|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
Blood, Vol. 91 No. 1 (January 1), 1998:
pp. 154-164
By
From the Department of Microbiology/Immunology, Northwestern
University Medical School, Chicago, IL; Vascular Research Division, the
Department of Pathology, Brigham and Women's Hospital and Harvard
Medical School, Boston, MA; and the Department of Pathology, Stanford
University Medical School, Palo Alto, CA.
Interactions between P-selectin and P-selectin glycoprotein ligand-1
(PSGL-1) mediate the earliest "rolling" of leukocytes on the
lumenal surface of endothelial cells at sites of inflammation.
Previously, PSGL-1 has been shown to be the primary mediator of
interactions between neutrophils and P-selectin, but studies on the
ability of PSGL-1 to mediate interactions between P-selectin and other
subsets of leukocytes have yielded variable and conflicting results. A
novel IgG monoclonal antibody (MoAb) to human PSGL-1 was generated, and
the specificity of this MoAb was confirmed by both flow cytometric
analysis and Western blotting of cells transfected with human PSGL-1.
This newly developed MoAb, KPL1, inhibited interactions between
P-selectin expressing COS cells and either HL60 cells, neutrophils, or
lymphocytes. Furthermore, KPL1 completely inhibited interactions
between P-selectin and either purified CD4 T cells or neutrophils in a
flow assay under physiological conditions, but had no effect on
interactions of T cells or neutrophils with E-selectin. In addition,
KPL1 blocked interactions between lymphoid cells transfected with
L-selectin and COS cells expressing PSGL-1. The KPL1 epitope was mapped
to a site within a consensus tyrosine sulfation motif of PSGL-1,
previously shown to be essential for interaction with P-selectin and
now shown to be essential for interaction with L-selectin, and to be
distinct from the epitope identified by the PL1 function blocking
anti-PSGL-1 MoAb. Two-color flow cytometry of normal leukocytes showed
that while natural killer (NK) cells (CD16+),
monocytes, CD4 and CD8 T cells, and
THE SELECTIN FAMILY of adhesion molecules
mediates the initial rolling of leukocytes on lumenal surfaces of
vascular endothelium.1 L-selectin is constitutively
expressed on all blood neutrophils, monocytes, the majority of T and B
cells, eosinophils, and most bone marrow cells. E-selectin in expressed
on endothelium in response to inflammatory mediators such as
interleukin-1 (IL-1), tumor necrosis factor (TNF- A glycoprotein ligand for P-selectin (PSGL-1) has been identified and
cloned.2,3 PSGL-1 is a mucinlike disulfide-linked homodimer
consisting of two identical 120-kD glycoprotein chains. The sequence of
human PSGL-1 contains a cleavage site for paired basic amino acid
converting enzymes (PACE).3 Three potential tyrosine
sulfation sites are located in a consensus sequence just downstream of
the PACE cleavage site, followed by 15 decamer repeats high in proline,
serine, and threonine. The extracellular portion of the molecule
contains three potential N-linked glycosylation sites. The remaining
C-terminal sequence consists of a single transmembrane spanning domain
followed by a 69-residue cytoplasmic tail. PSGL-1 has numerous
sialylated fucosylated O-linked oligosaccharides branches, many of
which terminate in the sialyl Lewis x (sLex) epitope.4-6 In
addition to fucose and sialic acid, interactions between PSGL-1 and
P-selectin require at least one tyrosine sulfate located in the amino
terminal consensus sequence.7-9
PSGL-1 is expressed by essentially all blood leukocytes including
neutrophils, monocytes, and lymphocytes, and has been shown to mediate
the rolling of human neutrophils on P-selectin.10 In
addition, PSGL-1 can serve as a ligand for L-selectin to mediate
neutrophil-neutrophil interactions.11,12 However, data
describing the function and expression of PSGL-1 on peripheral T and B
cells are conflicting.13-17 Although some of these
disparate observations may be related to expression of nonfunctional
PSGL-1 by the majority of T cells, some of these discrepancies may be
related to the properties of different anti-PSGL-1 reagents or
differences in posttranslational modifications characteristic of
different cell lineages.
