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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 95 No. 7 (April 1), 2000:
pp. 2413-2419
PHAGOCYTES
Polarization and interaction of adhesion molecules P-selectin
glycoprotein ligand 1 and intercellular adhesion molecule 3 with moesin
and ezrin in myeloid cells
José L. Alonso-Lebrero,
Juan M. Serrador,
Carmen Domínguez-Jiménez,
Olga Barreiro,
Alfonso Luque,
Miguel A. del Pozo,
Karen Snapp,
Geoffrey Kansas,
Reinhard Schwartz-Albiez,
Heinz Furthmayr,
Francisco Lozano, and
Francisco Sánchez-Madrid
From the Servicio de Inmunología, Hospital de la Princesa,
Madrid, Spain; Northwestern University Medical School, Chicago, IL;
Molecular Mechanisms of Disease Laboratories, Department of Pathology,
Stanford University Medical Center, Stanford, CA; Tumor Immunology
Program, German Cancer Research Center, Heidelberg, Germany; and Servei
d'Immunologia, Hospital Clinic, Barcelona, Spain.
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Abstract |
In response to the chemoattractants interleukin 8, C5a,
N-formyl-methionyl-leucyl-phenylalanine, and interleukin 15, adhesion molecules P-selectin glycoprotein ligand 1 (PSGL-1),
intercellular adhesion molecule 3 (ICAM-3), CD43, and CD44 are
redistributed to a newly formed uropod in human neutrophils. The
adhesion molecules PSGL-1 and ICAM-3 were found to colocalize with the
cytoskeletal protein moesin in the uropod of stimulated neutrophils.
Interaction of PSGL-1 with moesin was shown in HL-60 cell lysates by
isolating a complex with glutathione S-transferase fusions of the
cytoplasmic domain of PSGL-1. Bands of 78- and 81-kd were identified as
moesin and ezrin by Western blot analysis. ICAM-3 and moesin also
coeluted from neutrophil lysates with an anti-ICAM-3 immunoaffinity
assay. Direct interaction of the cytoplasmic domains of ICAM-3 and
PSGL-1 with the amino-terminal domain of recombinant moesin was
demonstrated by protein-protein binding assays. These results suggest
that the redistribution of PSGL-1 and its association with
intracellular molecules, including the ezrin-radixin-moesin
actin-binding proteins, regulate functions mediated by PSGL-1 in
leukocytes stimulated by chemoattractants.
(Blood. 2000;95:2413-2419)
© 2000 by The American Society of Hematology.
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Introduction |
Leukocytes emigrating from the bloodstream toward an
inflammatory focus follow different chemoattractant signals that guide their path.1 An early response of these cells to such
stimuli is a transition from a spherical to a polarized morphologic
conformation, which occurs as soon as the cell begins to
move.2 This response also occurs with several chemokines
that induce both leukocyte activation and migration.3,4
Interleukin 8 (IL-8) belongs to the subfamily of CXC chemokines that
mainly attract neutrophils. It is released in response to inflammatory
stimuli and induces the transmigration of neutrophils across vascular
endothelium.5 CXC chemoattractants induce the full range of
neutrophil activation events, including oxygen free-radical production
and degranulation. In contrast, other neutrophil-activating agents,
such as tumor necrosis factor  (TNF-) do not act as primary
chemotactic factors and induce degranulation only after priming with
other agents.6,7 Similar to the classic leukocyte
chemoattractants C5a and N-formyl peptides, chemokines bind to
7-trans-membrane spanning receptors coupled to a heterotrimeric
G-protein signaling pathway.8-10
On activation, endothelial cells express specific adhesion molecules
that are able to bind free-flowing neutrophils. The initial contact and
rolling of neutrophils along the endothelium are predominantly mediated
by E- and P-selectins, which are expressed by activated endothelium,
and by L-selectin, which is constitutively expressed on
leukocytes.11 All 3 selectins bind, in a calcium-dependent manner, to sialylated and fucosylated oligosaccharides such as sialyl
Lewisa and its isomer sialyl
Lewisx.11,12 P-selectin glycoprotein ligand 1 (PSGL-1) has been identified as a ligand for all 3 selectins.13 This molecule is expressed on leukocytes and
