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Blood, Vol. 91 No. 3 (February 1), 1998:
pp. 1067-1075
Functional Characterization of L-Selectin Ligands on Human
Neutrophils and Leukemia Cell Lines: Evidence for Mucinlike Ligand
Activity Distinct From P-Selectin Glycoprotein Ligand-1
By
Carroll L. Ramos,
McRae J. Smith,
Karen R. Snapp,
Geoffrey S. Kansas,
George W. Stickney,
Klaus Ley, and
Michael B. Lawrence
From the Department of Biomedical Engineering, University of Virginia
Health Sciences Center, Charlottesville; and the Department of
Microbiology-Immunology, Northwestern University Medical School,
Chicago, IL.
 |
ABSTRACT |
Recent reports have shown that leukocyte-leukocyte adhesion is
dependent on L-selectin and that leukocyte recognition of L-selectin may be mediated by P-selectin glycoprotein ligand-1 (PSGL-1). We show
that the specific attachment and rolling of human neutrophils and the
leukemia cell lines HL-60 and U937 on immobilized, purified L-selectin
under continuous shear stress is only partially inhibited by treatment
with the PSGL-1 monoclonal antibody (MoAb), KPL1 (41% to 53%
inhibition), suggesting that L-selectin ligand activity in addition to
PSGL-1 may mediate myeloid cell rolling on L-selectin. K562 cells
cotransfected with cDNAs encoding  (1,3)fucosyltransferase-VII (FucT-VII) and PSGL-1 rolled on L-selectin. Adhesion of FucT-VII-PSGL-1 transfectants to L-selectin was completely blocked by MoAb KPL1, indicating that both L-selectin and P-selectin bind similar sites on
PSGL-1. In support of existence of a non-PSGL-1 L-selectin ligand
activity on leukocytes, an HL-60 membrane preparation immunodepleted of
PSGL-1 supported rolling of L-selectin, but not P-selectin transfectants. Treatment of HL-60 cells with O-sialoglycoprotein endopeptidase inhibited attachment and rolling on L-selectin and P-selectin. However, neuraminidase treatment completely blocked HL-60
rolling on L-selectin, but not P-selectin, suggesting L-selectin and
P-selectin ligand activities have different contributions of sialic
acid. These findings indicate that myeloid cells express sialylated,
O-linked glycoprotein ligand activity independent of PSGL-1 that
supports L-selectin-mediated rolling.
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INTRODUCTION |
THE SELECTIN CLASS of adhesion molecules
plays a critical role in the migration of leukocytes to sites of
inflammation by mediating the initial attachment and rolling of
leukocytes on vascular endothelium prior to integrin-dependent arrest
and extravasation.1,2 L-selectin (CD62L), which is
constitutively expressed by circulating leukocytes,3 serves
as a homing receptor for lymphocyte binding to peripheral lymph node
high endothelial venules.4 L-selectin is also required for
leukocyte adhesion in response to trauma and inflammation as shown by
L-selectin monoclonal antibody (MoAb) inhibition of neutrophil rolling
on cytokine-stimulated endothelial monolayers in vitro5,6
and leukocyte rolling in mesenteric venules in vivo.7,8
Furthermore, leukocyte recruitment during inflammation is impaired in
L-selectin-deficient mice.9,10
In addition to leukocyte-endothelial interactions, it also appears that
L-selectin contributes to leukocyte-leukocyte adhesive interactions
between leukocytes. L-selectin MoAbs inhibit formyl peptide-induced
neutrophil aggregation11 and transient adhesive interactions between flowing neutrophils and adherent neutrophils in
vitro.12 In addition, monolayers of KG1a hematopoietic
progenitor cells support L-selectin-dependent adhesion of human
peripheral blood lymphocytes.13
Leukocyte recognition of L-selectin appears to depend on the expression
of sialylated and/or fucosylated glycoprotein ligands. Neuraminidase treatment of neutrophil monolayers blocks
L-selectin-dependent interactions with flowing
neutrophils.12 Similarly, treatment of adherent KG1a cells
inhibits the ability of these cells to support adhesion of
lymphocytes.13 Incubation of neutrophils with
O-sialoglycoprotein endopeptidase (OSGE), which cleaves mucinlike glycoproteins containing clustered, O-linked serine or threonine residues,14 inhibits aggregation with untreated
neutrophils.15 Furthermore, specific binding of recombinant
L-selectin-IgM to neutrophils is blocked by treatment with
neuraminidase and OSGE.16
Recent reports have suggested that the sialylated and
O-glycosylated P-selectin glycoprotein ligand-1
(PSGL-1)17,18 may also serve as an L-selectin ligand.
Immobilized PSGL-1 supports neutrophil adhesion under flow conditions
which is completely blocked by an L-selectin MoAb.19 In
addition, L-selectin-IgM binding to myeloid cells is inhibited by
treatment with a polyclonal antibody against PSGL-116 and
monolayers of L-selectin transfectants support HL-60 adhesion that is
blocked by the PSGL-1 MoAb, PL1.20 However, the combination
of an L-selectin MoAb and PL1 only partially inhibits formyl
peptide-induced neutrophil aggregation.21 Similarly, PL1
only partially blocks attachment of flowing neutrophils to fixed,
adherent neutrophil monolayers,19 implying the existence of
leukocyte L-selectin ligands distinct from PSGL-1. Furthermore, in some
assays, PSGL-1 does not appear to be required for leukocyte adhesion to
L-selectin. PL1 does not disrupt L-selectin-dependent neutrophil-neutrophil interactions in flow22 and an
OSGE-insensitive, non-mucinlike component of neutrophil adhesion to
recombinant L-selectin-IgG under flow conditions has been
shown.23
The objective of this study was to determine whether leukocyte ligand
structures distinct from PSGL-1 mediate leukocyte recognition of
L-selectin. By studying leukocyte adhesion directly to purified L-selectin immobilized on the wall of a flow chamber, potential artifacts associated with leukocyte-leukocyte adhesion assays, such as
incomplete MoAb saturation of PSGL-1, Fc receptor cross-linking, L-selectin shedding, and the heterogeneous surface presented by adherent leukocytes could be minimized. We show that the specific binding of leukocytes to L-selectin under flow conditions appears to be
dependent on the expression of mucinlike glycoprotein ligand activity
and that adhesion can be mediated by both PSGL-1-dependent and
-independent mechanisms.
