* Division of Hematology, the Hematology Central Laboratory of the University of Lausanne, 1011-CHUV Lausanne; and Biomedical Engineering Laboratory of the Swiss Federal Institute of Technology, 1015-PSE-Ecublens, Switzerland
This study examines the role of L-selectin in
monocyte adhesion to arterial endothelium, a key
pathogenic event of atherosclerosis. Using a nonstatic
(rotation) adhesion assay, we observed that monocyte
binding to bovine aortic endothelium at 4°C increased four to nine times upon endothelium activation with
tumor necrosis factor (TNF)-. mAb-blocking experiments demonstrated that L-selectin mediates a major
part (64 ± 18%) of monocyte attachment. Videomicroscopy experiments performed under flow indicated
that monocytes abruptly halted on 8-h TNF-
-activated aortic endothelium, ~80% of monocyte attachment being mediated by L-selectin. Flow cytometric
studies with a L-selectin/IgM heavy chain chimeric protein showed calcium-dependent L-selectin binding to
cytokine-activated and, unexpectedly, unactivated aortic cells. Soluble L-selectin binding was completely inhibited by anti-L-selectin mAb or by aortic
cell exposure to trypsin. Experiments with cycloheximide, chlorate, or neuraminidase showed that protein
synthesis and sulfate groups, but not sialic acid residues, were essential for L-selectin counterreceptor
function. Moreover, heparin lyases partially inhibited
soluble L-selectin binding to cytokine-activated aortic
cells, whereas a stronger inhibition was seen with unstimulated endothelial cells, suggesting that cytokine
activation could induce the expression of additional ligand(s) for L-selectin, distinct from heparan sulfate
proteoglycans. Under flow, endothelial cell treatment
with heparinase inhibited by ~80% monocyte attachment to TNF-
-activated aortic endothelium, indicating a major role for heparan sulfate proteoglycans in
monocyte-endothelial interactions. Thus, L-selectin
mediates monocyte attachment to activated aortic endothelium, and heparan sulfate proteoglycans serve as
arterial ligands for monocyte L-selectin.
L-selectin plays a major role in the regulation of the
inflammatory response by mediating the initial attachment of leukocytes along endothelial cells lining postcapillary venules (4, 42, 43, 44, 85, 89). L-selectin shares common structural features with P- and E-selectin,
including an NH2-terminal C-type lectin domain, an EGFlike domain, short consensus repeats, a transmembrane domain, and a short cytoplasmic tail (38, 39, 83, 84). L-selectin, which is expressed by most leukocytes (1, 16, 27, 39), supports leukocyte tethering and rolling along vascular endothelium by interacting with carbohydrates presented by specific endothelial cell ligands (38, 41, 42, 53,
79, 84, 89, 90). P-selectin is rapidly expressed by activated
platelets and endothelial cells exposed to thrombin or histamine (26, 37, 45, 51, 52). E-selectin is expressed by endothelial cells upon activation by interleukin-1, tumor necrosis factor (TNF)1- Selectins bind to various carbohydrate ligands (2, 5, 38,
53, 65, 79, 84, 88), most of them containing a lactosamine
backbone and carrying sialylated, sulfated, and/or fucosylated sequences. Some complex carbohydrates, such as the tetrasaccharide sialyl Lewisx, are ligands for all three selectins; other carbohydrates interact only with one or two of
them (23, 88). Selectins have also been shown to bind to
complex sulfated carbohydrates that do not contain sialic
acid or fucose residues, for example, heparin, sulfatide, or
the HNK-1-reactive sulfoglucuronyl glycolipids (5, 55, 56,
88). Monovalent carbohydrates have low affinity for selectins, and their role in supporting leukocyte rolling is unclear (17, 33, 53). However, when oligosaccharides are
presented by a protein backbone, high affinity multivalent interactions can be observed (19, 53, 65, 88). Several glycoproteins have high affinity for selectins. Most of them are sialylated or sulfated mucin-like glycoproteins with many
serine and threonine residues that are potential sites for
attachment of O-linked glycans. Four mucinlike ligands
for L-selectin have been identified on high endothelial
venules of mouse lymph nodes: GlyCAM-1, MadCAM-1,
CD34, and gp 200, a glycoprotein that has not yet been
cloned (9, 11, 30, 40). GlyCAM-1 is secreted and might
serve to modulate L-selectin-mediated attachment of lymphocytes to peripheral lymph node high endothelial venules (15, 40). MadCAM-1 is present on mesenteric lymph
nodes as a multifunctional ligand recognized by both Although in vitro and in vivo studies support the existence of carbohydrate ligands for L-selectin on activated
nonlymphoid vascular endothelium, the identity of these
ligands has not been established (34, 35, 42, 43, 44, 48, 71,
73, 76, 77, 85, 89, 90, 92). Staining of calf pulmonary artery endothelial cell line or human umbilical vein endothelial
cells with an L-selectin/IgG1 heavy chain chimera has revealed the presence of an intracellular pool of heparin-like
ligands for the chimeric protein (57). Additional studies
have indicated that L-selectin interacts with heparan sulfate proteoglycans associated with or secreted by cultured
endothelial cells (58). However, the capacity of these proteoglycans to support leukocyte attachment to the vascular endothelium has not been examined.