To approach these questions, a new monoclonal antibody (MoAb) to human
PSGL-1, KPL1, was generated. This MoAb inhibited interactions between
PSGL-1 and P-selectin and between PSGL-1 and L-selectin, but had no
effect on leukocyte binding to E-selectin. The KPL1 epitope was mapped
to the tyrosine sulfation consensus motif of PSGL-1. Using two-color
flow cytometry, all T cells (CD4+, CD8+,
Production of MoAbs to PSGL-1.
A cDNA for human PSGL-110 (kindly provided by Henri
Lichenstein, Amgen Inc, Thousand Oaks, CA) was subcloned into a
modified SR Flow cytometry.
For one-color analysis, 0.5 × 106 cells were incubated in
100 µL of phosphate-buffered saline (PBS)/1% fetal calf serum
(FCS)/NaN3 containing pretitered amounts of the indicated
PSGL-1 MoAb, washed, and incubated in goat anti-mouse IgG-FITC, diluted
1:100. For two-color flow cytometry, 0.5 × 106 cells were
incubated in 100 µL of PBS/1% FCS/NaN3 containing
anti-PSGL-1 MoAb conjugated to FITC or biotin plus a leukocyte subset
specific antibody conjugated to either biotin or FITC. After washing,
cells were stained with Streptavidin-PE (Phycoerythrin;
Fischer Scientific Co, Pittsburgh, PA). Antileukocyte
subset antibodies included MoAb directed against CD4, CD8, CD3, CD16,
CD19, and CD14. MoAbs against T-cell receptor (TCR)- Western blotting.
HL60, 300.19, 300.19/PSGL-1 cells, or COS cells transiently transfected
with either human PSGL-1 cDNA or a control plasmid were washed twice in
RPMI. HL60, 300.19, and 300.19/PSGL-1 cells were resuspended at 2 ×
107 cells/mL in lysis buffer (1% Triton-X-100 [Sigma; St
Louis, MO]; 150 mmol/L NaCl; 10 mmol/L Tris-HCl, pH 7.6;
1 mmol/L CaCl2; 1 mmol/L MgCl2; 1 mmol/L
aprotinin; 1 mmol/L phenylmethylsulfonyl fluoride (PMSF); 1 mmol/L
leupeptin; and 1 mmol/L pepstatin A) and incubated on ice for 30
minutes. COS cells were resuspended at 2 × 106 cells/mL
in lysis buffer containing 2% Triton-X-100 on ice for 30 minutes.
Samples were clarified by centrifugation at 14,000g for 30
minutes at 4°C, and supernatants were transferred to fresh tubes. For
certain experiments, aliquots of lysates were treated overnight at
37°C with 1 U of Aerobacter aerogenes arylsulfatase (Sigma).
Samples were boiled for 5 minutes in sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer
with or without 6.5% 2-mercaptoethanol, electrophoresed on a 6%
polyacrylamide gel, and transferred to nitrocellulose. The
nitrocellulose was blocked with 2% gelatin (Bio-Rad) in Tris-buffered
saline-Tween (TBS-T; 20 mmol/L Tris HCl, pH 7.6; 137 mmol/L NaCl; and
0.1% Tween-20) for 2 hours at room temperature. Blots were probed with
KPL1 or PL2 in TBS-T plus 2% gelatin for 30 minutes, washed five times
in TBS-T, and then incubated with goat anti-mouse IgG conjugated to
horseradish peroxidase (Biosource) for 30 minutes. After five washes in
TBS-T, blots were visualized by chemiluminescence generated after the
addition of enhanced chemiluminescence (Amersham,
Arlington Heights, IL) and exposed to Hyperfilm (Amersham).
Generation and expression of full-length FFFE/PSGL-1 cDNA.