belongs to the family of heavily O-glycosylated sialomucins
rich in serine and threonine.14-16 PSGL-1 requires sulfation for high-affinity binding to both L- and
P-selectin.16,17 P-selectin binds to PSGL-1 on all myeloid
cells but only in a subset of T lymphocytes corresponding to memory
cells.15,16
Chemokines cause polarization of lymphocytes and redistribution of
several adhesion molecules to the uropod, a cytoplasmic projection that
forms at the rear end of the moving cell.18-20 Similarly,
activation of neutrophils with platelet-activating factor or the
N-formyl-methionyl-leucyl-phenylalanine (fMLP) causes the
redistribution of PSGL-1 to the uropod and diminished adhesion to
P-selectin.21 However, adhesion molecules that are
concentrated in the uropod facilitate binding to other cells. This has
been described for PSGL-1 in interactions between neutrophils and
platelets22 and for ICAMs in lymphocyte cell-cell
interactions.23
A similar redistribution has been observed for members of the closely
related ezrin-moesin-radixin (ERM) family of proteins in locomoting
lymphocytes. Moesin is codistributed and interacts with ICAM-3, CD43,
and CD44 adhesion molecules in the distal portion of
uropods.24,25 The ERM proteins function as linking proteins between the plasma membrane and the actin cytoskeleton26-28
and are generally associated with cell-surface protrusions such as microvilli, filopodia, microspikes, retraction fibers, and membrane ruffling.29-32 Suppression of their expression has profound
effects on cell functions.33 Ezrin and moesin are also
expressed in neutrophils,34 and because activation with
different chemoattractants and movement depend on adhesion and on the
actin cytoskeleton, we explored the distribution of a variety of
adhesion molecules and moesin and possible interactions between them.
We found that several receptors were distributed to the uropod of
migrating neutrophils, where they were codistributed with moesin.
Moreover, we observed that, in vitro, the amino-terminal domain of
moesin interacted directly with the cytoplasmic domain of at least 2 of
these receptors ICAM-3 and PSGL-1.
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Materials and methods |
Antibodies, chemokines, and reagents
The anti-ICAM-3 HP2/19 and TP1/25, anti-ICAM-1 MEM 111, anti-CD44
HP2/9, anti-CD43 TP1/36, anti-CD45 D3/9, anti-PSGL-1 KPL1, and
antimoesin/antiradixin 38/37 monoclonal antibodies (mAbs) were
described previously.17,25,35,36,37 Antimoesin 95/2 and the
antiezrin 90/3 polyclonal antisera (pAb) were raised in rabbits by
immunization with recombinant human moesin and purified human ezrin,
respectively.29,38 Antivinculin mAb was purchased from
Sigma Chemical Co (St Louis, MO). Goat anti-GRP78 (HSP78) pAb was
purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA).
Aprotinin, iodoacetamide, C5a, human recombinant TNF-, and fMLP were
purchased from Sigma. IL-8 and interleukin 15 (IL-15) were purchased
from Peprotech EC Ltd (London, UK). The 80-kd fibronectin fragment
(FN80) was a gift of A. García Pardo (Centro de Investigaciones Biológicas, Madrid, Spain). A mixture of protease inhibitors (Complete Protease Inhibitors) was from Boehringer Mannheim Corp (Indianapolis, IN). Sodium orthovanadate was purchased from Sigma.
Cells
Neutrophils were isolated from fresh human blood by Ficoll-Hypaque
density gradient centrifugation, which was followed by sedimentation at
1g in dextran (Sigma) at room temperature. The neutrophil-enriched fraction was further purified by hypotonic lysis of
erythrocytes, which yielded a purity of greater than 98%. Neutrophils
were resuspended in 0.05% heat-denatured bovine serum albumin (BSA)
RPMI 1640 medium. Human umbilical vein endothelial cells were obtained
as described previously.39 The umbilical vein was
cannulated, washed, and incubated with 0.1% collagenase P (Boehringer
Mannheim) for 20 minutes at 37°C. Detached cells were then seeded
into flasks and cultured in M199 medium (BioWhittaker, Walkersville, MD) supplemented with 20% fetal-calf serum, 50 µg/mL of endothelial cell growth supplement, and 100 µg/mL of
heparin (Sigma).