| |
MATERIALS AND METHODS |
Antibodies and selectin chimeras.
L-selectin MoAb DREG56 (IgG1)24 was a gift of T.K.
Kishimoto (Boehringer Ingelheim Pharmaceuticals, Ridgefield, CT).
P-selectin MoAb G1 (IgG1)25 and PSGL-1 MoAbs PL1 and PL2
(IgG1)26 were gifts of R.P. McEver (University of Oklahoma,
Oklahoma City). Additional PSGL-1 MoAbs were KPL1 and KPL2 (IgG1)
(Snapp, K.R.; Ding, H.; Atkins, K.; Luscinskas, F.W.; Warnke, R.; and
Kansas, G.S.; submitted). The CD18 MoAb, TS1/18, was
purified from hybridoma supernatant as described.27 CSLEX-1
(IgM)28 and recombinant human L-selectin-IgG and
P-selectin-IgG chimeras29 were gifts of L.A. Lasky and
S.A. Watson (Genentech, Inc, South San Francisco, CA). Fluorescein
isothiocyanate-conjugated rabbit anti-mouse immunoglobulin was
purchased from Dako (Carpinteria, CA).
Isolation of L-selectin and P-selectin.
Human L-selectin was purified from human tonsil (provided by R.S.
Larson, University of New Mexico, Albuquerque) homogenized in 20 mmol/L
Tris, pH 8.0; 140 mmol/L NaCl; and 0.025% azide (TSA, pH 8.0) with 5 mmol/L EDTA, 10 µmol/L leupeptin, 0.1 U/mL aprotinin, and 1% Triton
X-100. After centrifugation of the lysate at 500g and
100,000g, the supernatant was passed over a column of
CNBr-activated Sepharose 4B (Pharmacia Biotech, Piscataway, NJ) coupled
to DREG56 (2 mg/mL). The column was washed with TSA, pH 8.0; containing 1% octylglucopyranoside (OG; Sigma, St Louis, MO) and eluted with acetate buffer, pH 3.0, 1% OG. The eluate was neutralized with 1 mol/L
Tris, pH 9.0; 1% OG (15% vol/vol). Human P-selectin was purified from
outdated platelets (American Red Cross, Richmond, VA) as previously
described.30
Cell lines and neutrophil isolation.
The human leukemia cell lines HL-60, U937, SKW3, and K562 were
maintained in RPMI 1640 supplemented with 10% fetal bovine serum (FBS;
Atlanta Biologicals, Norcross, GA), glutamine (2 mmol/L), penicillin
(100 U/mL), and streptomycin (100 µg/mL) (GIBCO-BRL, Grand Island,
NY). K562 cells were stably transfected with cDNA encoding for
(1,3)fucosyltransferase-VII (FucT-VII), PSGL-1, or both FucT-VII and
PSGL-1.31 The murine pre-B lymphocytic cell line, 300.19, was maintained in RPMI 1640 supplemented with 10% FBS and 10 µmol/L
2-mercaptoethanol (GIBCO-BRL). 300.19 cells stably transfected with
cDNA encoding human L-selectin or P-selectin have been
described.32 For adhesion assays, cell lines were suspended
in RPMI 1640 with 10 mmol/L HEPES, pH 7.4; and 1% human serum albumin
(HSA) at room temperature. Human neutrophils were isolated from
heparinized venous blood by density separation over Mono-Poly Resolving
Medium (ICN Biochemicals, Aurora, OH).11 Neutrophils were
suspended in Hanks' balanced salt solution (HBSS) without calcium and
magnesium, supplemented with 10 mmol/L HEPES, pH 7.4; and 0.1% HSA,
and placed on ice. Before use in experiments, neutrophils were washed
into HBSS with 2 mmol/L CaCl2; 10 mmol/L HEPES, pH 7.4; and
0.1% HSA at room temperature.
Enzymatic digestion of cell surface glycoproteins.
O-Sialoglycoprotein endopeptidase (OSGE) (P. haemolytica), E.C.
3.4.24.57, was purchased from Accurate Chemical & Scientific Corp
(Westbury, NY). Neuraminidase (V. cholerae), E.C. 3.2.1.18, was
purchased from Calbiochem (La Jolla, CA). Chymotrypsin (type 1-S), E.C.
3.4.21.1 was purchased from Sigma. HL-60 cells (5 × 106
cells in 1 mL) were incubated at 37°C with either 120 µg OSGE in
RPMI 1640, pH 7.4; and 10% FBS for 2 hours; 200 mU neuraminidase in
phosphate-buffered saline, pH 6.4; and 10% FBS for 2 hours; or 10 U
chymotrypsin in RPMI 1640, pH 7.4; for 15 minutes, followed by
resuspension at 0.5 × 106/mL in RPMI 1640, pH 7.4; and
1% HSA. Enzymatic activities of OSGE and neuraminidase were verified
by flow cytometry using MoAbs against CD43 (IgG1) (Biodesign
International, Kennebunk, ME) for OSGE-treated cells and CSLEX-1 for
neuraminidase-treated cells. Cell surface CD43 and sialyl
Lewisx were reduced by greater than 95% after OSGE and
neuraminidase treatment, respectively (data not shown). Enzyme
treatments did not reduce cell viability based on trypan blue
exclusion.
