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INTRODUCTION |
The selectins are a family of three calcium-dependent
lectins that mediate adhesive interactions between leukocytes and the endothelium during normal and abnormal inflammatory conditions such as
rheumatoid arthritis (RA)1 or
atherosclerosis (1, 2). E-selectin is a single-chain 115-kDa
glycoprotein with a lectin-like N-terminal domain, an epidermal growth
factor (EGF)-like motif, and a variable number of repeat units
homologous to the consensus repeats of complement binding proteins (3).
The lectin domain of E-selectin plays a major role in ligand
recognition and binds to sialyl LeX on leukocytes as well
as on endothelial cells (4-6). Soluble isoforms of these adhesion
molecules are rapidly shed from the cellular surfaces on cellular
activation (1). We have found elevated levels of soluble E-selectin
(sE-selectin) in patients with RA (7). In contrast to the earlier
hypothesis that soluble adhesion molecules serve an anti-inflammatory
role, our laboratory has shown a unique role for sE-selectin as a
proinflammatory, angiogenic mediator (8).
The infiltration of monocytes into the synovial tissue is a key
factor in amplifying and perpetuating RA. The functions of elevated
levels of sE-selectin and the mechanism of action of sE-selectin are
still poorly understood. One of the primary mechanisms by which
leukocytes are activated might be through the activation of protein
tyrosine kinases at the cell surface (9, 10). The Src family of
tyrosine kinases is activated during a number of biological functions
including monocyte interaction with endothelial cells (11), endothelial
cell differentiation (12), and cardiac motility (13). A number of
pathways lead to the activation of mitogen-activated protein kinases
(MAPKs). MAPKs are a family of 38-45 kDa proteins that exist in a
dephosphorylated form in quiescent cells and can be activated in
response to various growth factors and chemoattractants (14, 15). MAPKs
are activated by MAPK/ERK kinase (MEK), which in turn is activated by
MEK kinase (MEKK). One of the extensively studied MEKKs is Raf kinase
(16). Raf kinase is in turn activated by binding to Ras-GTP (17), and
Src is an important upstream kinase linked to Ras-Raf activation (12,
18).
In this study, we examined the role and mechanism of action of
sE-selectin in mediating monocyte recruitment. We report that sE-selectin is a potent chemotactic factor for monocytes, and its neutralization in RA synovial fluids, led to a significant decrease in RA synovial fluid-mediated monocyte migration. Furthermore, our results suggest that sE-selectin mediates signaling in monocytes through a Src-Ras-MAPK pathway. The inhibition of Src kinase by the
Src-specific inhibitor, PP2, abolished sE-selectin-mediated monocyte
chemotaxis. These results suggest that sE-selectin mediates chemotaxis
through the Src pathway, and this could thus be a potential target for
modulating monocyte recruitment-driven diseases.
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EXPERIMENTAL PROCEDURES |
Reagents--
Recombinant human sE-selectin was purchased from
R&D Systems (Minneapolis, MN). sE-selectin contained less than 0.1 ng
of endotoxin/1 µg of protein content (as per the manufacturer).
Accu-Prep was purchased from Accurate Chemical and Scientific
Corporation (Westbury, NY). Percoll and Hanks' balanced salt solution
(HBSS) were obtained from Life Technologies, Inc. Orthovanadate,
paranitrophenylphosphate, leupeptin, aprotinin, phenylmethylsulfonyl
fluoride, dimethyl sulfoxide (Me2SO), bovine serum
albumin, and N-formyl-Met-Leu-Phe (fMLP) were obtained from
Sigma. Protease inhibitor mixture tablets were obtained from Roche
Molecular Biochemicals. PP2 was purchased from Calbiochem. Mouse
anti-human E-selectin monoclonal antibody BB11 (IgG1) was a
gift from Biogen (Cambridge, MA). Mouse IgG1 antibody
(negative control) was purchased from Coulter (Hialeah, FL). Mouse
monoclonal anti-phosphotyrosine antibody (4G10), polyclonal rabbit
anti-human pERK1/2 antibody, polyclonal rabbit anti-human Lyn, mouse
monoclonal anti-EGF-receptor (Tyr(P)1173), and the
Ras activation detection kit were purchased from Upstate Biotechnology
(Lake Placid, NY). Polyclonal rabbit anti-human Hck was purchased from
Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Polyclonal
rabbit anti-human pSrc antibody (Tyr(P)418) and polyclonal
rabbit anti-human focal adhesion kinase (Tyr(P)576) were
obtained from BIOSOURCE (Camarillo, CA), and
polyclonal rabbit anti-human p38 antibody was purchased from New
England Biolabs (Beverly, MA). Polycarbonate filters with 5-µm pores
were obtained from Poretics Corp. (Livermore, CA). Protein estimation reagents (BCA kit) were from Pierce. Enhanced chemiluminescence (ECL)
Western blotting detection reagents and mouse horseradish peroxidase-conjugated antibody were obtained from Amersham Pharmacia Biotech.
