. Time-lapse videomicroscopy analysis was then
performed as described in the Materials and Methods. Sequential time
frames are shown. White arrowheads point to uropods of cells participating in the formation of small and large aggregates. (ii) T
lymphoblast aggregation was induced by using the anti-CD43 HP2/21 MoAb,
and upon cell incubation at 37°C for 5 (A), 10 (B), and 30 (C)
minutes, cells were fixed and stained for CD43. (iii) Similarly, 5 (A) and 30 (B)
minutes after the triggering of cell aggregation with the anti-CD43
HP2/21 MoAb, cells were fixed and stained for moesin by using the
biotinylated antimoesin 38/87 MoAb. The same cells were photographed
under epifluorescent (above) and bright field (below) conditions.
Arrows point to cellular uropods.
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Myosin disruption prevents CD43-induced homotypic T-cell aggregation.
Polarized T cells express linear arrays of myosin at the uropod
neck,43 and we have described that the disruption of this structure by the butanedione monoxime prevents uropod formation and
redistribution of both CD43 and moesin.26,44 To investigate the role of myosin in the induction of T lymphoblast aggregation triggered through CD43, we performed time-lapse videomicroscopy of
T-cell aggregation in the presence of butanedione monoxime. At short
times, although cell aggregation was observed in the absence of this
agent (Fig 6i), the addition of this drug prevented the acquisition of
polarized morphology of T cells adhered to FN80 and the formation of
cell clusters (Fig 7A). At longer times, when the butanedione effect start to vanish, some cells become polarized participating in small aggregates through its uropods (Fig 7A
and B). We have also tested the role of colchicine and cytochalasin D
on cell aggregation induced by the anti-CD43 HP2/21 MoAb.
Interestingly, whereas cytochalasin D inhibit cell aggregation, no
significant inhibition was observed under colchicine treatment (Fig
7B). These results indicate that myosin plays a central role in the
establishment of cell-cell interactions required for aggregate formation induced by CD43.
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| Fig 7.
Myosin-disruption prevents CD43-mediated cell
aggregation. (A) T lymphoblasts were pretreated during 30 minutes with
20 mmol/L of butanedione monoxime. Cells were then allowed to adhere to FN80 in the presence of the proaggregatory anti-CD43 HP2/21 MoAb. Cells
were filmed with a time-lapse videocassete recorder for 10 hours. Note
the lack of cell polarization and aggregation in the first 2 hours
recorded. White arrowheads point to cellular uropods displayed by cells
after 9 hours of anti-CD43 treatment. (B) T lymphoblasts were incubated
for 30 minutes at 37°C in the presence of 10 mmol/L butanedione
monoxime ( ), 20 µmol/L colchicine ( ), or 20 µmol/L
cytochalasin D ( ) or in the absence of any cytoskeletal drug ( ),
before the addition of anti-CD43 HP2/21 MoAb. The percentage of
aggregation was calculated at different times as described in the
Materials and Methods.
|
|
Redistribution of CD43 to the uropods enhance recruitment and
transport of T cells.
We have recently described the involvement of T-cell uropods in the
transport and recruitment of other lymphocytes as well as the role of
ICAM-3 and chemokines in the induction of this phenomenon.40 To investigate whether similar phenomena take place upon CD43 engagement, we studied this issue by time-lapse videomicroscopy. A first layer of T cells was allowed to attach to
ICAM-1-Fc-coated surface plates and then induced to develop uropods
with the HP2/21 anti-CD43 MoAb. A second layer of T cells was then
added and cellular interactions were filmed. Interestingly, cells of
the second layer (phase-bright cells) were contacted, trapped, and
transported by cells of the first layer (phase-dark cells) locomoting
on the ICAM-1-Fc-coated surface (Fig 8A).
Quantification of cells of the second layer recruited and transported
by the adhered cells of the first layer showed that the treatment with anti-CD43 induced a 10-fold increase in cell recruitment compared with
unstimulated cells (Fig 8B).