Laminar flow adhesion assay.
Purified human selectins or selectin-IgG chimeras were diluted as
indicated in 50 mmol/L Tris, pH 9.5; and 0.025% azide, followed by
adsorption to polystyrene slides cut from bacteriological petri dishes
(Falcon 1058) for 2 hours at room temperature. Site densities of
purified human L-selectin (1:5) and P-selectin (1:60) were determined
to be 170 and 270 sites/µm2, respectively, by
radioimmunoassay as previously described using MoAbs DREG56 and
G1.30 The slides were blocked with 3% HSA for 1 hour at
room temperature or overnight at 4°C and fitted into a parallel plate
laminar flow chamber33 which was mounted on the stage of an
inverted phase-contrast microscope (Diaphot-TMD; Nikon, Garden City,
NY). Adhesive interactions between cellular Fc receptors and the Fc
domain of selectin-IgG were eliminated by incubation of the adsorbed
selectin substrate with polyclonal goat F(ab )2 (10 µg/mL) against human IgG Fc (Biodesign International). In some
experiments, the adsorbed selectin substrate or cell suspensions were
incubated with MoAbs (10 µg/mL) for 15 minutes at room temperature prior to initiation of flow. Cell suspensions
(0.5-1 × 106/mL) were drawn through the flow chamber at
room temperature using a syringe pump (Harvard Apparatus, South Natick,
MA) and the number of bound cells quantitated from videotape recordings
of 10-20 fields of view obtained while scanning the lower plate of the flow chamber using a 10× or 20× objective after 2 to 3 minutes of
flow. Wall shear stress (dyne/cm2) was calculated assuming
a viscosity of assay buffer equal to water at room temperature (1.0 centipoise; 24°C). For cell tracking analysis, video images were
captured using public domain NIH Image v.1.57 and the displacement of
individual leukocytes under shear conditions was determined at
0.03-second intervals. Critical velocity was defined as the velocity of
a noninteracting leukocyte in a shear flow near the wall of the flow
chamber.33 Leukocytes with a translational velocity lower
than critical velocity were defined as rolling.
HL-60 lysate and immunodepletion of PSGL-1.
HL-60 cell pellets (2 × 108 cells) were incubated with
ice cold lysis buffer (20 mmol/L Tris, pH 8.0; 140 mmol/L NaCl; 0.025% sodium azide; 5 mmol/L EDTA; 10 µmol/L leupeptin; 0.1 U/mL aprotinin; and 1% Triton X-100). After centrifugation of the lysate at
500g and 100,000g, the supernatant was passed over a
wheat germ agglutinin (WGA)-Sepharose column prepared by coupling WGA
(Pharmacia Biotech, Piscataway, NJ) at 2 mg/mL to CNBr-activated
Sepharose 4B. The column was washed with lysis buffer containing 1% OG
and eluted with 100 mmol/L N-acetyl-D-glucosamine (Sigma) in lysis
buffer with 1% OG. Protein-containing fractions were identified under nonreducing conditions by 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by silver staining. PSGL-1 was
immunodepleted from the WGA affinity-purified HL-60 lysate using
protein G-Sepharose (Pharmacia Biotech) saturated with KPL1 (10 mg
IgG/mL). A mock immunoprecipitation was performed by incubating lysate
with protein G-Sepharose alone. Immunodepletion of PSGL-1 from the
lysate was verified by subjecting supernatants to 7.5% SDS-PAGE and
transfer of separated proteins to nitrocellulose. The membrane was
probed for PSGL-1 by incubating with KPL1 followed by horseradish
peroxidase (HRP)-conjugated goat anti-mouse IgG (Pierce, Rockford, IL).
The blot was developed using enhanced chemiluminescence detection
reagents and exposed to Hyperfilm ECL (Amersham, Arlington Heights, IL)
for 2 to 3 minutes.
| |
RESULTS |
Rolling of neutrophils and leukemia cell lines on L-selectin.
Purified human L-selectin, adsorbed to the lower wall of a parallel
plate flow chamber, supported attachment and rolling of human
neutrophils under flow conditions (Fig 1).Neutrophils formed stable rolling interactions with immobilized
L-selectin at velocities below the critical velocity determined for
nonadherent cells traveling adjacent to the lower wall of the flow
chamber (Fig 1A). The ability of neutrophils to attach to L-selectin
under flow was dependent on the level of wall shear stress. Neutrophil
attachment significantly increased as the wall shear stress was lowered
from 5.0 to 2.0 dyne/cm2 (Fig 1B). However, the efficiency
of neutrophil attachment decreased as the wall shear stress was further
reduced to 0.5 dyne/cm2, which is consistent with previous
work showing that a threshold level of shear stress is required to
support the selectin receptor-ligand interaction.30,34
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| Fig 1.