Patient Sample Preparation--
Synovial fluids were obtained
from patients with RA who met the American College of Rheumatology
criteria for RA (19). All specimens were obtained with Institutional
Review Board approval.
Isolation of Human Mononuclear Cells (MN) and Monocytes--
MN
were isolated from the peripheral blood (PB) of normal healthy
volunteers using Accu-Prep. PB was collected from normal healthy adult
donors in heparinized tubes. After collecting a buffy coat, MN were
purified under sterile conditions by centrifugation on Accu-Prep at
400 × g for 30 min at room temperature. MN were collected at the interface, washed twice with phosphate-buffered saline, and resuspended in HBSS with calcium and magnesium at 2.5 × 106 cells/ml. Cell viability was determined by trypan
blue exclusion. The viability of MN used was >98%, and the purity was
>99%. The method of monocyte separation was described previously
(20). Briefly, 4 ml of MN (5 × 107 cells) was gently
layered over 8 ml of isolation buffer (1.65 ml of 10× HBSS in 10 ml of
Percoll, pH 7.0). After centrifugation at 400 × g for
25 min at room temperature, monocytes at the interface were collected.
The viability of monocytes determined by trypan blue exclusion was
found to be >98%, and the purity was >90%.
Cell Lysis and Immunoblotting--
Monocytes (1 × 107 cells/ml) were incubated in 24-well plates for 3 h
in HBSS (with calcium and magnesium) prior to stimulation with
sE-selectin for various time points. At the end of each time period,
supernatants were gently aspirated, and cells were lysed in extraction
buffer containing 100 mM Tris, pH 7.4, 100 mM
NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20 mM NaP2O4, 2 mM Na3VO4, 1% Triton X-100, 10%
glycerol, 0.1% SDS, 0.5% deoxycholate, 1 mM phenylmethylsulfonyl fluoride, and protease inhibitors (protease inhibitor mixture tablets, 1 tablet/10 ml) (Roche Molecular
Biochemicals). For experiments with signaling inhibitors, monocytes
were preincubated with the respective inhibitor before activation with
sE-selectin. The protein content of different samples was quantitated
using a bicinchoninic acid (BCA) protein assay kit. Cell lysates were mixed at 1:1 with Laemmli's sample buffer and boiled for 5 min. 15 µg of each sample was subjected to 10% SDS-polyacrylamide gel electrophoresis. Separated proteins were electrophoretically
transferred from the gel onto nitrocellulose membranes using a
Tris-glycine buffer. To block nonspecific binding, membranes were
incubated with 3% bovine serum albumin in Tris-buffered saline
containing 0.1% Tween 20 (TBST) for 1 h at room temperature. The
blots were incubated in respective primary antibody in TBST + 3%
bovine serum albumin at 4 °C overnight. After washing with TBST, the
blots were incubated with horseradish peroxidase-conjugated sheep
anti-mouse IgG (1:10,000) or with goat anti-rabbit IgG (1:10,000) for
45 min at room temperature. An enhanced chemiluminescence detection (ECL) system (Amersham Pharmacia Biotech) was used to detect specific protein bands. The different bands were then scanned and quantitated using an imaging densitometer (Bio-Rad).