The plasmid pED.FFFE.148.Fc9 was kindly provided by Gray
Shaw (Genetics Institute, Cambridge, MA). This chimeric protein
contains the extracellular 148 amino acids of PSGL-1 fused to the heavy
chain CH2-CH3 region of IgG, and replaces the
three tyrosines at positions 5, 7, and 10 of PSGL-1 with phenylalanine
and the aspartic acid at position 11 with glutamic acid.9 A
423-bp Xma I/Stu I insert containing the FFFE mutation
was ligated into the full-length PSGL-1 cDNA in pBluescript, and the
full-length FFFE/PSGL-1 was inserted into a modified SR Neutrophil, peripheral blood mononuclear cell (PBMC), and
CD4+ T-cell purification.
Human neutrophils were isolated from heparinized blood by dextran
sedimentation, Ficoll density gradient centrifugation (Histopaque-1077;
Sigma), and hypotonic lysis of the neutrophil-rich pellet. PBMCs were
isolated from heparinized whole blood by dextran sedimentation followed
by Ficoll density gradient centrifugation. CD4 T cells were purified
from single donor human platelet pheresis residues by sequential
density gradient centrifugation and elutriation followed by culture
overnight and positive selection on Dynabeads (model M-450 CD4) and
Dynal DETACHaBEAD (Dynal, Great Neck, NY) as described.16
Low shear force COS cell adhesion assay.
This assay was performed as previously described.19-22
Briefly, COS cells were transfected with either P-selectin or
E-selectin cDNA by the DEAE-dextran method in 100-mm tissue culture
grade petri dishes. For binding of 300.19/L-selectin (300.19/L) or
300.19/P-selectin (300.19/P) cells,19,23 COS cells were
cotransfected with plasmids encoding PSGL-1 or FFFE/PSGL-1, FucT-VII,
and C2GnT. The following day, COS cells were replated on 35-mm dishes
(assay plates), and allowed to readhere overnight. The next day, HL60
cells, freshly purified human neutrophils, or PBMC were washed twice in
RPMI 1640 and resuspended at 2 × 106 cells in a total
volume of 100 µL containing saturating amounts of either KPL1 ascites
or control antibody and placed on ice for 15 minutes. Alternatively,
for 300.19/L or 300.19/P cells binding to transfected COS cells, the
COS cells were incubated with saturating amounts of KPL1 or control
ascites. After cells had been washed and resuspended in 0.6 mL of RPMI,
each petri dish was washed three times with unsupplemented RPMI 1640,
followed by the addition of appropriate cells, and incubated on a
constantly rocking platform for 15 minutes at 4°C. The plates were
washed five times with RPMI 1640 followed by fixation with cold 0.37%
formaldehyde/RPMI 1640. Mean number of cells bound per COS cell was
determined by counting the number of cells bound/COS cell on ~125 COS
cells in multiple 40× fields using a standard inverted light
microscope.
Parallel plate flow chamber adhesion assay.
The flow chamber apparatus used in these studies has been described
previously.15,24,25 Chinese hamster ovary (CHO)/P-selectin
or CHO/E-selectin monolayers were grown to confluence on glass
coverslips and inserted into the flow chamber. A defined flow level of
1.8 dynes/cm2 was obtained by drawing media containing the
desired cell population through the chamber using a syringe pump.
Purified CD4+ T cells or neutrophils, 1 ×
106, were assayed in each experiment. The flow chamber was
mounted on an inverted microscope (Nikon Diaphot; Melville, NY) and
each 5-minute perfusion period was recorded on videotape by a video
camera and video cassette recorder. Leukocyte adhesion was
determined as previously described.15,24,25 In some
experiments, monolayers of CHO/P cells were preincubated with HDPG2/3,
a blocking anti-P-selectin antibody3 at 10 µg/mL for 30
minutes at 37°C. Monolayers of CHO/E were incubated with HEL3/2, a
blocking anti-E-selectin antibody.26 W6/32 was used as an
isotype control for both monolayers. In other experiments, cells
(1 × 106) were incubated with either KPL1 ascites or
control ascites at a 1:200 dilution for 15 minutes at 4°C before use
in assays.