Human neutrophils roll on immobilized L-selectin under
flow conditions. Human neutrophils (1 × 106/mL) were
infused through a parallel plate flow chamber in which dilutions of
human purified L-selectin (1:5; 170 sites/µm2) were
adsorbed to the lower wall. (A) Displacement as a function of time for
neutrophils rolling on L-selectin under a continuous wall shear stress
(1 dyne/cm2). Tracings represent positions of five
independent cells measured at 0.03-second intervals. Bold solid line
(arrow) represents the tracking of a noninteracting neutrophil
traveling at critical velocity near the wall of the flow chamber. (B)
Neutrophil attachment to purified L-selectin under continuous flow
conditions is shear stress-dependent. Adhesion to L-selectin was
specific based on inhibition of neutrophil attachment after inclusion
of EDTA (5 mmol/L) in the perfusion media and treatment of the adsorbed
L-selectin substrate with L-selectin MoAb, DREG56 (10 µg/mL).
Metabolic inhibition with 0.06% azide and 50 mmol/L 2-deoxy-D-glucose
for 30 minutes at room temperature or incubation of neutrophils with
CD18 MoAb, TS1/18 (10 µg/mL), did not block attachment to L-selectin.
Treatment of neutrophils with PSGL-1 MoAbs PL1 (Fab fragments) (10 µg/mL) or KPL1 (10 µg/mL), but not KPL2 ascites (1:500), partially
inhibited attachment to L-selectin. Mean ± SEM of bound
cells/mm2 in multiple fields from two to four independent
experiments.
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Neutrophil attachment and rolling on L-selectin was completely calcium-
and L-selectin-dependent as shown by inhibitory effects of EDTA and
treatment of the adsorbed substrate with the L-selectin MoAb, DREG56
(Fig 1B). Cellular metabolism and/or  2-integrin expression were not required for rolling on L-selectin because treatment of the neutrophil suspension with the metabolic inhibitor combination azide/deoxyglucose or the function-blocking
2-integrin MoAb, TS1/18,27 did not reduce
neutrophil adhesion to L-selectin (Fig 1B). Treatment of neutrophils
with the PSGL-1 MoAb, KPL1, which recognizes an epitope within the
tyrosine sulfate motif of human PSGL-1 and blocks leukocyte adhesion to
P-selectin (Snapp, K.R.; Ding, H.; Atkins, K.; Luscinskas, F.W.;
Warnke, R.; and Kansas, G.S.; submitted), resulted in
significant inhibition of neutrophil adhesion to L-selectin under
continuous flow conditions (73% ± 2% inhibition; Fig 1B). In
contrast, the nonblocking, isotype-matched PSGL-1 MoAb, KPL2 (Snapp,
K.R.; Ding, H.; Atkins, K.; Luscinskas, F.W.; Warnke, R.; and Kansas,
G.S.; submitted), failed to inhibit neutrophil rolling on
L-selectin (Fig 1B). Incubation of neutrophil suspensions with Fab
fragments of PL1, a PSGL-1 MoAb which blocks neutrophil rolling on
P-selectin,26 also partially inhibited attachment of
neutrophils to L-selectin (27% ± 5% inhibition; Fig 1B).
To distinguish between MoAb effects on cell attachment under flow and
inhibitory effects of the PSGL-1 MoAb on rolling, neutrophils and the
human leukemia cell lines HL-60, U937, and SKW3 were treated with
PSGL-1 MoAbs and allowed to settle onto immobilized L-selectin before
the onset of flow (Fig 2). This
experimental design selectively tests for MoAb effects on leukocyte
rolling independent of potential indirect effects such as
activation-induced shape changes that may alter initial cell
attachment.35 Incubation of cell suspensions with KPL1
resulted in a complete inhibition of SKW3 rolling, but only partial
reductions in the number of rolling neutrophils (41% ± 3% inhibition), HL-60 promyelocytes (53% ± 3% inhibition), and U937 cells (42% ± 2% inhibition) on L-selectin (Fig 2A) at 1.0 dyne/cm2 wall shear stress. In contrast, KPL1 treatment
completely blocked neutrophil, HL-60, U937, and SKW3 rolling on
purified P-selectin, indicating the presence of functionally saturating
concentrations of antibody (Fig 2B). Incubation of cell suspensions
with the nonblocking, isotype-matched MoAb KPL2 failed to inhibit
rolling of neutrophils and cell lines on L-selectin or P-selectin (data not shown). These results indicate that neutrophil, HL-60, and U937
cell lines express both PSGL-1-dependent and -independent L-selectin
ligand activity.
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| Fig 2.
Effect of PSGL-1 MoAb on human neutrophil and leukemia
cell line rolling on L-selectin and P-selectin. Neutrophils
(1 × 106/mL) and the leukemia cell lines HL-60, U937,
and SKW3 (0.5 × 106/mL) were treated with either MoAb
KPL1 (10 µg/mL) or KPL1 ascites (1:500) and allowed to bind to
purified L-selectin (1:5; 170 sites/µm2) (A) or
P-selectin (1:60; 270 sites/µm2) (B) under static
conditions for 90 seconds, followed by initiation of flow at a wall
shear stress of 1 dyne/cm2. Mean ± SEM of bound
cells/mm2 in multiple fields from three independent
experiments.
|
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To determine the relative role of PSGL-1-independent and -dependent
interactions with L-selectin at distinct wall shear stresses, neutrophils were perfused over purified L-selectin under continuous flow conditions in the presence and absence of MoAb KPL1.
Neutrophil-neutrophil interactions that may influence the apparent
capture rate and total accumulation of cells on
L-selectin22 were minimized by using more dilute
suspensions of cells and decreasing the time of flow before
quantitating adhesion. PSGL-1 appears to contribute more to neutrophil
capture on L-selectin as wall shear stresses are increased (Fig
3). At 4.0 dyne/cm2 wall shear
stress, MoAb KPL1 significantly inhibited capture of neutrophils on
L-selectin, but had less inhibitory effects as shear stress
was lowered to 2.0 and 1.0 dyne/cm2 (Fig 3). This finding
suggests that the PSGL-1-independent L-selectin ligand activity on
neutrophils may have lower association rates and may play a greater
role at relatively low levels of shear stress.