Immunofluorescence microscopy
Purified neutrophils (7 × 105) were incubated in
flat-bottomed, 24-well plates (Costar Corp, Cambridge, MA) in a final
volume of 400 µL of RPMI 1640 with 0.05% heat-denatured BSA on
coverslips coated with FN80 (20 µg/mL). After 10 minutes, 2 nmol/L of
a chemoattractant was added, except for IL-15, which was used at 10 nmol/L. The cells were incubated at 37°C in a 5% carbon dioxide
atmosphere for 5 minutes, fixed with 1% (wt/vol) paraformaldehyde in
phosphate-buffered saline (pH 7.4) at room temperature, and rinsed in
triethanolamine-buffered saline (TBS). Cells were then incubated with
the selected mAb, washed, and stained with a rabbit
F(ab')2 antimouse IgG (Pierce Montlucon, France)
labeled with fluorescein-isothiocyanate, conjugated (FITC). The number
of uropod-bearing cells was determined in each experiment by direct
counting of the total cells (300-400) and the uropod-bearing cells in
10 randomly selected fields (60 × objective).
For double immunofluorescence analysis of neutrophils, fixed cells were
incubated with the 38/87 mAb and then with a tetrarhodamine isothiocyanate (TRITC)-labeled rabbit antimouse IgG. Subsequently, coverslips were saturated with normal mouse serum and incubated with
FITC-conjugated TP1/24 anti-ICAM-3 mAb. In some experiments, neutrophils were incubated for 5 minutes with confluent monolayers of
endothelial cells previously activated with TNF- (20 ng/mL) for 4 hours. For double staining of endothelial cells and neutrophils, coverslips were incubated with anti-ICAM-1 mAb MEM 111 followed by
TRITC-labeled rabbit antimouse IgG. Coverslips were then saturated with
normal mouse serum and incubated with FITC-conjugated TP1/24 anti-ICAM-3 mAb. Cells were observed with 60 × and 100 ×
oil-immersion objectives and a photo-microscope (Nikon Labophot-2;
Nikon, Inc, Melville, NY). Images were recorded on Ektachrome 400 film
(Eastman Kodak Co, Rochester, NY).
Construction, expression, and purification of glutathione
S-transferase (GST) fusion proteins
Complementary DNA fragments for expression of the cytoplasmic
regions of ICAM-3 and PSGL-1 were obtained by polymerase chain reaction amplification and cloned as SalI-NotI
fragments into pGEX-4T (Pharmacia LKB Biotechnology, Uppsala, Sweden).
Primers for amplification of the PSGL-1 cytoplasmic region were
SALCYPSGL5'-TCCGCCGTCGACCGCCTCTCCCGCAAGGGCCACA-3' and NOTCYPSGL
5'-ATAAGAATGCGGCCGCCTAAGGGAGGAAGCTGTGCA-3'; those for ICAM-3 were SALCY50TH
5'-CAACGCGTCGACCAGGGAGCACCAACGG-3' and NOTCY50TH
5'-ATAAGAATGCGGCCGCTCACTCACTCAGCTCTGGA-3'.
Constructs for expression of GST fusions of full-length moesin (pGhuMo)
and its amino-terminal region containing amino acid residues 1 to 310 (pKG-MSNn) were described previously.40
Expression of GST-fusion proteins in DHIOB cells and purification were
carried out by following the manufacturer's instructions. Proteins
were stored at 4°C on glutathione-Sepharose 4B beads as a 50%
slurry in 10 mmol/L of Tris (pH 7.4), 140 mmol/L of sodium chloride
(NaCl), 0.5% Triton X-100, 0.02% sodium azide (NaN3), and
protease inhibitors (Boehringer Mannheim). The concentration of fusion
proteins was estimated by Coomassie-blue staining of polypeptide bands
after sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE). BSA was used as the standard protein for estimating
concentrations of recombinant proteins.
Isolation of moesin by ICAM-3 antibody-affinity chromatography and
Western blot analysis
ICAM-3 was isolated as described previously.37 Briefly,
human neutrophils were lysed in 20 mmol/L of Tris-hydrochloric acid (HCl) (pH 8.0), 0.5% Triton X-100 buffer, 5 mmol/L of iodoacetamide, protease inhibitors, and 0.025% NaN3. Cell lysates were
centrifuged at 2500g for 60 minutes, and the supernatants were
passed through a Sepharose CL-4B anti-ICAM-3 mAb column. The column was
washed with 50 mmol/L of ethanolamine (pH 10), 0.5 mol/L of NaCl, and 1% N-octylglucoside, and bound proteins were eluted in a
step-wise manner with the same buffer, adjusted to pH 11, pH 11.5, and
pH 12. Eluted fractions were identified by 8% SDS-PAGE, and proteins were visualized with use of silver staining. Fractions containing ICAM-3 were pooled and concentrated 30-fold by using microconcentrators (Centricon-100; Amicon, Danvers, MA).