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| Fig 8.
CD43-stimulated T lymphocytes recruit additional cells
through the uropods. (A) T lymphoblasts adhered to petri dishes coated with ICAM-1-Fc were treated with the anti-CD43 HP2/21 MoAb. A second
layer of untreated T lymphoblasts (bright cells) was then added and
cell-cell interactions were recorded by videomicroscopy for 30 minutes.
Black arrowheads point to transported cells; white arrowheads point to
cellular uropods of cells from the first cohort, whereas white arrows
point from cellular leading edges. (B) Quantitation of T-cell
lymphocyte recruitment mediated by anti-CD43 polarized cells. T
lymphoblasts were allowed to adhere to plastic petri dishes coated with
ICAM-1-Fc for 30 minutes at 37°C in the presence of the
proaggregatory anti-CD43 (HP2/21 and TP1/36) and anti-ICAM-3 (HP2/19)
MoAb or the control anti-HLA-A,B W6/32 MoAb. After the addition of a
second layer of cells, cell-cell interactions were recorded for 1 hour,
and the recruitment index was calculated as the number of cells of the
second layer contacted per cell number adhered to the substrate.
|
|
| |
DISCUSSION |
CD43 is a heavily charged transmembrane sialomucin that provides
stimulatory signals inducing lymphocyte proliferation and IL-2
secretion24,45 and costimulates T lymphoblasts in a
CD28-independent manner.25 In contrast, a negative
regulation of T-cell activation and adhesion through CD43 has also been
reported.21,22 However, it has been described that
anti-CD43 MoAb are able to trigger homotypic leukocyte aggregation in
both CD18-dependent and CD18-independent manner.19,20,41,46
Because cell aggregation and polarization studies have become useful
assays for analyzing leukocyte motility and activation in vitro, we
decided to study the role of CD43 in these assays to further understand
the biological function of this molecule.
Previously, we have reported that proaggregatory and activating
anti-CD43 MoAbs enhance  1 and 2 integrin-mediated T-cell adhesion to both endothelial and ECM proteins and induce
T-cell polarization with redistribution of CD43 to cellular
uropods.26 We demonstrate herein that CD43 plays an
important regulatory role in T-cell polarization, inducing the
appearance of specialized cellular domains with redistribution of the
CCR2 chemokine receptor to the leading edge and ERM actin-binding
proteins to the uropod of polarized T lymphocytes. For the ERM proteins
analyzed, ezrin and moesin colocalized with CD43 at the uropod tips,
whereas radixin was detected at the uropod neck. It is worth mentioning
that the data regarding redistribution of CD43 and ezrin contrast with our previous results showing no colocalization of ezrin with ICAM-3 at
the uropods of T cells treated with chemokines.44 This
discrepancy seems to be due to the different polyclonal antibodies used
in both studies; whereas 90/3 pAb was generated against purified human
ezrin, the affinity-purified pAb 464 was raised against an unique
peptide of mouse ezrin.44 Therefore, it is evident that the
bright immunostaining of cell uropods produced by the 90/3 pAb indeed
corresponds to the redistribution of ezrin to this structure.
Furthermore, the CD43/ezrin codistribution found by us agrees with
previous data from other investigators regarding the presence of ezrin
in the uropods of mouse T cells and with the colocalization of ERM
proteins and CD43 in the cleavage furrows during cytokinesis of mouse
thymocytes in division.47,48 Interestingly, our data
demonstrate that ezrin and moesin physically interact with CD43 in
unstimulated T lymphocytes, yet only moesin/CD43 interaction was
increased after CD43 stimulation. Moreover, the coprecipitation studies
with the GST-CyD7CD43 fusion protein demonstrate a direct interaction
of moesin and ezrin with the cytoplasmic region of CD43. In this
regard, other adhesion molecules, such as CD44 and ICAM-3, have been
reported to interact with moesin in motile T cells, and this
association is increased under conditions that induce cell polarization
such as the treatment with chemokines.44 Thus, it appears
that the process of moving membrane molecules towards the trailing edge
of the cell is directed by the submembranous cytoskeleton.49 By linking adhesion molecules to
submembranous cytoskeleton, moesin could promote the redistribution of
membrane complexes towards the cellular uropod.