Immunohistology.
Immunohistology was performed using an indirect biotin-streptavidin
method.27 Incubation with primary antibody was followed by
incubation with a biotinylated goat anti-mouse second stage antibody
(Jackson Immunoresearch Laboratories, Inc, West Grove, PA) followed by
peroxidase-conjugated streptavidin (Jackson Immunoresearch
Laboratories). Diaminobenzedene was used as a substrate for the
horseradish peroxidase. Frozen sections were fixed in acetone for 10
minutes at 4°C before staining. Formalin-fixed deparaffinized
sections were stained without pretreatment or microwaved for 15 minutes
in 0.01 mol/L citrate buffer at pH 6.0 before staining.
Development and characterization of a new MoAb to human PSGL-1.
Balb/c mice were immunized with 300.19/PSGL-1 cells as described in
Materials and Methods. After routine immunization and fusion protocols,
hybridoma supernatants were screened by flow cytometry for negative
staining on untransfected 300.19 cells and positive staining on
300.19/PSGL-1 cells (Fig 1A). One MoAb,
KPL1, was identified. The KPL1 MoAb stained the 300.19/PSGL-1 cells at
high levels but did not stain untransfected 300.19 cells (Fig 1A). To
further confirm the specificity of KPL1, Western blot analysis of cells
known to express PSGL-1 or transfected with PSGL-1 cDNA was performed.
PSGL-1 is a homodimer of ~240 to 250 kD under
nonreducing conditions and ~120 kD under reducing
conditions.2,10 PSGL-1 is difficult to completely reduce,
allowing visualization of both reduced and nonreduced forms of PSGL-1.
KPL1 reacted with bands of ~120 and ~240 kD in lysates from HL60
cells (a myeloid cell line known to express PSGL-1), 300.19/PSGL-1
transfectants, or COS/PSGL-1 transfectants, but not in lysates from
untransfected 300.19 cells or COS cells transfected with a control
plasmid (Fig 1B). The slight differences in molecular weight between
PSGL-1 expressed by HL60 and the two transfectants were most likely
caused by differences in glycosylation in these different cell types.
Finally, KPL1 stained HL60 cells, freshly purified neutrophils, and
peripheral blood lymphocytes by flow cytometry (see
below).
KPL1 recognizes a unique epitope within the tyrosine sulfation motif
of PSGL-1.
Because of apparent differences in staining results with different
anti-PSGL-1 antibodies (see Introduction and below), the epitope
defined by KPL1 was explored in some detail. Neuraminidase treatment of
HL60 cells did not affect expression of the KPL1 epitope, but removed
all surface sLex carbohydrates as measured by lack of HECA452 staining
(data not shown). In addition, KPL1 stained cells regardless of the
presence (neutrophils, HL60 cells) or absence (300.19/PSGL-1 cells) of
either FucT-VII or C2GnT (Fig 1 and data not shown; by
RT-PCR,21,22,28 300.19 cells do not detectably express mRNA
for either FucT-VII or C2GnT). Thus, the KPL1 epitope appears to be
independent of carbohydrate modifications, including terminal sialic
acid residues, branched O-linked glycans, or fucosylation.
KPL1 MoAb inhibits binding of normal leukocytes and HL60 cells to
P-selectin but not E-selectin.
Adhesion assays were performed to determine if KPL1 could inhibit
interactions between normal leukocytes or HL60 cells and P- or
E-selectin. In the absence of antibody, HL60 cells, PBMCs, and
neutrophils each bound well to COS cells transfected with either P- or
E-selectin, with neutrophils binding slightly better to both P- and
E-selectin compared with PBMC or HL60 cells (Fig
3). Consistent with a requirement for
tyrosine sulfation of PSGL-1 for interaction with P-selectin,
preincubation of leukocytes with KPL1 virtually completely blocked
(>95%) interactions of HL60 cells and neutrophils with P-selectin
and almost completely (>90%) inhibited interactions of PBMCs with
P-selectin (Fig 3A). In contrast, KPL1 had no effect on binding to
E-selectin (Fig 3B).