Concentrated fractions were subjected to 8% SDS-PAGE under reducing
conditions and transferred onto a nitrocellulose membrane (Millipore
Corp, Bedford, MA) in Tris-glycine-methanol buffer for 30 minutes at 17 V by using a transfer cell (Transfer-Blot SD Semi-Dry Transfer Cell;
Bio-Rad Laboratories, Hercules, CA). To detect moesin or ezrin,
membranes were blocked overnight in TBS containing 3% BSA, washed 3 times with TBS and 0.1% Tween 20 for 15 minutes, and incubated for 1 hour with a 1:1000 dilution of the rabbit antimoesin pAb 95/2 or with a
1:4000 dilution of the rabbit antiezrin pAb 90/3. After 3 washes, blots
were incubated with a peroxidase-conjugated goat antirabbit IgG (Pierce
Montlucon), and proteins were visualized with use of an enhanced
chemiluminescence detection system (Amersham, Little Chalfont, UK).
Purification of recombinant moesin and in vitro binding assay
Recombinant full-length moesin and the N-terminal domain of moesin
containing amino acid residues 1 to 310 were expressed in
Escherichia coli and isolated and purified as GST-fusion
proteins as described previously.40,41 Cleavage of the
fusion protein bound to glutathione-Sepharose 4B beads (Pharmacia LVB
Biotech) preequilibrated with 50 U/mL of thrombin (Pharmacia LVB
Biotech) in Tris buffer (50 mmol/L of Tris-HCl [pH 7.5] and 150 mmol/L of NaCl) occurred after shaking at room temperature for 12 hours. The supernatant was loaded onto a 0.8 × 4-cm column
(Pharmacia LVB Biotech) preequilibrated with washing buffer (50 mmol/L
of Tris-HCl [pH 7.5], 1 mmol/L of phenylmethylsulfonyl fluoride, and
4 µg of leupeptin); the column included 6 mL of heparin and Sepharose. Proteins were eluted by using a step gradient of 0.2 mol/L,
0.4 mol/L, 0.5 mol/L, and 1 mol/L of NaCl. All purification procedures
were performed at 4°C. Fractions containing full-length and
N-terminal moesin were pooled and dialyzed against Tris buffer (50 mmol/L of Tris-HCl [pH 7.5] and 150 mmol/L of NaCl). The amount of
recombinant moesin obtained was estimated by SDS-PAGE with use of known
amounts of BSA.
Recombinant moesin was assayed for binding activity by incubation with
glutathione- Sepharose-linked GST-ICAM-3 and GST-PSGL-1 fusion
proteins in binding buffer (150 mmol/L of NaCl, 20 mmol/L of Tris-HCl
[pH 8.3] and 0.2% Triton X-100) at 4°C. After 1.5 hours, the
beads were washed 6 times in binding buffer. GST fusion proteins
and their associated proteins were eluted by using 50 mmol/L of Tris buffer containing 20 mmol/L of glutathione. Bound moesin
was assessed by SDS-PAGE and immunoblotting using moesin 95/2 pAb.
In vitro translation and binding assay
The pCR3 plasmids carrying the N-terminal domain of moesin
containing inserts of amino acid residues 1 to 310 (MSNn/pCR3) were
described previously.40 These plasmids were transcribed and
translated in vitro by using a TNT-coupled rabbit
reticulocyte lysate system (Promega, Madison, WI) in the presence of T7
RNA polymerase and methionine labeled with sulfur 35 (35S), according to the manufacturer's instructions.
Isotope-labeled moesin was incubated in binding buffer (20 mmol/L of
Tris-HCl [pH 8.3], 150 mmol/L of NaCl, and 0.2% Triton X-100) at
4°C with GST-ICAM-3 and PSGL-1 fusion proteins that had been
immobilized on glutathione-Sepharose beads. After 1.5 hours, the beads
were washed 6 times in binding buffer, resolved on SDS-PAGE,
and subjected to autoradiography.