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| Fig 3.
Shear stress-dependent contribution of PSGL-1 to
neutrophil capture by L-selectin. Neutrophils
(0.5 × 106/mL) were treated with either MoAb KPL1 (10 µg/mL) or KPL2 ascites (1:500) and perfused over a range of wall
shear stresses through a parallel plate flow chamber in which dilutions
of human purified L-selectin (1:5; 170 sites/µm2) were
adsorbed to the lower wall. Numbers of bound cells were quantitated
from 30 fields of view after 1 minute of continuous flow at each shear
stress. Mean ± SD of bound cells/mm2 in multiple fields
from two independent experiments.
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FucT-VII/PSGL-1 transfectants roll on L-selectin.
To determine whether PSGL-1 is sufficient to mediate attachment and
rolling on immobilized L-selectin under flow conditions, K562 cells
transfected with cDNAs encoding FucT-VII and PSGL-1 (FucT-VII/PSGL-1)31 were infused into the flow chamber
containing either adsorbed purified L-selectin or P-selectin. As shown
in Fig 4, FucT-VII/PSGL-1 transfectants
attached and rolled on both P-selectin and L-selectin under flow
conditions. Adhesion of FucT-VII/PSGL-1 transfectants on L-selectin and
P-selectin was completely inhibited by MoAb KPL1 (Fig 4). K562 cells
transfected with FucT-VII cDNA alone were observed to bind to purified
L-selectin at a level more than 20-fold less (1 to 3 transient
interactions/mm2) than FucT-VII/PSGL-1 transfectants,
despite a 4- to 6-fold higher level of sialyl Lewisx
expression,31 suggesting a highly specific interaction of
PSGL-1 with L-selectin (Fig 4).
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| Fig 4.
FucT-VII/PSGL-1 transfectants roll on
L-selectin. K562-FucT-VII and K562-FucT-VII/PSGL-1
transfectants (0.5 × 106/mL) were infused through a
parallel plate flow chamber at a wall shear stress of 1 dyne/cm2 in which purified human L-selectin (1:5; 170 sites/µm2) or P-selectin (1:60; 270 sites/µm2) were adsorbed to the lower wall of the
chamber. Nontransfected K562 cells did not bind to L-selectin or
P-selectin. Mean ± SEM of bound cells/mm2 in multiple
fields from three independent experiments.
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An HL-60 lysate supports rolling of L-selectin transfectants.
To determine whether the presentation of L-selectin on a cellular
surface modifies the leukocyte L-selectin ligand specificity and to
further examine the requirement for PSGL-1 expression in myeloid cell
recognition of L-selectin, a reciprocal adhesion assay was designed in
which an HL-60 cell lysate, partially purified by WGA-Sepharose
chromatography, was incorporated as an adhesive substrate in the flow
chamber. Suspensions of L-selectin and P-selectin transfectants served
as specific probes for L-selectin and P-selectin ligand activity in the
lysate. As shown in Fig 5, the WGA-purified HL-60 lysate supported calcium-dependent rolling of both 300.19 P-selectin and L-selectin transfectants, but not nontransfected 300.19 cells. Adhesion of P-selectin and L-selectin transfectants was
completely inhibited by treatment with the P-selectin MoAb, G1,25 and DREG56, respectively (Fig 5). Incubation of the
lysate with PL1 completely abolished adhesion of P-selectin
transfectants, but did not significantly inhibit binding of
L-selectin transfectants (Fig 5), which was consistent with
the inability of PSGL-1 MoAbs to completely block rolling of
neutrophils, HL-60, and U937 cells on L-selectin.
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| Fig 5.
L-selectin transfectants roll on an HL-60 lysate
partially purified by WGA affinity chromatography. Nontransfected
300.19 cells, 300.19 L-selectin, or 300.19 P-selectin transfectants
(1 × 106/mL) were infused through a parallel plate flow
chamber at a wall shear stress of 0.64 dyne/cm2 in which a
dilution of an HL-60 lysate partially purified by WGA affinity
chromatography was adsorbed to the lower wall of the chamber.
L-selectin and P-selectin-dependent adhesion were shown by incubation
of L-selectin transfectants with MoAb DREG56 (10 µg/mL) and
P-selectin transfectants with MoAb G1 (10 µg/mL). PSGL-1-dependent
adhesion was shown by treating the adsorbed lysate with MoAb PL1 (10 µg/mL). Mean ± SEM of bound cells/mm2 in multiple
fields from two to three independent experiments.
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L-selectin transfectants roll on lysates immunodepleted of PSGL-1.
To determine the relative contribution of PSGL-1 to L-selectin
transfectant rolling on the WGA-purified HL-60 lysate versus other
membrane glycoproteins, the lysate was immunodepleted of PSGL-1 using
KPL1 bound to protein G-Sepharose beads. Western blot analysis showed
significant depletion of PSGL-1 from the lysate under these conditions,
based on a reduction in intensity of the prominent 220- to 250-kD band
corresponding to PSGL-1 dimer36 (Fig
6A). Immunodepleted lysates were then
incorporated into the flow chamber and assayed for L-selectin and
P-selectin ligand activity. Compared to a mock-immunodepleted control,
in which the lysate was incubated with protein G-Sepharose alone,
adhesion of P-selectin transfectants was almost completely inhibited
after immunodepletion of PSGL-1 (Fig 6B). Only a small number of
transient attachments (1 second in duration) and no stable rolling
interactions were observed. This result confirmed the requirement of
PSGL-1 to support P-selectin-dependent rolling in this system. In
contrast, L-selectin transfectants attached and formed stable rolling
interactions on lysates immunodepleted of PSGL-1, indicating that
L-selectin ligand activity independent of PSGL-1 was present in the
lysate. However, the number of bound L-selectin transfectants was
partially reduced relative to the mock-immunodepleted control (Fig 6B), apparently caused by the contribution of PSGL-1 to the L-selectin ligand activity in the HL-60 lysate. A second round of immunodepletion of the lysate completely eliminated residual skipping interactions with
P-selectin transfectants, but only reduced adhesion of L-selectin transfectants by an additional 10% (data not shown).