KPL1 MoAb inhibits rolling of freshly isolated human neutrophils and
CD4 T cells on P-selectin but not E-selectin.
The results from the low shear stress COS cell adhesion assay were
extended to analysis of rolling under defined shear stress (Figs 4 and
5). Purified human neutrophils
or purified CD4
T cells were incubated with media containing either KPL1 or control
antibody, or the monolayers were incubated with either HDPG2/3, an
anti-P-selectin antibody (CHO/P monolayer); HEL3/2, an
anti-E-selectin antibody (CHO/E monolayer); or an isotype control
antibody (W6/32). Preincubation of the monolayers with MoAb to the
appropriate selectin completed blocked rolling of neutrophils.
Preincubation of neutrophils with KPL1 also completely inhibited
rolling of neutrophils on P-selectin (Fig 4A), but had no effect on
interactions with E-selectin (Fig 4B). Similarly, preincubation of
transfected monolayers with the appropriate anti-selectin antibody
completely inhibited rolling of CD4 T cells, and, as observed with
neutrophils, KPL1 completely inhibited rolling of CD4 T cells on CHO/P
but had no effect on CHO/E (Fig 5A and B).
KPL1 inhibits interactions between L-selectin and PSGL-1.
PSGL-1 has recently been identified as a leukocyte ligand for
L-selectin, and appears to mediate neutrophil-neutrophil interactions
which may be important in amplifying an inflammatory
response.11,12 Because normal neutrophils express ligands
for L-selectin in addition to PSGL-1,11,31 we analyzed the
effect of KPL1 on L-selectin/PSGL-1 interactions using L-selectin
transfectants binding to PSGL-1 transfectants, which isolates the
molecular interaction of interest. COS cells were cotransfected with
individual plasmids containing cDNA encoding PSGL-1, FucT-VII, and
C2GnT. For the assay, these COS cells were preincubated with either
KPL1 or control antibody, washed, and incubated with 300.19 cells
stably transfected with either L-selectin (300.19/L) or P-selectin
(300.19/P). Preliminary studies (data not shown) demonstrated that
binding of either 300.19/P or 300.19/L was absolutely dependent on
expression of both PSGL-1 and FucT-VII, and that binding of 300.19/L
was enhanced by cotransfection with C2GnT cDNA. Binding of 300.19/P was
not significantly enhanced by transfection with C2GnT cDNA, presumably
because of the endogenous expression of C2GnT by COS cells.
The KPL1 epitope is expressed at high levels on circulating T cells,
monocytes, and natural killer (NK) cells, but at low levels on B cells.
All circulating leukocyte subsets have been reported to express PSGL-1,
although different researchers have reported variable levels of
expression, or lack of expression, on specific subsets. Some of these
differences may be caused by properties of different PSGL-1 specific
antibodies. Staining of PBMCs with KPL1 in one-color flow cytometry
showed a small but distinct subpopulation expressing lower levels of
PSGL-1 (data not shown; see below), whereas most anti-PSGL-1 MoAb
exhibit a single peak on lymphocytes10,17 (data not shown).
To identify this KPL1lo subpopulation, two-color flow
cytometry was performed with a leukocyte lineage marker in one color
and KPL1 in a second color. Appropriate electronic scatter gates were
set for either monocytes or lymphocytes. All NK cells
(CD16+), monocytes (CD14+ ), and T
cells (CD3+) (Fig 7A),
including all CD4+, CD8+,
Expression of PSGL-1 in lymphoid tissues.