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Results |
Induction by chemoattractants of redistribution of adhesion
receptors and moesin to the uropod in neutrophils
In response to chemoattractants, neutrophils begin to migrate and to
form uropods, and we decided to analyze this response in detail. ICAM-3
and other adhesion molecules were evenly distributed in the plasma
membrane in unstimulated neutrophils (Figure
1i and data not shown). When treated with
the chemokine IL-8, the adhesion molecules ICAM-3, PSGL-1, CD43, and
CD44, but not CD45, redistributed to uropods. Other chemoattractants
(fMLP and C5a) and the chemotactic cytokine IL-15 also triggered this
response for ICAM-3 and PSGL-1 (Figure 1ii and data not shown). IL-8
and C5a were the most potent chemoattractants in inducing receptor polarization. In contrast, TNF- did not act as a primary chemotactic factor, and it caused neither uropod formation nor receptor
redistribution (Figure 1ii).
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| Fig 1.
Redistribution of adhesion receptors to the uropod of
neutrophils stimulated by chemoattractants.
(i) Neutrophils were allowed to bind to coverslips coated with 20 µg/mL of an 80-kd fibronectin (FN80) fragment for 10 minutes at
37°C and stimulated with interleukin 8 (IL-8) for an additional 5 minutes. Neutrophils were then fixed and stained with the following
monoclonal antibodies (mAb): anti-intercellular adhesion molecule 3 (ICAM-3) TP1/25 (B), anti-P-selectin glycoprotein ligand 1 (PSGL-1)
KPL-1 (C), anti-CD43 TP1/36 (D), anti-CD44 HP2/9 (E), and anti-CD45
D3/9 (F). The control was unstimulated neutrophils stained with the
anti-ICAM-3 TP1/25 mAb (A). (ii) Neutrophils were stimulated in the
presence of 2 nmol/L of C5a (A, B, and C),
N-formyl-methionyl-leucyl-phenylalanine (D, E, and F), or tumor
necrosis factor (TNF-) (G, H, and I) for 5 minutes at 37°C.
Cells were then fixed and stained with the following mAb: anti-ICAM-3
TP1/25 (A, D, and G), anti-PSGL-1 KPL1 (B, E, and H), and anti-CD45
D3/9 (C, F, and I).
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Polarization and redistribution of PSGL-1 were also observed in
neutrophils that migrated toward a gradient of C5a (Figure 2A). Similarly, PSGL-1 and ICAM-3
redistributed in neutrophils that transmigrated across a monolayer of
endothelial cells activated by TNF- (Figure 2B and 2C).
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| Fig 2.
Polarization of PSGL-1 in neutrophils migrating toward a
chemoattractant gradient or on endothelial cells.
(A) Neutrophils were allowed to adhere to FN80-coated coverslips for 10 minutes. C5a was then deposited at one edge of the bottom of the well
(lower right-hand corner of photograph) and cells were allowed to
migrate for 5 minutes. Cells were fixed and stained with anti-PSGL-1
KPL-1 mAb (green fluorescence). Epifluorescence and bright-field
conditions were photographed on the same frame by means of double
exposure. (B) Neutrophils were allowed to adhere to an endothelial cell
monolayer activated by TNF- for 5 minutes at 37°C. Cells were
then fixed and double stained with anti-ICAM-3 TP1/25 mAb (green
fluorescence) and anti-ICAM-1 MEM111 (red fluorescence) mAb. (C)
Neutrophils were allowed to adhere to an endothelial cell monolayer
activated by TNF- for 5 minutes at 37°C. Cells were then fixed
and stained with anti-PSGL-1 KPL-1 mAb. Epifluorescence and
bright-field conditions were photographed on the same frame by means of
double exposure.
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In studying possible mechanisms for the redistribution of these
receptors, we found that moesin colocalized with PSGL-1 and ICAM-3 and
was apparently also concentrated in uropods of neutrophils activated
with IL-8 (Figure 3).
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| Fig 3.
Redistribution of PSGL-1, ICAM-3, and moesin to the
uropod of polarized neutrophils.
Neutrophils adhering to FN80 were stimulated with IL-8 for 5 minutes.
Cells were then fixed and stained. (A) ICAM-3. (B) PSGL-1. Inset shows
colocalization of PSGL-1 (a) and ICAM-3 (b). (C) Moesin. Inset shows
colocalization of moesin (c) and ICAM-3 (d).