Immunoprecipitation--
Cell lysates were precleared with
rabbit nonimmune serum prebound to sheep anti-rabbit IgG-agarose
(Sigma). Lyn and Hck kinases were immunoprecipitated from 100 µg of
each precleared sample by incubation at 4 °C for 16 h with 2 µg of polyclonal rabbit anti-Lyn (Upstate Biotechnology) or 3 µg of
polyclonal rabbit anti-Hck antibody (Santa Cruz Biotechnology),
respectively. The immune complexes were further incubated for 2 h
at 4 °C after the addition of sheep anti-rabbit IgG-agarose
conjugate. Immunopellets were washed three times with lysis buffer,
reconstituted in 40 µl of 2× Laemmli sample buffer, and boiled for 5 min. These samples were subsequently run on 10% SDS-PAGE as described
above. The presence of activated Lyn and Hck kinases was detected by
probing the blots with mouse monoclonal anti-phosphotyrosine antibody (4G10 clone) (Upstate Biotechnology). The immunoblots were stripped for
30 min at 55 °C in 67 mM Tris at pH 6.7, 2% SDS, and
100 mM 2-mercaptoethanol and reprobed with rabbit
polyclonal anti-Lyn or rabbit polyclonal anti-Hck antibody, respectively.
Ras Activation Assay--
Ras activation was studied using a Ras
activation kit (Upstate Biotechnology). Monocytes were stimulated with
sE-selectin (50 nM) for different time periods. At each
time point monocytes were extracted with cell lysis buffer, and the
protein content in each sample was quantitated. The Ras activation
assay involved two steps. In the second step, the presence of
activated Ras was immunoprecipitated from 100 µg of each monocyte
lysate sample with an immobilized Raf-1-Ras binding domain and
subsequently run on 10% SDS-PAGE as described above. The presence of
activated Ras in samples was then detected by probing with a specific
mouse monoclonal anti-Ras antibody (1 µg/ml). The different bands
were then scanned and quantitated using an imaging densitometer.
Monocyte Chemotaxis--
Monocyte chemotaxis was performed using
48-well chemotaxis chambers (Neuroprobe, Cabin John, MD) with a
polyvinylpyrolidone-free polycarbonate filter as described previously
(21). In brief, 25 µl of stimulant or buffer was added to the bottom
wells of the chambers. A 5-µm membrane was placed in the assembly,
and 40 µl of monocytes at 2.5 × 106 cells/ml was
placed in the top of wells. The chemotaxis chambers were incubated for
2 h at 37 °C with 5% CO2. The filters were removed, and the membranes were fixed in methanol and stained with
Diff-Quik (VWR Scientific Products, Chicago, IL). Each test group was assayed in quadruplicate. Three high power (×400) microscope fields were counted in each replicate well, and the results were expressed as cells per high power field. In synovial fluid
neutralization studies, 1:2 diluted synovial fluid was preincubated
with anti-sE-selectin antibody (5 µg/ml) (Biogen) or corresponding
control antibody (mouse IgG1) for 1 h at 37 °C. In
the signaling inhibitor studies, monocytes (2.5 × 106
cells/ml) were preincubated with their respective inhibitors for 1 h at 37 °C. After washing twice with HBSS containing calcium and
magnesium, the cells were adjusted to 2.5 × 106
cells/ml and then assayed in response to different concentrations of
sE-selectin. The results are expressed as the mean and S.E. of three
high power fields (×400) per replicate well, and each test group was
assayed in quadruplicate. The incubation of monocytes with the Src
inhibitor PP2 at 1 µM did not alter cell viability. For
all assays, controls included HBSS (negative control), fMLP (positive
control), and Me2SO (a vehicle for the Src inhibitor).
Statistical Analysis--
Data were analyzed using Student's
t test, and p values less than 0.05 were
considered significant.
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RESULTS |
sE-selectin Is Chemotactic for Monocytes in Vitro--
Our
laboratory has shown that sE-selectin levels are up-regulated in
synovial fluids from patients with RA (7). To examine the hypothesis
that sE-selectin might be chemotactic for monocytes, we studied
monocyte chemotaxis in response to sE-selectin in a modified Boyden
chamber. A representative assay from five experiments is shown in Fig.
1. sE-selectin-induced chemotaxis in a
concentration-dependent manner and was significantly higher
at 10, 50, and 100 nM sE-selectin concentrations as compared with the negative control (p < 0.05).

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Fig. 1.
sE-selectin-mediated monocyte
chemotaxis. Monocytes (2.5 × 106
cells/ml) in the upper chemotaxis chamber were incubated with various
concentrations of sE-selectin in the lower chamber for 2 h at
37 °C. Results are expressed as the mean and S.E. of three high
power fields (×400) per replicate well. Each test group was assayed in
quadruplicate. This is a representative assay from five independent
experiments. HBSS was used as a negative control, and fMLP (100 nM) was used as a positive control. sE-selectin showed a
dose-dependent increase (p < 0.05) in
monocyte chemotaxis as compared with the negative control HBSS.