It has been described that cell aggregation can be induced in T
lymphoblasts upon engagement of a large array of cell adhesion molecules, including CD43.33,50,51 We have undertaken a
dynamic assessment of cell aggregation by time-lapse video recording of CD43-stimulated T lymphocytes. Upon treatment with anti-CD43 MoAb, the
formation of large cell aggregates takes place by congregation of small
ones. In small aggregates, cells contact each other mainly through
their uropods, where moesin and CD43 colocalize, suggesting that
moesin/CD43 interaction may have a role in the initial phase of the
establishment of cell-cell contacts, as it has been described for
ICAM-3.33 In accordance, a similar mechanism of cell-cell interactions has been described in NK-resistant cells, which after ezrin transfection, display an uropod where ICAM-2 is redistributed, allowing cellular interactions and becoming NK-sensitive.47 Similarly, uropod formation, moesin/CD43 interaction, and CD43 redistribution during T-cell polarization induced through CD43 would
increase molecular density and accessibility of CD43 in the cellular
uropod enabling interactions with putative ligands, facilitating
homotypic cell aggregation. In this regard, the existence of cellular
ligands for CD43 involved in leukocyte endothelial cell interactions
has recently been postulated on the basis of in vivo inhibition of
lymphoid cell homing with an anti-CD43 MoAb.27
In previous studies using the myosin-disrupting drug
butanedione monoxime, we found that myosin is involved in uropod
formation, cell aggregation induced by anti-ICAM-3 MoAb, and adhesion
receptors and moesin redistribution to the
uropod.11,26,43,44 Our data indicate that myosin is also
necessary for the cell-cell interactions that occur during the
homotypic T-cell aggregation induced through CD43, reinforcing the
issue that uropod formation is required to optimize cell-cell
interactions.
We previously reported that cellular uropods induced by both
chemokines and activating anti-ICAM-3 MoAb are able to mediate the
recruitment of lymphocytes and transports them through extracellular matrix layers or endothelial cells specialized in lymphocyte
extravasation.40 Likewise, CD43-mediated cell polarization
and CD43 and moesin redistribution towards uropods are involved in the
migration of T lymphoblasts on ICAM-1 as well as in the capture and
transport of additional bystander cells. Because the phenotype of T
lymphoblasts used in these studies is similar to memory and effector T
cells (95% CD45RO, 98% CD3, and 96% CD43high), our work
agrees with a previous report showing that the high expression of CD43
by memory T cells facilitate the extravasation of these
cells.20 In this regard, it is conceivable that the highly
polarized morphology of TIL, with a pronounced uropod where most of
CD43 molecules are redistributed, facilitates the motility of these
lymphocytes through cellular layers amplifying the recruitment of
leukocytes as well as its interaction with tumoral cells. Altogether, our findings on cell aggregation induced through CD43 suggest that this molecule is a proadhesive rather than antiadhesive molecule and provide evidence for the role of CD43 as a membrane receptor able
to interact with putative counter-receptor(s), mediating cell-cell
contacts through uropods in leukocyte intercellular interactions.
Recently, it has been postulated the involvement of CD43 in
the selective binding and recruitment of T lymphocytes to lymphoid organs as well as in the existence of an unidentified vascular ligand
for CD43.27 In neutrophils, the interaction of P-selectin glycoprotein ligand-1 (PSGL-1) with P-selectin is uncoupled upon cell
activation with the concomitant redistribution of PSGL-1 towards
the cellular uropod.52 On the other hand, PSGL-1
redistribution towards the uropod of neutrophils after chemotactic
activation could be responsible for cell-cell interactions between
neutrophils and activated platelets.53 Because CD43, as
PSGL-1, is a sialylated mucin, it is feasible that, in T lymphocytes,
the redistribution of CD43 to the cellular uropod by its interaction
with ERM-mediated cytoskeleton would facilitate transendothelial
migration and recruitment of other T lymphocytes by interaction through
their uropods.
| |
FOOTNOTES |
Submitted September 10, 1997;
accepted February 7, 1998.