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Coelution of ICAM-3 and moesin from human neutrophils
To determine whether ICAM-3 is associated with moesin in
neutrophils, ICAM-3 was isolated from lysates by antibody-affinity chromatography. A protein of 78 kd eluted at approximately pH 11 and
was partly separated from ICAM-3, which eluted at pH 11.5 as a broad
band of nearly 125 kd (Figure 4A). When the
pooled fraction containing ICAM-3 was analyzed by immunoblotting,
moesin was detected (Figure 4B). This immunoreactive polypeptide
corresponded to the 78-kd band on the gel (Figure 4A). The original
lysate of neutrophils reacted with antibodies to both moesin and
ezrin,34 although higher amounts of moesin than of ezrin
were detected with specific antibodies (data not shown). Ezrin was not
detected in the ICAM-3 fraction (Figure 4B). To exclude a possible
nonspecific effect, the pooled fractions were also analyzed in
additional control experiments by immunoblotting with antibodies to
GRP78, an immunoglobulin-binding and possible contaminating protein
with a molecular mass similar to that of moesin. No reactivity was observed (data not shown).
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| Fig 4.
Association of ICAM-3 and moesin in neutrophils.
ICAM-3 was isolated from human neutrophil lysates by antibody-affinity
chromatography. (A) A polypeptide of 78 kd (arrow) eluted in fractions
at pH 11.0 (lanes 1-2) while ICAM-3 coeluted at pH 11.0 (lane 2) and
separated from the 78-kd polypeptide at pH 11.5 (lanes 3-4). (B)
Western blot analysis of purified recombinant ezrin (lane 1) and the
pooled ICAM-3 fraction (lane 2) with ezrin antibodies and the same
pooled ICAM-3 fraction (lane 3) and recombinant moesin (lane 4) with
moesin antibodies. A strong signal for moesin showed that the 78-kd
polypeptide coeluting with ICAM-3 (Figure 4A) corresponded to moesin.
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Interaction of ERM proteins with the cytoplasmic region
of PSGL-1
To investigate further the association between adhesion proteins and
moesin, GST-fusion proteins of the cytoplasmic domains of PSGL-1 and
ICAM-3 were prepared. Instead of neutrophils, HL-60 cells were
metabolically labeled with 35S met-cys to
search for labeled proteins in the lysate that would bind to the
cytoplasmic domains of these 2 membrane proteins. Several radiolabeled
polypeptides were recovered from the GST-PSGL-1- and
ICAM-3-glutathione-Sepharose but not from control beads containing either GST or no ligand that included polypeptides of 75-78 kd (data
not shown). In parallel experiments, lysates of unlabeled HL-60 cells
were incubated with the GST-PSGL-1 cytoplasmic-domain fusion protein,
and bound proteins were analyzed by Western blot assessment using
moesin and ezrin antibodies. As shown in Figure 5, the cytoplasmic region of PSGL-1 bound
78- and 81-kd polypeptides that were identified as moesin and ezrin,
respectively (Figure 5A). In contrast, none of the proteins bound to
PSGL-1 corresponded to vinculin (Figure 5B).
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| Fig 5.
Western blot analysis of proteins interacting with the
cytoplasmic tail of PSGL-1 in HL-60 cells.
HL-60 cell lysates were incubated with equivalent amounts of
glutathione-Sepharose, glutathione S-transferase (GST), and
GST-CyPSGL-1 fusion protein bound to glutathione-Sepharose 4B beads.
Each immunoprecipitate was immunoblotted after 8% sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with antimoesin
95/2 polyclonal antibody (pAb), antiezrin 90/3 pAb (A), or antivinculin
mAb (B). Cell lysate blotted with antivinculin was included as the
positive control.
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Interaction of PSGL-1 and ICAM-3 with the amino-terminal
domain of moesin
Several studies have shown that the binding of moesin and related
proteins to membrane proteins requires additional factors that may
include a conformational change to expose potential binding sites.28,40,41 When purified soluble recombinant
full-length moesin or the isolated amino-terminal domain proteins were
added to beads containing GST-fusion proteins of the cytoplasmic
domains of PSGL-1 and ICAM-3, we found only trace amounts of
full-length moesin (Figure 6); however,
there were much stronger signals with smaller immunoreactive
polypeptides (~60 kd). In contrast to the weak binding observed for
full-length moesin, the N-terminal domain of moesin bound strongly to
the cytoplasmic domain of both PSGL-1 and ICAM-3 (Figure 6C). This
result suggests that the bound immunoreactive polypeptides shown in
Figure 6B represented enriched degradation products that contaminated
the purified full-length moesin preparation.
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| Fig 6.
Interaction of recombinant moesin with the cytoplasmic
region of PSGL-1 and ICAM-3.