Monocyte attachment to arterial endothelium is considered to be a key event of the early phase of atherosclerosis. However, little information is available on the molecular
mechanisms that mediate monocyte-endothelial interactions.
Earlier reports have shown that L-selectin is the major receptor for monocyte attachment to activated venous endothelium in nonstatic adhesion assay (76) and under flow
conditions (48, 49). The study described here was designed
to investigate the role of L-selectin and aortic ligands in
mediating monocyte attachment to resting and activated arterial endothelium.
Endothelial Cell Culture
Bovine aortic endothelial cells (BAEC; provided by J.-A. Haeffliger, Department of Internal Medicine, University Hospital, Lausanne), isolated
by collagenase treatment of bovine aorta, were established as primary culture and serially passaged in RPMI 1640 medium (Gibco Laboratories,
Paisley, Scotland) supplemented with 10% FCS (Gibco Laboratories). For
adhesion studies, BAEC (passages 3-5) were plated on 60 × 15-mm tissue
culture dishes (Becton Dickinson, Basel, Switzerland) and grown within a
2-cm-diam circle delineated by a ring of 12 M polysiloxane as described elsewhere (72, 73). For use in the parallel flow chamber, BAEC were
plated on 25-mm circular glass coverslips (Bellco Glass, Inc., Vineland,
NJ). Because a lower reactivity of BAEC with L-selectin/µ chimera was
observed with endothelial passages >8, only passages 3-5 were used to
perform immunofluorescence analysis or adhesion assays.
Monocyte Isolation
Human monocytes were prepared from blood buffy coats obtained from
healthy blood donors. Monocytes were isolated by centrifugation on Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) and adhesion on gelatine
1% (Sigma Chemical Co., St. Louis, MO) at 37°C. Nonadherent cells were
removed by three washes with HBSS (Gibco Laboratories). Adherent
cells were then detached with PBS containing 5 mM EDTA and washed
again in RPMI 1640 (Gibco Laboratories). The cell suspension obtained
by this method contained >95% monocytes as determined by Giemsa
stain and immunostaining with phycoerythrin-conjugated anti-CD14 mAb
Leu-M3 (Becton Dickinson). L-selectin and CD14 expression by whole
blood and isolated monocytes was evaluated by double immunofluorescence and flow cytometry (see below). Monocyte isolation caused a 40-
50% loss of L-selectin expression. Isolated monocytes were kept on ice
and used immediately after isolation.
mAbs
Anti-L-selectin mAbs anti-LAM1-3, -4, and -11 and anti-VCAM-1 mAb
HAE2 (all IgG1) were produced as described (72, 73). mAbs were purified from hybridoma culture supernatants on Affigel protein A (Bio-Rad,
Glattbrugg, Switzerland). For cell adhesion-blocking experiments, purified mAb IgG was used at 10 µg/ml. For chimeric protein-binding inhibition experiments, purified mAb IgG was used at 50 µg/ml.
Monocyte-Endothelial Interactions under Rotation
Cell attachment assays were carried out under rotation as previously described (73, 76, 77). BAEC, grown to confluence on tissue culture dishes,
were stimulated for 8 h with 100 U/ml TNF- Monocyte-Endothelial Interactions under Flow
Well-defined laminar flow was produced over confluent endothelial cell
monolayers on 25-mm circular glass coverslips introduced in a parallel
plate flow chamber (70). Monocytes were suspended at 0.5 × 106/ml in
RPMI 1640 medium and perfused at room temperature (18°C) through
the chamber at a shear stress of 1.8 dynes/cm2 via a syringe pump (model
22; Harvard Apparatus, Indulab AG, Switzerland). Monocyte-endothelial
interactions were visualized using a phase-contrast videomicroscope (Axiovert; Carl Zeiss, Lausanne, Switzerland) and CCD videocamera (model XC73CE; Sony, Japan) and videotaped (Panasonic s-VHS recorder; TSA
Telecom, Lausanne, Switzerland). Endothelial cell monolayers were cultured for 8 h in medium or in medium containing 100 U/ml TNF- Production of L-Selectin/µ Chimeric Protein
The L-selectin/µ chimeric protein was prepared by a method described in
detail elsewhere (78). Briefly, sequences encoding the lectin domain, the
EGF-like domain, and the first two short consensus repeats of L-selectin
were amplified by PCR using synthetic oligonucleotides. An artificial splice
donor site was introduced at the 3 Immunofluorescence Analysis
Indirect immunofluorescence analysis was performed using suspended
BAEC, which had been detached from plastic flasks with PBS/5 mM EDTA.