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| Fig 6.
Immunodepletion of PSGL-1 from the WGA affinity purified
HL-60 lysate inhibits binding of P-selectin transfectants, but not L-selectin transfectants. (A) Western blot analysis showing
immunodepletion of PSGL-1 from the HL-60 lysate. Lane 1: intact
WGA-purified HL-60 lysate (220 to 250-kD band corresponding to PSGL-1);
lane 2: lysate after immunodepletion with KPL1/protein G-Sepharose;
lane 3: lysate after mock immunodepletion (incubation with protein
G-Sepharose alone). PSGL-1 was detected by probing the blot with KPL1
ascites (1:2,500), followed by HRP-conjugated goat anti-mouse IgG and development using enhanced chemiluminescence. (B) Adhesion of 300.19 P-selectin or 300.19 L-selectin transfectants
(1 × 106/mL) to PSGL-1-depleted HL-60 lysates under
flow conditions (0.64 dyne/cm2) after immunodepletion of
PSGL-1. Mean ± SD of bound cells/mm2 in multiple fields
from two independent experiments.
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To investigate the possibility that domains of PSGL-1 independent of
the KPL1 epitope may have remained in the lysate after immunodepletion,
Western blots were stripped and reprobed with MoAb PL2, which
recognizes residues 188-235 in the consensus repeat domain of human
PSGL-137 and does not block neutrophil adhesion to
P-selectin.26 The resulting blot was nearly identical to the KPL1-probed blot shown in Figure 6A, in that the intensity of the
band representing 220-250-kD PSGL-1 dimer in the
PSGL-1-immunodepleted lysate was strongly reduced compared with the
intact and mock-immunodepleted lysates (data not shown). This finding
suggests that epitopes on PSGL-1 distinct from the P-selectin-binding
site were not likely to be responsible for mediating the L-selectin
transfectant rolling observed after immunodepletion with MoAb KPL1.
Effect of protease and neuraminidase treatment on myeloid cell
L-selectin ligand activity.
The L-selectin ligand activity on myeloid cells was characterized by
assessing the effect of enzyme treatments on HL-60 rolling on
immobilized L-selectin or P-selectin under flow conditions. Several
groups have reported conflicting observations on the protease sensitivity of L-selectin ligand activity expressed by
leukocytes.13,15,22 OSGE specifically cleaves glycoproteins
that contain clustered O-glycosylated serine or threonine
residues,14 including PSGL-1.17 Incubation of
HL-60 cells with OSGE resulted in complete inhibition of attachment and
rolling on immobilized L-selectin and P-selectin (Table
1), indicating that ligand activity for
these selectins requires the expression of an O-glycosylated, mucinlike
protein. Chymotrypsin treatment also completely blocked HL-60 rolling
on L-selectin and P-selectin (Table 1), which confirmed the protease sensitivity of L-selectin and P-selectin ligand activity.
View this table:
[in this window]
[in a new window]
|
Table 1.
Effect of Protease and Neuraminidase Treatment on HL-60
Adhesion to L-Selectin and P-Selectin Under Flow Conditions
|
|
HL-60 cell suspensions were incubated with neuraminidase to establish
whether sialylation is required for myeloid cell recognition of
L-selectin. Ligand activity for P-selectin appears to be relatively resistant to neuraminidase, since radiolabeled P-selectin binding to
purified PSGL-117,36 and P-selectin-IgM binding to
neutrophils16 is only partially inhibited by neuraminidase
treatment. As shown in Table 1, treatment of HL-60 cells with
neuraminidase resulted in almost complete inhibition of attachment and
rolling on L-selectin, but only a partial decrease in binding to
P-selectin. The differential neuraminidase sensitivity of HL-60 cell
adhesion to L-selectin and P-selectin supports the existence of
distinct carbohydrate requirements for L-selectin and
P-selectin ligand activity on myeloid cells.
| |
DISCUSSION |
Previous reports of L-selectin mediated leukocyte-leukocyte adhesion
have produced conflicting conclusions regarding the identity of the
leukocyte ligand for L-selectin. A candidate ligand for L-selectin
appears to be PSGL-1, based on the demonstration of neutrophil rolling
on purified PSGL-1 and its inhibition by an L-selectin
MoAb.19 However, other findings suggest that PSGL-1 may be
only one of several L-selectin ligands expressed by leukocytes. PSGL-1
MoAb treatment only partially inhibits formyl peptide-induced homotypic
neutrophil aggregation21 and does not completely block transient neutrophil interactions with fixed neutrophil
monolayers.19 Furthermore, the failure of a PSGL-1 MoAb to
inhibit L-selectin-dependent neutrophil-neutrophil interactions in
flow22 and the demonstration of OSGE-resistant neutrophil
adhesion to L-selectin23 suggest that PSGL-1 may
not always be required for leukocyte adhesive interactions with
L-selectin. Overall, these observations are analogous to conflicting
reports on whether PSGL-1 serves as a leukocyte ligand for
E-selectin22,31,38-40 and underscore the need to further
characterize leukocyte L-selectin ligand activity.