* represents a significant difference (p < 0.05) between the sE-selectin group and the negative control
HBSS.
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Contribution of sE-selectin to RA Synovial Fluid Chemotactic
Activity for Monocytes--
We have shown earlier that RA synovial
fluid is potently chemotactic for monocytes (22), and we have also
demonstrated that high levels of sE-selectin are present in the RA
synovial fluid (7). To examine the contribution of sE-selectin present
in the RA synovial fluid to the chemotaxis of normal PB
monocytes, a 1:2 dilution of RA synovial fluid was preincubated with
isotype-matched control antibody or anti-E-selectin antibody for 1 h at 37 °C. The incubation of RA synovial fluid with anti-E-selectin
antibody significantly decreased monocyte chemotaxis (129 ± 3.2, 193 ± 4.4, and 90 ± 7.5) as compared with the isotype
control antibody in the respective samples (225 ± 5.9, 270 ± 13.7, and 134 ± 4.1) (Table
I). These data indicate that sE-selectin
contributes significantly to the RA synovial fluid chemotactic activity
for monocytes.
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Table I
Percent suppression of normal human PB monocyte migration in response
to RA synovial fluid (SF) incubated with anti-E-selectin antibody
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Signaling Mechanism Involved in sE-selectin-mediated
Chemotaxis--
We next investigated the signaling pathways involved
in the sE-selectin-mediated monocyte chemotaxis. sE-selectin (50 nM) induced protein tyrosine phosphorylation in monocytes
in a time-dependent manner showing two peaks. Western blot
analysis showed a first peak at 30 s and a second peak at 30 min.
Proteins phosphorylated at tyrosine residues corresponding to molecular
sizes of 130-140, 70, 60-65, 50-55, 42-44, 35-40, and 25 kDa were
detected (Fig. 2) by using a mouse
monoclonal antibody (4G10), which specifically reacts with tyrosine
phosphorylated proteins.

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Fig. 2.
Protein tyrosine phosphorylation pattern in
monocytes stimulated with sE-selectin (A) or monocytes
pretreated with the Src inhibitor PP2 prior to sE-selectin stimulation
(B). Monocytes were stimulated with sE-selectin
(50 nM) for various amounts of time as indicated
(NS, nonstimulated; m, minutes) or first
pretreated with PP2 (1 µg) for 30 min and then stimulated with
sE-selectin. Cells were lysed with lysis buffer, and the protein
content of each sample was quantitated. 15 µg of each sample was
resolved by 10% SDS-PAGE and probed with mouse monoclonal antibody
4G10 (750 ng/ml) to detect the phosphorylated proteins. Experiments
shown in A and B were repeated five and three
times, respectively, with essentially identical results. sE-selectin
induced the tyrosine phosphorylation of a number of proteins. An
increase in tyrosine phosphorylation was observed as early as 30 s, peaked at 30 min, and then tapered off by 1 h. The pretreatment
of monocytes with PP2 prior to sE-selectin stimulation showed the
inhibition of the tyrosine phosphorylation of proteins in the Src
region (50-60 kDa) and MAPK region (35-45 kDa).
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As the predominant bands (50-65 kDa) observed with the 4G10 antibody
corresponded to the molecular sizes of the Src family kinases, we next
studied the role of Src family kinases Src, Hck, and Lyn in
sE-selectin-mediated signaling in monocytes. We first performed a time
course study of Src kinase activation followed by a Src inhibition
study with the Src inhibitor PP2. Western blot analysis of activated
Src with a phosphorylation state-specific antibody
(Tyr(P)418) showed a biphasic activation of Src with the
first peak at 30 s and a second peak at 30 min (Fig.
3). The pretreatment of monocytes with
the Src inhibitor PP2 (1 µM) for 30 min prior to
stimulation with sE-selectin partially inhibited Src phosphorylation
(Fig. 4). We next studied the role of two
other Src family kinases (Hck and Lyn) in sE-selectin-mediated
signaling in monocytes. Hck and Lyn kinases were immunoprecipitated
from sE-selectin-stimulated monocytes by polyclonal rabbit anti-Hck or
polyclonal rabbit anti-Lyn antibodies, respectively. The activation of
Hck and Lyn kinases was determined by subjecting these
immunoprecipitates to SDS-PAGE and Western blotting with
anti-phosphotyrosine antibody (4G10). Hck kinase showed a
time-dependent increase in tyrosine phosphorylation (Fig.