(A) Coomassie-blue-stained 12% SDS-PAGE results with recombinant
full-length moesin (moesin) and the amino-terminal domain of moesin
(N-moesin) purified from bacteria. Small amounts of contaminating
peptides are present. (B) Glutathione-Sepharose (Glut-Seph), GST, or
GST-fusion proteins containing the cytoplasmic domain of PSGL-1
(CyPSGL-1) or ICAM-3 (CyICAM-3) linked to glutathione-Sepharose beads
were incubated with purified recombinant full-length moesin. GST,
GST-fusion proteins, and other bound proteins were eluted together from
the beads with Tris buffer containing 20 mmol/L of glutathione. Eluted
proteins were resolved by 10% SDS-PAGE followed by immunoblotting with
95/2 pAb specific for moesin. (C) Binding assay of the amino-terminal
region of moesin (N-moesin) to the cytoplasmic tail of PSGL-1 and
ICAM-3 was performed as described above (Figure 6B); proteins were
separated by 10% SDS-PAGE and immunoblotted with the 95/2 pAb. To
detect N-terminal moesin, a chemiluminescence system and a longer
exposure than that used to obtain the results shown in Figure 6B were
required because of the lower reactivity of the 95/2 pAb for the
amino-terminal fragment compared with the full-length form of moesin.
Molecular masses in kilodaltons are indicated on the left in Figure 6A
and 6C and on the right in Figure 6B.
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The amino-terminal domain of moesin made in bacteria contains several
additional amino acid residues that remain attached after cleavage by
thrombin. To exclude a contribution of this additional structure to
binding, we tested the same domain of moesin made by in vitro
translation. The amino acid sequence of this fragment corresponded
precisely to the sequence of moesin from amino acid residues 1 to 310. As shown in Figure 7, the radiolabeled probe associated with PSGL-1 and ICAM-3 but did not bind to the control
GST.
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| Fig 7.
Association of in vitro translated amino-terminal moesin
with PSGL-1 and ICAM-3.
In vitro translated amino-terminal moesin (N-moesin) was incubated with
GST or GST-fusion proteins containing the cytoplasmic domain of PSGL-1
or ICAM-3 linked to glutathione-Sepharose beads. After incubation,
Sepharose beads were centrifuged and washed and bound proteins were
boiled in sample buffer and analyzed by means of 10% SDS-PAGE and
autoradiography. N-moesin labeled with sulfur 35 was loaded as a
control in the last lane. Autoradiography showed nonspecific binding of
a polypeptide of about 50 kd and specific binding of a labeled
polypeptide of the size of N-moesin to PSGL-1 and ICAM-3. Molecular
masses in kilodaltons are indicated on the left.
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Discussion |
During acute inflammatory responses, selectins mediate initial
rolling of neutrophils along the endothelial surface.42,43 This is thought to facilitate juxtacrine signaling by positioning leukocytes near locally generated chemoattractants. These mediators, which are either synthesized or presented by activated endothelial cells, activate leukocyte integrins. The signals generated through selectins, combined with those triggered by chemoattractants, result in
the integrin-mediated tight adhesion of leukocytes, followed by their
transendothelial migration.42,43 Free-flowing leukocytes
can also attach to and roll on monolayers of adherent leukocytes under
hydrodynamic shear stress, and PSGL-1 can serve as the ligand for
L-selectin in neutrophil-neutrophil interactions.44,45 The
redistribution of PSGL-1, ICAM-3, and other adhesion molecules to the
uropod may therefore enable activated cells to make contact with
additional leukocytes and to increase neutrophil recruitment. The
concentration of PSGL-1 in the uropod may also facilitate interaction
with and recruitment of platelets.22
L-selectin and other adhesion receptors involved in initiation of the
leukocyte-endothelial cell adhesion cascade, such as PSGL-1, are
concentrated in microvillous tips.21,46 This preferential localization in microvilli is thought to facilitate contact with the
endothelium.46 Studies have shown that the uropod of
leukocytes is highly enriched in microvilli and
microspikes.47 This suggests that accumulation of adhesion
molecules, such as PSGL-1, in the uropod of neutrophils
stimulated by chemoattractants serves to capture other leukocytes.