Under flow conditions, resting human neutrophils were observed to form
specific and stable rolling interactions with purified native
L-selectin immobilized on the wall of a flow chamber, similar to those
formed by neutrophils on recombinant L-selectin-IgG.23 L-selectin ligand activity on leukocytes appears to be constitutive, because treatment of neutrophils with metabolic inhibitors did not
inhibit adhesion to L-selectin. In contrast to leukocyte interactions with the vascular selectins, neutrophils were observed to attach to
L-selectin at shear stresses that were significantly higher than
corresponding interactions reported for either P-selectin or E-selectin
at similar site densities.26,39,41 The relatively high
capture rate of neutrophils to purified L-selectin is consistent with
its proposed role in mediating leukocyte capture from flow in
vivo.42,43
In agreement with studies reporting partial inhibitory effects of
PSGL-1 MoAbs in leukocyte-leukocyte adhesion assays,19,21 neutrophil and myeloid cell line attachment to purified
L-selectin under flow conditions was only partially inhibited
after treatment with PSGL-1 MoAbs. Partial PSGL-1 MoAb inhibition of
L-selectin-mediated neutrophil-neutrophil interactions under flow may
be caused by either incomplete saturation of PSGL-1, a consequence of
adherent neutrophil spreading,44 and/or levels of
shear stress that may favor or preclude a non-PSGL-1 L-selectin
interaction. PSGL-1 does appear to contribute significantly to
neutrophil-neutrophil interactions under static conditions or low shear
stress and when at least one of the interacting neutrophil populations
has been chemotactically stimulated.16,19,21 Because of
these considerations, we elected to study leukocyte adhesion directly
to immobilized L-selectin, which removes complications of L-selectin
shedding, PSGL-1 redistribution,44 and simplifies the flow
field in which the adhesive interaction takes place. The residual
binding of neutrophils, HL-60, and U937 cells to native L-selectin in
the presence of shear stress and MoAb KPL1 suggests that structures in
addition to PSGL-1 contribute to recognition of L-selectin under flow
conditions. In contrast to the myeloid cells tested, rolling of the
SKW3 lymphoid cell line on L-selectin was completely inhibited by
PSGL-1 MoAb treatment, indicating that PSGL-1 serves as the principal
L-selectin ligand in this cell line. Therefore, the relative
contribution of PSGL-1 to L-selectin ligand activity may be
at least partly dependent on leukocyte type.
To address the possibility that PSGL-1 has binding sites for L-selectin
that are distinct from the P-selectin binding site, which may be the
case for PSGL-1 E-selectin interactions,45 K562 cells
transfected with cDNAs encoding PSGL-1 and FucT-VII were evaluated for
binding to L-selectin in the presence of MoAb KPL1. MoAb KPL1
recognizes an epitope which contains a sulfated tyrosine (Snapp, K.R.;
Ding, H.; Atkins, K.; Luscinskas, F.W.; Warnke, R.; and Kansas, G.S.;
submitted) previously shown to be critical for binding of
PSGL-1 to P-selectin.45-48 Adhesion of FucT-VII/PSGL-1
transfectants to immobilized L-selectin was completely abolished by
KPL1, suggesting that PSGL-1 interacts with L-selectin at a binding
site shared by P-selectin. Consistent with this observation, binding of
L-selectin transfectants to PSGL-1 expressing COS cells is completely
blocked by MoAb KPL1 (Snapp, K.R.; Ding, H.; Atkins, K.; Luscinskas,
F.W.; Warnke, R.; and Kansas, G.S.; submitted). Therefore,
the PSGL-1 recognition of L-selectin may depend on the
N-terminal sulfated tyrosine domain shown to be required for recognition of P-selectin.
To characterize interactions between L-selectin ligand activity on
leukocytes and L-selectin expressed on a cell surface rather than
immobilized on a substrate, HL-60 cell membrane preparations were
isolated using WGA-lectin affinity chromatography. Rolling of
L-selectin expressing transfectants on HL-60 membrane preparations was
only partially reduced after significant immunodepletion of PSGL-1 from
the lysate, whereas P-selectin transfectant binding was completely
inhibited. This finding showed that ligand activity reconstituted from
a myeloid cell lysate supported L-selectin-dependent adhesion under
conditions in which P-selectin recognition was completely abolished.
The partial reduction in L-selectin binding to the
PSGL-1-immunodepleted lysate appears to represent the contribution of
PSGL-1 to L-selectin ligand activity in this membrane preparation.
The ligand activity mediating myeloid cell adhesion to L-selectin
appears to belong to a subset of mucinlike glycoproteins bearing
O-linked carbohydrate side chains, which include PSGL-1,17 leukosialin (CD43),49 and CD34.50 The OSGE
sensitivity of the myeloid cell L-selectin ligand activity
characterized in the present study sets it apart from that of KG1a
hematopoietic progenitor cells, because treatment with OSGE does not
block L-selectin-dependent lymphocyte adhesion to KG1a
monolayers.13 A nonmucinlike component of L-selectin ligand
activity on leukocytes has also been described in a recent report
showing OSGE-insensitive adhesion of myeloid cells to recombinant
L-selectin.23 However, the OSGE-insensitive adhesion was
only observed with high concentrations of L-selectin-IgG substrate,
which may have obscured OSGE effects because of either low affinity
interactions with sialylated glycoproteins29 or background
adhesion mediated by Fc receptor interactions with the selectin-IgG
chimera.