5A), whereas Lyn kinase showed
a minor peak at 30 s and a major peak at 30 min (Fig.
5B). The pretreatment of monocytes with PP2 markedly
inhibited the phosphorylation of both Hck and Lyn kinases (Fig. 5). The
total protein phosphotyrosine profile analysis in monocytes pretreated
with PP2 prior to sE-selectin stimulation showed inhibition of the
phosphorylation of proteins in the MAPK region (35-45 kDa) in addition
to the inhibition of Src family kinase region (60-70 kDa) (Fig.
2B).

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Fig. 3.
Time-dependent activation of Src
in monocytes by sE-selectin. Monocytes were stimulated with
sE-selectin (50 nM) for various time points as indicated in
the figure (m, minutes). Cell extracts were prepared with
cell lysis buffer, and the protein content of each sample was
quantitated. 5 µg of each sample was subjected to 10% SDS-PAGE and
probed with rabbit polyclonal anti-pSrc antibody (0.5 µg/ml).
sE-selectin induced the tyrosine phosphorylation of Src in a
time-dependent manner showing a biphasic activation
pattern. The first activation peak occurred at 30 s, and the
second peak occurred at 30 min. This blot is representative of three
independent experiments.
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Fig. 4.
Inhibition of sE-selectin-mediated Src
activation by the Src-specific inhibitor PP2. Monocytes were
pretreated with the Src-specific inhibitor PP2 (1 µM) for
30 min at 37 °C before being stimulated with sE-selectin for
different time points. Cell extracts were prepared with lysis buffer at
each time point, and the protein concentration in each sample was
quantitated. 15 µg of each sample was subjected to 10% SDS-PAGE and
probed with rabbit polyclonal anti-pSrc antibody (0.5 µg/ml). The
pretreatment of monocytes with the Src-specific inhibitor PP2 markedly
decreased Src phosphorylation by sE-selectin. This blot is
representative of three independent experiments.
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Fig. 5.
Hck and Lyn activation in monocytes
stimulated with sE-selectin. Monocytes, untreated or preincubated
with 1 µM PP2 for 30 min, were stimulated with
sE-selectin (50 nM). Cell extracts were prepared at various
time points, and the protein concentration in each sample was
quantitated. Each sample (100 µg) was immunoprecipitated
(IP) with anti-Hck or anti-Lyn antibodies and
Western-blotted (WB) with anti-phosphotyrosine antibody
(4G10) to detect activated Hck or Lyn kinase. Alternatively, the blots
were probed for total Hck or Lyn kinase. sE-selectin induced the
tyrosine phosphorylation of Hck (A) and Lyn (B)
in a time-dependent manner, which was markedly inhibited by
pretreatment with PP2. These results are representative of three
independent experiments.
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To test the hypothesis that the sE-selectin signal from Src family
kinases might be acting through MAP kinases, we studied the time course
of extracellular signal-related kinase (ERK1/2) and p38 MAPK activation
in monocytes. ERK1/2 showed a biphasic activation profile as observed
with Src kinase with the first peak occurring at 1 min and the second
peak at 30 min (Fig. 6). However, p38
MAPK did not show phosphorylation until 5 min after stimulation and
showed a single peak at 30 min (Fig. 7).
Next, we studied the role of the Src inhibitor PP2 on ERK1/2 and p38 MAPK activation. The pretreatment of monocytes with the Src inhibitor PP2 prior to stimulation with sE-selectin revealed 89 and 83% inhibition of ERK1/2 and p38 MAPK, respectively, as compared
with sE-selectin-stimulated monocytes (Figs.
8 and 9) at 30 min. Similar levels of the inhibition of ERK1/2 and
p38 MAPK were observed at other time points.

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Fig. 6.