Both paraffin-embedded tissues and frozen sections (with or without
heat antigen retrieval) were prepared for immunohistology as described
in Materials and Methods. KPL1 stained numerous T cells in the T-zone
(TZ) of a human tonsil (Fig 8A), but did
not stain B cells in the mantle zone (M) or germinal center (GC) of the
secondary follicles. Macrophages in the germinal center stained but
follicular dendritic cells did not (Fig 8A). Just outside the germinal
center, intense KPL1 staining of overlying plasma cells was observed. A
high-power magnification (Fig 8B) shows this intense staining of plasma
cells which surround the germinal centers. Subepithelial plasma cells
also stained with KPL1 (data not shown), as did plasma cells around
vessels in the skin (Fig 8C); these plasma cells costained with the
plasma cell marker VS38 (Fig 8D). Thus, B cells located in germinal
centers either lack the KPL1 epitope, or express it at levels which are
not detected by these methods, whereas plasma cells in numerous sites
express high levels. Similarly, four IL-6-dependent human myeloma cell
lines expressed high levels of the KPL1 epitope (Diane Jelinek,
personal communication, March 1997). Both cortical and medullary
lymphocytes in the thymus stained with KPL1 (data not
shown). Langerhans cells, bone marrow-derived antigen
presenting cells which reside in the suprabasilar region of the
epidermis, were also positive for KPL1 (Fig 8E). Langerhans cells in
the tonsillar epithelium also stained with KPL1 (data not shown).
Langerhans cells in soft tissue also stain with both KPL1 (Fig 8F) and
CD1a (Fig 8G).
(E) Paraffin section of skin stained with KPL1.
The Langerhans cells in epidermis and superficial dermis stain
intensely in this example of Langerhans cell histiocytosis. The stained
dendritic processes are apparent in the epidermis. (F) Langerhans cells
in soft tissues also stain with KPL1. (G) Identification of Langerhans
cells in (F) is confirmed by costaining with CD1a (immunoperoxidase
with hematoxylin counterstain).
We have generated a novel MoAb against PSGL-1, designated KPL1. The
specificity of the KPL1 MoAb for PSGL-1 was confirmed by both flow
cytometry and Western blotting of multiple PSGL-1-expressing
transfectants. The epitope defined by KPL1 is independent of
carbohydrate modifications, including the presence or absence of
sialylation, fucosylation, or branched O-linked structures, because
KPL1 stains cells equivalently regardless of whether the cells express
no or high levels of FucT-VII or C2GnT, and KPL1 staining was not
affected by treatment of cells with neuraminidase. In addition, this
epitope is independent of PACE processing. However, the KPL1 epitope
requires sulfation of at least one tyrosine contained within a
consensus tyrosine sulfation motif (YEYLDYD)29 at the
extreme amino terminus of PSGL-1. Deletion of these seven amino acids
eliminated binding of KPL1,30 as did simultaneous mutation
of all three tyrosines to phenylalanine (Fig 2A) or sulfatase treatment
of PSGL-1 (Fig 2B). Thus, the KPL1 epitope maps to the tyrosine
sulfation motif of PSGL-1.
Submitted April 7, 1997;
accepted August 29, 1997.
The authors gratefully acknowledge Kevin Moore (University of Oklahoma
Health Sciences Center, Oklahoma City, OK) for supplying PL2 MoAb;
Henri Lichenstein (Amgen, Inc, Boulder, CO) for PSGL-1 cDNA; and Gray
Shaw, Ray Camphausen, and Gloria Vachino (Genetics Institute, Inc,
Cambridge, MA) for PSL275 MoAb, CHO/PACE cells, and the FFFE PSGL-1
mutant and helpful discussions; and M. Snapp for
inspiration.
1.
Kansas GS:
Selectins and their ligands: Current concepts and controversies.
Blood
88:3259,
1996
2.
Moore KL,
Stults NL,
Diaz S,
Smith DF,
Cummings RD,
Varki A,
McEver RP:
Identification of a specific glycoprotein ligand for P-selectin (CD62) on myeloid cells.
J Cell Biol
118:445,
1992
3.
Sako D,
Chang X-J,
Barone KM,
Vachino G,
White HM,
Shaw G,
Veldman GM,
Bean KB,
Ahern TJ,
Furie B,
Cumming DA,
Larsen GA:
Expression cloning of a functional glycoprotein ligand for P-selectin.