Chemoattractants induce the full range of neutrophil activation events,
including chemotaxis, oxygen free-radical production, and
degranulation.6,7 An early event is a change in morphologic conformation and, as we observed in this study, polarization and redistribution to the uropod of several adhesion molecules and members
of the ERM protein family, which link membrane F-actin. Activated and adherent neutrophils move on the endothelial
surface and extravasate by migrating through endothelial clefts. Our
data show that both crawling neutrophils and those migrating through a
monolayer of endothelial cells have polarized morphologic features, with ICAM-3 and PSGL-1 redistributed to the uropod. These results concur with those of previous studies, which found that neutrophils stimulated by fMLP transmigrated across the endothelium and engaged other neutrophils through uropod-mediated contacts.48
Protrusions of the plasma membrane in lymphocytes and neutrophils,
described morphologically as microvilli or microspikes, have not been
studied in detail. These structures, unlike microvilli on the apical
surface of brush-border epithelia in the gut or kidney, do not contain
villin or fimbrin. They are presumably more closely related to
filopodia, microspikes, and retraction fibers of fibroblasts in
culture.29 It is well established that these
microextensions contain actin filaments and one or more members of a family of proteins (the ERM proteins) that include moesin,
ezrin, and radixin. At least one of these proteins appears to be
necessary for either the formation or maintenance of such microextensions.33 All 3 proteins bind F-actin at a site in the C-terminal domain that must be activated or unmasked, since no
interaction has been observed with intact, undenatured full-length molecules.40,41,49
In cells, moesin, ezrin, and radixin form complexes with CD44, CD43,
ICAM-1, ICAM-2, and ICAM-3,24,25,50-53 and direct
low-affinity associations between recombinant full-length proteins and
CD44 and ICAM-1 have been studied in vitro.52,53 The in
vitro data in this study show that PSGL-1 and ICAM-3 bind poorly to
full-length moesin but strongly to the isolated amino-terminal domain.
This result is reminiscent of the strong interaction between actin filaments and the isolated C-terminal domain40 and may
indicate that a conformational change is necessary to expose
functionally important binding sites in the N-terminal domain. In the
native configuration, N- and C-terminal domains of moesin associate in a manner that prevents F-actin binding.26-28,40,41 The same
intramolecular association may prevent interactions with membrane
proteins, as we observed in this study.
The binding of ICAM-3 and PSGL-1 to moesin from cell lysates but not
from bacteria may be explained by the fact that, in neutrophils and
HL-60 cells, a fraction of the moesin molecules can be structurally altered by phosphorylation or by formation of a complex with
polyphosphoinositides. For example, phosphorylation by Rho-dependent
kinase or protein kinase C disrupts the intramolecular association
between N- and C-terminal domains, suggesting regulation by small
GTPases of the Rho family and other signaling
pathways,49,54,55 and cosedimentation of highly purified
moesin from human platelets with F-actin was observed in vitro when the
molecule was both phosphorylated at thr558 and when it formed a complex
with phospholipids.40,41 A study has found that tyrosine
phosphorylation is required for the interaction between the N-terminal
domain of ezrin and phosphatidylinositol 3-kinase,56 suggesting yet other mechanisms
for activation. Although our in vitro binding data are consistent with
this simple mechanism and a direct interaction between receptor
proteins and moesin or ezrin, another possibility has not been
excluded. Links between transmembrane proteins and the actin
cytoskeleton in cells also develop by insertion of additional proteins
containing the PDZ domain that interact with membrane
proteins on one hand and moesin or ezrin on the
other.57,58 This provides additional possibilities for
regulation. Further work is required to determine whether ICAM-3 or
PSGL-1 are among the receptors that use this mechanism.
| |
Acknowledgments |
We thank R. González-Amaro, M. Montoya, and M. Vicente-Manzanares
for critical reading of the manuscript and E. Fernández Villareal, M. J. Borque, and Lourdes Herreros for help in providing reagents and technical advice.
| |
Footnotes |
Submitted April 26, 1999; accepted December 16, 1999.
Supported by grants SAF 99/0034-CO1 and 2FD97-0680-C02-02 from the
Ministerio de Educación y Cultura (to F.S.-M.) and 08/0011/1997 from the Comunidad Autónoma de Madrid. G.K. is an Established Investigator of the American Heart Association, funded by grant CB-204
from the American Cancer Society.
Reprints: Francisco Sánchez-Madrid, Hospital de la
Princesa, Servicio de Inmunología, C/ Diego de León 62, Madrid 28006, Spain; e-mail: smadrid/princesa{at}hup.es.
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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Top
Abstract
Introduction