ERK1/2 activation in monocytes stimulated
with sE-selectin. Monocytes were stimulated with sE-selectin (50 nM) at various time points as indicated (m,
minutes). Cells were extracted with cell lysis buffer, and the protein
content of each sample was quantitated. 15 µg of each sample was
subjected to 10% SDS-PAGE and probed with rabbit polyclonal
anti-pERK1/2 antibody (0.5 µg/ml). The sE-selectin stimulation of
monocytes induced enhanced the tyrosine phosphorylation of ERK1/2 in a
time-dependent manner as compared with the control
nonstimulated monocytes. This blot is representative of three
independent experiments.
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Fig. 7.
p38 MAPK activation in monocytes stimulated
with sE-selectin. Monocytes were stimulated with sE-selectin (50 nM) at various time points as indicated in Fig. 6
(m, minutes). Cells were extracted with cell lysis buffer,
and the protein content of each sample was quantitated. 15 µg of each
sample was subjected to 10% SDS-PAGE and probed with rabbit polyclonal
anti-p38 antibody (1:1000 dilution). The sE-selectin stimulation of
monocytes induced the enhanced tyrosine phosphorylation of p38 MAPK in
a time-dependent fashion as compared with the control
nonstimulated monocytes. This blot is representative of three
independent experiments.
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Fig. 8.
Inhibition of sE-selectin-mediated ERK1/2
activation by the Src inhibitor PP2. Monocytes were preincubated
with the Src inhibitor PP2 (1 µM) for 30 min at 37 °C
prior to stimulation with sE-selectin (50 nM) for various
time periods. At each time point, cells were extracted with lysis
buffer, and the protein concentration was quantitated. 15 µg of each
sample was loaded on 10% SDS-PAGE and probed with rabbit polyclonal
anti-pERK1/2 antibody (0.5 µg/ml). The pretreatment of monocytes with
the Src-specific inhibitor PP2 markedly inhibited ERK1/2
phosphorylation in monocytes in response to sE-selectin. This blot is
representative of three independent experiments.
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Fig. 9.
Inhibition of sE-selectin-mediated p38 MAPK
activation by Src inhibitor PP2. Monocytes were preincubated with
the Src inhibitor PP2 (1 µM) for 30 min at 37 °C
before being stimulated with sE-selectin (50 nM) for
various time points. At the end of each time point, cells were
extracted with lysis buffer, and the protein concentration was
quantitated. 15 µg of each sample was loaded on 10% SDS-PAGE and
probed with rabbit polyclonal anti-p38 MAPK antibody (1:1000 dilution).
The pretreatment of monocytes with the Src-specific inhibitor PP2
markedly inhibited p38 MAPK phosphorylation in monocytes in response to
sE-selectin. This blot is representative of three independent
experiments.
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sE-selectin is a glycoprotein and contains an EGF-like motif (3), so we
tested whether sE-selectin mediates its action through the EGF
receptor. Our results show that sE-selectin does not induce the
phosphorylation of the EGF receptor (data not shown). We also
studied the phosphorylation profile of focal adhesion kinase, a
substrate for the Src family kinases. However, we failed to detect the
phosphorylation of focal adhesion kinase in sE-selectin-stimulated monocytes (data not shown). These data indicate that in contrast to Src
family kinases (Src, Hck, and Lyn) and MAPKs (ERK1/2 and p38), the EGF
receptor and focal adhesion kinase pathways are not involved in
sE-selectin-mediated monocyte signaling.
A number of studies have shown that Raf kinase is a predominant MEK
kinase (16, 23) that in turn is activated by binding to Ras-GTP (17).
Src family kinases have also been shown to be linked to MAPK through
Ras/Raf kinases (12, 18). To study the activation of Ras/Raf kinases,
we first immunoprecipitated activated Ras with a Raf-1-Ras binding
domain conjugated to agarose beads and subsequently probed with mouse
monoclonal anti-Ras antibody. sE-selectin stimulation of
monocytes showed a time-dependent activation of the Ras/Raf
pathway, which peaked at 30 min (Fig.
10).

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Fig. 10.
Time-dependent Ras activation in
monocytes stimulated with sE-selectin. Monocytes were stimulated
with sE-selectin for various time points as indicated (m,
minutes). Cells were extracted with cell lysis buffer, and the protein
content in each sample was estimated. 100 µg of each sample was
immunoprecipitated with a Raf-1-Ras binding domain-agarose conjugate
and subsequently subjected to 10% SDS-PAGE. The presence of activated
Ras in the samples was then detected by probing with a specific mouse
monoclonal anti-Ras antibody (1 µg/ml). The sE-selectin stimulation
of monocytes induced a time-dependent increase in Ras
activation with a peak activation at 30 min. This blot is
representative of three independent experiments.