Cell
75:1179,
1993[Medline]
[Order article via Infotrieve]
4.
Moore KL,
Eaton SF,
Lyons DE,
Lichenstein HS,
Cummings RD,
McEver RP:
The P-selectin glycoprotein ligand from human neutrophils displays sialylated, fucosylated, O-linked poly-N-acetyllactosamine.
J Biol Chem
269:23318,
1994
5.
Norgard KE,
Moore KL,
Diaz S,
Stults N,
Ushiyama S,
McEver RP,
Cummings RD,
Varki A:
Characterization of a specific ligand for P-selectin on myeloid cells. A minor glycoprotein with sialylated O-linked oligosaccharides.
J Biol Chem
268:12764,
1993
6.
Wilkins PP,
McEver RP,
Cummings RD:
Structure of the O glycans on P-selectin glycoprotein ligand-1 from HL60 cells.
J Biol Chem
271:18732,
1996
7.
Wilkins PP,
Moore KL,
McEver RP,
Cummings RD:
Tyrosine sulfation of P-selectin glycoprotein ligand-1 is required for high affinity binding to P-selectin.
J Biol Chem
270:22677,
1995
8.
Pouyani T,
Seed B:
PSGL-1 recognition of P-selectin is controlled by a tyrosine sulfation consensus at the PSGL-1 amino terminus.
Cell
83:333,
1995[Medline]
[Order article via Infotrieve]
9.
Sako D,
Comess KM,
Barone KM,
Camphausen RT,
Cumming DA,
Shaw GD:
A sulfated peptide segment at the amino terminus of PSGL-1 is critical for P-selectin binding.
Cell
83:323,
1995[Medline]
[Order article via Infotrieve]
10.
Moore KL,
Patel KD,
Breuhl RE,
Fugang L,
Johnson DL,
Lichenstein HS,
Cummings RD,
Bainton DF,
McEver RP:
P-selectin glycoprotein ligand-1 mediates rolling of human neutrophils on P-selectin.
J Cell Biol
128:661,
1995
11.
Walchek B,
Moore KL,
McEver RP,
Kishimoto TK:
Neutrophil-neutrophil interactions under hydrodynamic shear stress involve L-selectin and PSGL-1.
J Clin Invest
98:1081,
1996[Medline]
[Order article via Infotrieve]
12.
Guyer GA,
Moore KL,
Lynam EB,
Schammel CMG,
Rogelj S,
McEver RP,
Sklar LA:
P-selectin glycoprotein ligand-1 (PSGL-1) is a ligand for L-selectin in neutrophil aggregation.
Blood
88:2415,
1996
13.
Alon R,
Rossiter H,
Wang X,
Springer TA,
Kupper TS:
Distinct cell surface ligands mediate T lymphocyte attachment and rolling on P and E-selectin under physiological flow.
J Cell Biol
127:1485,
1994 |
Abstract
Introduction
/
and 
/
T cells were
uniformly positive for PSGL-1, B cells expressed low levels of the KPL1
epitope. This low level of KPL1 staining was also observed
immunohistologically in germinal centers, which had no detectable KPL1
staining, whereas T-cell areas (interfollicular region) were positive
for KPL1. Interestingly, plasma cells in situ and
interleukin-6-dependent myeloma cell lines were KPL1+.
Thus, PSGL-1 is expressed on essentially all blood neutrophils, NK
cells, B cells, T cells, and monocytes. Variation in tyrosine sulfation
during B-cell differentiation may affect the ability of B cells to
interact with P- and L-selectin.
Abstract
), neutrophils
(
), and PBMC (
) to COS cells transiently transfected with
P-selectin was performed as described in Materials and Methods. All
three cell types adhered well to P-selectin. KPL1 almost completely
blocked these interactions, whereas a control antibody had no effect on
adhesion. (B) HL60 cells (
cells were
KPL1lo (Fig 