After three washes in RPMI 1640/1% FCS medium, BAEC were incubated for 30 min at 4°C with L-selectin/µ or CD4/µ chimera. Chimeric
protein binding to suspended endothelial cells was revealed using FITCconjugated rabbit anti-human IgM heavy chain (Dako, Glostrup, Denmark). Flow cytometry was performed using a cytofluorimeter (EPICS Profile;
Coulter Corp., Hialeah, FL). Cells were gated by forward- and side-scatter
signals. 5,000 cells were analyzed in each experiment.
Glycosaminoglycan Characterization
Endothelial cells were incubated with various enzymes for 45 min at 37°C
in 25 µl RPMI 1640. Concentration curves were done for each enzyme.
Optimal inhibition of L-selectin/µ binding was observed at the chosen enzyme concentrations. Heparinase I (Sigma Chemical Co.) was used at 600 mU, and heparitinase II (Seikagaku Corporation, Tokyo, Japan) was used
at 4 mU. In other experiments, BAEC were incubated with chondroitinase ABC (200-800 mU; Sigma Chemical Co.) or hyaluronidase (200 mU;
Sigma Chemical Co.). In experiments investigating the role of sialic acid,
BAEC were incubated with Vibrio cholerae neuraminidase (750 mU/ml;
Boehringer Mannheim) or Arthrobacter ureafaciens neuraminidase (200 mU/ml; Oxford Glycosystems, Ltd., Abingdon, UK). At this concentration, neuraminidase completely inhibited CSLEX-1 mAb binding to KG-1
cells treated with this neuraminidase (100 U/ml). The role of sulfate was
evaluated by culturing trypsinized BAEC (5 µg/ml trypsin for 30 min at
37°C) for 24 h in RPMI 1640 medium/10% FCS in the presence of 10 mM sodium chlorate. In additional experiments, BAEC were cultured with cycloheximide (10 µg/ml) for 30 min before and during TNF- Statistical Analysis
Analysis of variance (ANOVA) and the Bonferroni multiple comparisons
test were used to assess statistical significance between the different treatments versus control when three or more groups were analyzed; the
Mann-Whitney test was used to compare the median of two unpaired
groups, and the Wilcoxon signed rank test was used for paired groups. P
values <0.05 were considered significant.
Role of L-Selectin in Mediating Monocyte Adhesion to
Cytokine-activated Aortic Endothelium
Monocyte adhesion assays were performed at 4°C under
rotation. In these conditions, where L-selectin shedding is
minimal and CD18-mediated adhesion is inactive (50, 73,
74, 76), few monocytes attached to unactivated BAEC
monolayers (84 ± 20 monocytes/field, mean ± SD, n = 6).
When BAEC were activated for 8 h with TNF-
The notion that L-selectin could play a major role in the
attachment of monocytes to cytokine-activated arterial endothelium was evaluated further in experiments comparing
the effect of L-selectin/µ and CD4/µ chimera on the monocyte-binding capacity of BAEC monolayers. Whereas monocyte binding was not inhibited by pretreatment of BAEC
monolayers with CD4/µ (30 µg/ml), strong inhibition (56 ± 9%, n = 3, P < 0.005) was observed when monolayers
were preincubated with L-selectin/µ (50 µg/ml) (Fig. 2).