Supported by Grant No. SAF96/0039 from Plan Nacional de
Investigación y Desarrollo, Grant No. 07/44/96 from Comunidad
Autónoma de Madrid, a grant from Fundación
Científica de la Asociación Española contra el
Cancer (to F.S.M.), by fellowship BAE FIS 97/5089 to M.A.P., and by
Grant No. AR41045 from the US Public Health Service (to H.F.)
Address reprint requests to Francisco Sánchez-Madrid, PhD,
Servicio de Inmunología, Hospital de la Princesa, Universidad Autónoma de Madrid, C/ Diego de Leon, 62, Madrid, Spain; e-mail: fsmadrid/princesa{at}hup.es.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Dr P. Lauzurica for critical
readings of the manuscript. We are greatfully indebted to M.C. Montoya
and E. Fernández-Villareal for their help and advice with
different techniques and to R. Tejedor for preparing recombinant
ICAM-1-Fc.
| |
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S. Sizemore, M. Cicek, N. Sizemore, K. P. Ng, and G. Casey
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G. Tamma, G. Procino, M. Svelto, and G. Valenti
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C. M. Finnerty, D. Chambers, J. Ingraffea, H. R. Faber, P. A. Karplus, and A. Bretscher
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G. Greicius, L. Westerberg, E. J. Davey, E. Buentke, A. Scheynius, J. Thyberg, and E. Severinson
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R. F. Thorne, J. W. Legg, and C. M. Isacke
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E. Layseca-Espinosa, G. Pedraza-Alva, J. L. Montiel, R. del Rio, N. A. Fierro, R. Gonzalez-Amaro, and Y. Rosenstein
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N. Da Silva, A. Bharti, and C. S. Shelley
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M. Wykes, K. P. A. MacDonald, M. Tran, R. J. Quin, P. X. Xing, S. J. Gendler, D. N. J. Hart, and M. A. McGuckin
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C. Barat and M. J. Tremblay
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K. R. Snapp, C. E. Heitzig, and G. S. Kansas
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N. D. L. Savage, S. L. Kimzey, S. K. Bromley, K. G. Johnson, M. L. Dustin, and J. M. Green
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E. Gubina, T. Chen, L. Zhang, E. F. Lizzio, and S. Kozlowski
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L. Cermak, S. Simova, A. Pintzas, V. Horejsi, and L. Andera
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J. Millan, M. C. Montoya, D. Sancho, F. Sanchez-Madrid, and M. A. Alonso
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V. L. Bonilha and E. Rodriguez-Boulan
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The Glomerular Epithelial Cell Anti-Adhesin Podocalyxin Associates with the Actin Cytoskeleton through Interactions with Ezrin
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D. M. Felschow, M. L. McVeigh, G. T. Hoehn, C. I. Civin, and M. J. Fackler
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D. Reczek and A. Bretscher
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Ezrin Immunoreactivity Is Associated with Increasing Malignancy of Astrocytic Tumors but Is Absent in Oligodendrogliomas
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M. Borset, O. Hjertner, S. Yaccoby, J. Epstein, and R. D. Sanderson
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C. Fratazzi, N. Manjunath, R. D. Arbeit, C. Carini, T. A. Gerken, B. Ardman, E. Remold-O'Donnell, and H. G. Remold
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J. R. Harrington
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L. Huang, T. Y. W. Wong, R. C. C. Lin, and H. Furthmayr
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M. Amieva, P Litman, L Huang, E Ichimaru, and H Furthmayr
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Abstract
Introduction
Abstract