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Next, to examine the functional role of Src kinase in
sE-selectin-induced monocyte chemotaxis, we performed monocyte
chemotaxis in the presence and absence of the Src inhibitor PP2 (1 µM). Monocytes were pretreated with PP2 for 30 min at
37 °C prior to performing the chemotaxis assay. fMLP served
as the positive control, and HBSS served as the negative control. Fig.
11 shows a representative assay from
three independent experiments. The pretreatment of monocytes with the
Src inhibitor PP2 significantly inhibited (p < 0.05)
monocyte chemotaxis as compared with sE-selectin alone, thereby
suggesting that sE-selectin could be mediating monocyte chemotaxis
through the Src pathway.

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Fig. 11.
Src inhibitor PP2 inhibits monocyte
chemotaxis mediated by sE-selectin. Monocytes were preincubated
with the Src inhibitor PP2 (1 µM) for 30 min at 37 °C
and then assayed in a 48-well chemotaxis chamber in response to various
concentrations of sE-selectin. Results are expressed as the mean and
S.E. of three high power fields (×400) per replicate well. Each test
group was assayed in quadruplicate. This is a representative
assay of three independent experiments. HBSS- and fMLP-induced
chemotaxis were tested as negative and positive controls, respectively.
Error bars represent S.E., and * represents a
significant difference (p < 0.05) between the
sE-selectin group and negative control. # represents a
significant inhibition (p < 0.05) of chemotaxis in the
sE-selectin + PP2 (Src inhibitor) group as compared with sE-selectin
alone.
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DISCUSSION |
Monocyte recruitment into the synovial tissue is a key step in RA
pathogenesis because activated monocytes and/or macrophages can
function as antigen-presenting cells, and these cells also have
the ability to secrete a variety of inflammatory mediators including
prostaglandins (24), nitric oxide (25), interleukin-1, tumor necrosis
factor-
(26), interleukin-8 (27), monocyte chemotactic protein-1
(28), and macrophage inflammatory protein-1
(29). We and others have
reported a number of putative monocyte/macrophage chemoattractants in
the RA synovial fluid, such as monocyte chemotactic protein-1 and
macrophage inflammatory protein-1
(29, 30), which may be involved in
monocyte/macrophage recruitment into the RA synovial tissue.
In this report, we show that sE-selectin is a potent chemotactic agent
for monocytes in the nanomolar range. We have previously shown elevated
levels of sE-selectin (in the nanomolar range) in the RA synovial fluid
(7). Thus, it is likely that at the concentrations found in the
RA joints, sE-selectin might be acting in large part to recruit and
activate monocytes. To address the contribution provided by the
sE-selectin to RA synovial fluid chemotactic activity for monocytes, we
immunodepleted RA synovial fluid sE-selectin by using neutralizing
mouse monoclonal anti-E-selectin antibodies. The neutralization of RA
synovial fluid sE-selectin significantly decreased monocyte chemotaxis
(mean of 31%, p < 0.05) as compared with the matched
isotype control antibody. Thus, these results suggest a novel function
for sE-selectin as a chemotactic factor for monocytes in the RA
synovial fluid.
Recent studies have shown that intact, transmembrane E-selectin can
transduce signals in endothelial cells during inflammatory leukocyte
recruitment (31). Hu et al. (23) showed that the cross-linking of endothelial cell surface E-selectin with antibodies leads to the up-regulation of mRNA for c-fos, an early
response gene, through the Ras-Raf-MAPK pathway. However, we are not
aware of any studies regarding the signaling pathway used by
sE-selectin to mediate its biological function. In this regard, we
investigated sE-selectin-mediated signaling in normal PB monocytes.
sE-selectin induced the tyrosine phosphorylation of a number of
monocyte proteins corresponding to molecular sizes of 130-140, 70-74,
60-65, 50-55, 42-44, 35-40, and 20-25 kDa in a
time-dependent manner. Marked increases in tyrosine
phosphorylation were detected as early as 30 s after stimulation
and peaked at 30 min, which is consistent with a typical time course of
protein tyrosine phosphorylation (13, 32). We next investigated the
role that Src family kinases play in sE-selectin-mediated signaling.