The role of L-selectin in mediating monocyte primary
adhesion was further examined in a parallel flow chamber
at a shear stress of 1.8 dynes/cm2 (70). All monocytes interacting with endothelial monolayers during the first 5 min of the experiment were counted. Most of these cells
were rolling before being abruptly halted and becoming stably adherent or detaching. The inhibition of L-selectin
with the function-blocking mAb anti-LAM1-3 reduced
monocyte primary adhesion to 8-h TNF-
Monocyte Adhesion to Cytokine-activated Aortic
Endothelial Cells: Kinetic Analysis
Monocyte adhesion to BAEC was determined under rotation before and after 2, 4, 6, and 8 h of endothelial cell incubation with TNF-
Unstimulated and Cytokine-activated BAEC Express
L-selectin Ligands
L-selectin ligand expression by suspended BAEC was detected by flow cytometry, L-selectin/µ being the probe and
CD4/µ being the control. L-selectin/µ was found to bind to
both unstimulated and cytokine-activated BAEC (Fig. 5,
top, solid lines) whereas CD4/µ did not (Fig. 5, top, dotted
lines). L-selectin/µ binding to BAEC was completely inhibited by the presence of 5 mM EDTA (Fig. 5, middle) or
100 µg/ml of function-blocking mAb anti-LAM1-3 or anti-
LAM1-4, which react with epitopes located on the lectin domain of L-selectin (Fig. 5, bottom). These latter results
demonstrate the calcium dependence of L-selectin binding
to aortic ligands and the involvement of the L-selectin lectin domain in this reaction.
Because activation of aortic endothelium with TNF-
L-selectin Binding to BAEC: Different
Ligand Characteristics on Unstimulated and
Cytokine-activated Endothelial Cells
The role of proteoglycans in supporting L-selectin-endothelial interactions was investigated in experiments examining the effect of glycosidase or trypsin treatment on L-selectin binding to aortic endothelium. As illustrated in Fig.
7, L-selectin binding to unstimulated BAEC was not affected by hyaluronidase (bottom left) or chondroitinase
ABC (middle right), whereas it was strongly inhibited by
incubation with heparinase I (top right), heparinase I or III
(not illustrated), and heparitinase II (middle left), and abrogated by cell exposure to trypsin (bottom right). Importantly, a quite different pattern was observed with BAEC
activated by 8 h of incubation with TNF-
Heparan sulfate proteoglycans are highly sulfated molecules, and sulfate residues are important for the function
of several selectin ligands (30, 32, 63, 68, 78). The role of
sulfate residues in L-selectin-BAEC interactions was assessed by experiments using unactivated or TNF-
Cycloheximide treatment also strongly inhibited L-selectin binding, indicating that protein synthesis is required for
ligand(s) expression by both unactivated and cytokine-
activated endothelium (Fig. 9, middle).
Intact sialic acid residues are required for interactions between L-selectin and mucinlike glycoproteins such as GlyCAM-1, CD34, or PSGL-1. To assess whether sialic acid
residues are involved in L-selectin binding to aortic endothelium, BAEC were pretreated for 45 min with V. cholerae (750 mU/ml) or A. ureafaciens neuraminidase (200 mU/
ml) before incubation with L-selectin/µ chimera (Fig. 9,
Vibrio Cholerae). Endothelial cell exposure to neuraminidase did not significantly affect L-selectin/µ binding to unactivated BAEC. Thus, 63 ± 15% (n = 8) of BAEC
treated with V. cholerae neuraminidase bound L-selectin/
µ, whereas 47 ± 26% (n = 8) of untreated cells bound the
chimera. Similarly, L-selectin/µ binding to activated
BAEC was not affected by neuraminidase. L-selectin/µ bound to 57 ± 19% (n = 6) of untreated cells and to 59 ± 16% (n = 6) of neuraminidase-treated cells. In contrast,
monocyte exposure to V. cholerae neuraminidase (100 mU/ml) abolished L-selectin/µ binding to monocyte
PSGL-1 (not illustrated) (78).
Role of Heparan Sulfates in Monocyte Adhesion to
Cytokine-activated Endothelium
The role of heparan sulfates in supporting monocyte attachment to TNF- Additional experiments were performed to examine the
contribution of heparan sulfate proteoglycans in mediating primary monocyte adhesion to activated endothelial
monolayers under flow. Monocyte attachment was very
significantly affected by the pretreatment of endothelial
monolayers with heparinase I. At 1.8 dynes/cm2, the total
number of interacting monocytes (primary adhesion) during the first 5 min of the videotaped experiments was significantly reduced (P < 0.001). Thus, 304 ± 43 monocytes/
mm2 (mean ± SD, n = 3) interacted with activated endothelial monolayers during this time, whereas 854 ± 72 interacting monocytes/mm2 (mean ± SD, n = 3) were observed with untreated endothelium. The number of stably
adherent monocytes was also considerably inhibited by
the pretreatment of activated endothelium with heparinase I. Adherent monocytes were counted during the last
2 min of the 12-min experiments. Stable monocyte adhesion was reduced by 88 ± 6% (mean ± SD, n = 4, P < 0.001) after the pretreatment of endothelial monolayers
with heparinase I (Fig. 10). Similar inhibition was obtained
by treating monocytes with the function-blocking mAb anti-LAM1-3 (83 ± 8%), whereas the control anti-L-selectin mAb anti-LAM1-11 had no significant inhibitory effect.