In contrast to the similar OSGE protease sensitivity of P-selectin and
L-selectin ligand activity on myeloid cells, studies with HL-60 rolling
on L-selectin and P-selectin showed a differential sensitivity to
neuraminidase treatment. Myeloid cell L-selectin ligand activity
appears to be considerably more dependent on the presentation of sialic
acid residues than PSGL-1 binding to P-selectin. This finding is
consistent with previous reports showing that neuraminidase treatment
blocks L-selectin-dependent interactions between flowing neutrophils
and adherent neutrophils12 and L-selectin-IgM binding to
myeloid cells.16 In contrast, PSGL-1 recognition of
P-selectin is relatively resistant to neuraminidase,16,17 which suggests distinct structural requirements for L-selectin and
P-selectin ligand activity.
There is currently a limited understanding of the physiological role of
L-selectin ligand expression by leukocytes. L-selectin-dependent interactions have been proposed as an initial step in neutrophil aggregation that enhances cell-cell contact time for formation of
larger, more stable aggregates through
2-integrin-mediated adhesion.21 Transient,
L-selectin-dependent adhesive interactions between flowing leukocytes
and leukocytes adherent to the vessel wall may serve as a mechanism to
sustain leukocyte recruitment at inflammatory sites.12 In
support of this hypothesis, capture of neutrophils in flow by adherent
neutrophils appears to amplify neutrophil accumulation on stimulated
endothelium and purified selectins in vitro.19,22 Our data
show that mucinlike ligands distinct from PSGL-1 support leukocyte
adhesion and rolling on L-selectin. Therefore, the expression of
multiple ligand structures for L-selectin by leukocytes may be
important for the efficiency of leukocyte capture from flow and the
accumulation of leukocytes at inflammatory sites.
| |
FOOTNOTES |
Submitted May 13, 1997;
accepted September 30, 1997.
Supported by National Institutes of Health (NIH) Grants No. HL54614 to
M.B.L., HL54136 to K.L., and NIH NRSA postdoctoral fellowship No.
HL09578 to C.L.R.; a Mitzutani Foundation grant to K.L.; and Grant No.
CB-204 from the American Cancer Society to G.S.K. G.S.K. is an
Established Investigator of the American Heart Association.
Address reprint requests to Michael B. Lawrence, PhD, Department of
Biomedical Engineering, University of Virginia, Box 377, Health
Sciences Center, Charlottesville, VA 22908.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely
to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank Drs R.P. McEver and K.L. Moore for the gift of PL1 and PL2, Dr
T.K. Kishimoto for the gift of DREG56, and Drs L.A. Lasky and S.R.
Watson for the gifts of selectin-IgG chimeras.
| |
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E. E. Eriksson, X. Xie, J. Werr, P. Thoren, and L. Lindbom
Importance of Primary Capture and L-Selectin-Dependent Secondary Capture in Leukocyte Accumulation in Inflammation and Atherosclerosis in Vivo
J. Exp. Med.,
July 16, 2001;
194(2):
205 - 218.
[Abstract]
[Full Text]
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J. Friederichs, Y. Zeller, A. Hafezi-Moghadam, H.-J. Gröne, K. Ley, and P. Altevogt
The CD24/P-selectin Binding Pathway Initiates Lung Arrest of Human A125 Adenocarcinoma Cells
Cancer Res.,
December 1, 2000;
60(23):
6714 - 6722.
[Abstract]
[Full Text]
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E. R. Hentzen, S. Neelamegham, G. S. Kansas, J. A. Benanti, L. V. McIntire, C. W. Smith, and S. I. Simon
Sequential binding of CD11a/CD18 and CD11b/CD18 defines neutrophil capture and stable adhesion to intercellular adhesion molecule-1
Blood,
February 1, 2000;
95(3):
911 - 920.
[Abstract]
[Full Text]
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L. Tu, P. G. Murphy, X. Li, and T. F. Tedder
L-Selectin Ligands Expressed by Human Leukocytes Are HECA-452 Antibody-Defined Carbohydrate Epitopes Preferentially Displayed by P-Selectin Glycoprotein Ligand-1
J. Immunol.,
November 1, 1999;
163(9):
5070 - 5078.
[Abstract]
[Full Text]
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C. L. Ramos, Y. Huo, U. Jung, S. Ghosh, D. R. Manka, I. J. Sarembock, and K. Ley
Direct Demonstration of P-Selectin– and VCAM-1–Dependent Mononuclear Cell Rolling in Early Atherosclerotic Lesions of Apolipoprotein E–Deficient Mice
Circ. Res.,
June 11, 1999;
84(11):
1237 - 1244.
[Abstract]
[Full Text]
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E. J. Kunkel, C. L. Ramos, D. A. Steeber, W. Muller, N. Wagner, T. F. Tedder, and K. Ley
The Roles of L-Selectin, {beta}7 Integrins, and P-Selectin in Leukocyte Rolling and Adhesion in High Endothelial Venules of Peyer's Patches
J. Immunol.,
September 1, 1998;
161(5):
2449 - 2456.
[Abstract]
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O. Dwir, Geoffrey. S. Kansas, and R. Alon
An Activated L-selectin Mutant with Conserved Equilibrium Binding Properties but Enhanced Ligand Recognition under Shear Flow
J. Biol. Chem.,
June 16, 2000;
275(25):
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[Abstract]
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T. S. Olson, K. Singbartl, and K. Ley
L-selectin is required for fMLP- but not C5a-induced margination of neutrophils in pulmonary circulation
Am J Physiol Regulatory Integrative Comp Physiol,
April 1, 2002;
282(4):
R1245 - R1252.
[Abstract]
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Abstract
Introduction
Abstract