The Src family of tyrosine kinases is reported to be activated during a
number of biological phenomena including monocyte adherence to
endothelial cells (11), integrin-mediated signaling in monocytes and/or
macrophages (33), endothelial cell differentiation (12), and cardiac
contractility (13). In our studies, sE-selectin up-regulated the
phosphorylation of Src family kinases (Src, Hck, and Lyn) in a
time-dependent manner. The pretreatment of monocytes with
PP2 for 30 min prior to sE-selectin stimulation partially inhibited Src
phosphorylation, whereas Hck and Lyn phosphorylation was markedly
inhibited. These results indicate that the Src family kinases may be an
important mediator in sE-selectin-mediated signaling in monocytes.
We next studied the role of PP2 on the total cellular phosphotyrosine
pattern. The pretreatment of monocytes with PP2 prior to stimulation
with sE-selectin showed the partial inhibition of a 60-kDa band.
However, other Src family kinases (50-60 kDa) were substantially
inhibited, thus corroborating our other findings that PP2 has a
partial effect on sE-selectin-mediated Src phosphorylation, whereas it
markedly inhibited Hck and Lyn kinases. In addition to the Src family
kinases, PP2 also showed substantial inhibition of the tyrosine
phosphorylation of proteins in the MAPK region (35-45 kDa). MAPK is a
key signaling central point at which a number of pathways converge
(34). MAPK is involved in a number of basic physiological phenomena,
including cell migration, cell cycle, and apoptosis, in addition to
playing an important role in the signaling of a number of important
genes such as chemokines (35) and adhesion molecules (36, 37). We next
determined whether sE-selectin-mediated signaling involves the
activation of MAPK. sE-selectin-stimulated monocytes showed the
time-dependent phosphorylation of ERK1/2 and p38 MAPK.
ERK1/2 activation was observed as early as 30 s and persisted
until 30 min. However, p38 activation was detectable from 5 min
onwards. To test the hypothesis that the signal from Src might be going
through MAPK, we pretreated monocytes with the Src inhibitor PP2 and
then studied the phosphorylation of ERK1/2 and p38. Pretreatment with
the Src inhibitor (PP2) showed 89 and 83% inhibition of ERK1/2 and
p38, respectively, at 30 min after sE-selectin stimulation. These
results suggest that MAPKs (ERK1/2 and p38) are activated in monocytes by sE-selectin through the Src kinase pathway. Schmid-Alliana et
al. (38) have demonstrated that microtubule depolymerization by
colchicine in human monocytes induces the selective production of
interleukin-1 through the Src, Ras, Raf-1, and MAPK pathway. A similar
signaling cascade involving Src, Ras, and MAPK was reported by Jalali
et al. (35) in vascular endothelial cells stimulated with
shear stress.
Raf kinase is one of the best characterized MEKKs (16). Raf kinase is
activated by binding to Ras-GTP, which then phosphorylates MEK. Ras
kinase has been shown to be activated by Src kinase (12, 35). To test
whether Ras and Raf kinases are also activated during the sE-selectin
activation of monocytes, we immunoprecipitated Ras with the Raf-1-Ras
binding domain linked to agarose beads and then probed with mouse
monoclonal anti-Ras antibody. sE-selectin stimulation showed a
time-dependent activation of Ras, thereby suggesting a
possible link between Src and MAPK in sE-selectin signaling through
Ras-Raf kinase.
We next determined whether this Src-MAPK signaling cascade has
functional relevance and whether it is essential for
sE-selectin-mediated monocyte chemotaxis. PB monocytes were pretreated
with the Src-specific inhibitor PP2 at a concentration of 1 µM for 30 min at 37 °C before performing chemotaxis in
response to sE-selectin. PP2 completely blocked sE-selectin-mediated
chemotaxis. These data suggest that sE-selectin uses the Src pathway to
mediate monocyte chemotaxis.
In summary, we have demonstrated a novel function for sE-selectin as a
monocyte chemotactic agent. sE-selectin accounted for a significant
proportion of the monocyte chemotactic activity in the RA synovial
fluid. We have further shown that sE-selectin uses the Src-Ras-MAPK
signaling pathway to mediate its biological function. Thus, sE-selectin
may be a potent recruiter of monocytes in the RA joint and may
contribute to RA pathogenesis through the Src-Ras-MAPK signaling pathway.