The following observations were made in this study: (a)
L-selectin plays a major role in monocyte adhesion to
TNF- Cell adhesion assays performed under rotation have
previously shown that L-selectin plays a major role in initiating monocyte attachment to cytokine-activated venous
endothelium in vitro (76). Subsequently, experiments made
with an in vitro flow system have confirmed that L-selectin
has a crucial role in initiating monocyte attachment, supporting monocyte rolling, and facilitating The cellular and molecular bases of monocyte attachment were further analyzed using an in vitro flow chamber
using function-blocking mAb and chimeric molecules. Observations made in videomicroscopy experiments showed
that freely flowing monocytes abruptly halted on 8-h TNF Interestingly, studies with adhesion-blocking mAbs have
shown that L-selectin cooperates with VCAM-1 to support monocyte attachment to activated arterial endothelium. Anti-VCAM-1 mAb HAE-2 (73) inhibited 38% of
monocyte adhesion (Fig. 1). The induction of VCAM-1 by
TNF- Adhesion-blocking mAb studies have previously suggested that an inducible ligand for monocytes is expressed
on human umbilical vein endothelial cells upon activation
with TNF- The lack of significant change in L-selectin/µ binding to
aortic endothelial cells after TNF- E-selectin is an inducible high affinity ligand that could
cooperate with heparan sulfate proteoglycans to mediate
monocyte attachment to activated endothelial cells. Expression of this adhesion molecule has been observed on
endothelial cells lining atherosclerotic lesions and in rabbits fed a hypercholesterolemic diet (24). PSGL-1 interacts
with E-selectin to mediate monocyte attachment to endothelial cells (6, 61, 78). In addition, L-selectin expressed by
human neutrophils binds to E-selectin through a carbohydrate ligand expressed by the lectin domain of L-selectin
(62). This latter interaction was studied in a control shear
adhesion assay by Lawrence et al. (41) and others (61), who observed an L-selectin-dependent neutrophil tethering to E-selectin. E-selectin and heparan sulfate could cooperate to mediate monocyte attachment to activated endothelium. Further studies will be required to determine if
additional ligands distinct from E-selectin could be involved in initiating monocyte attachment through L-selectin to activated endothelium. Finally, leukocyte recruitment in inflammatory lesions is not only dependent on the
interaction of neutrophils with endothelial cells but could
be considerably increased by the rolling of leukocytes on
already adherent leukocytes (3, 8, 25, 60, 73). Several studies demonstrated that a major part of leukocyte-leukocyte
interactions is regulated by L-selectin and its ligand PSGL-1
(8, 28, 60, 78, 87). Thus, L-selectin is critically involved in
promoting leukocyte recruitment at the site of inflammation by its capacity to regulate leukocyte interactions with
endothelial cell surface and leukocyte attachment to already adherent leukocytes.
The strong inhibition by cycloheximide of L-selectin
ligand expression by unstimulated and cytokine-activated
aortic endothelium indicated that protein synthesis is required for L-selectin binding (Fig. 9). Heparan sulfate proteoglycans involved in L-selectin binding are probably renewed in a continuous fashion. Earlier reports on heparan
sulfate proteoglycans have indicated that these species
have half-lives of between 3 and 8 h at the endothelial cell
surface, removal from the cell surface resulting from proteoglycan endocytosis and shedding into the extracellular
space (94).
The role of sulfates on L-selectin binding to aortic endothelium was evaluated because sulfate residues were
found to be necessary for the function of several selectin
ligands (9, 11, 29, 30, 40, 63, 68, 88, 93). Inhibition of ATP-
sulfurylase by chlorate (7) prevented most soluble L-selectin
binding, demonstrating that sulfation is critical for the interaction of L-selectin with arterial endothelial cell ligands
(Fig. 9). Inhibition of sulfation could abolish the interaction of L-selectin with highly sulfated molecules, like
heparan sulfate, thereby inhibiting most L-selectin reactivity with BAEC. It is also possible that other unidentified
sulfated ligands interact with L-selectin to support monocyte adhesion to activated aortic endothelium.
Several glycoprotein ligands for selectins require sialic
acid residues for function. In the present study, digestion
of activated and unactivated aortic endothelium with neuraminidase did not affect significantly L-selectin binding or
monocyte attachment, under rotation, to aortic endothelium. In this regard, aortic endothelium L-selectin ligands
behave quite differently from GlyCAM-1, CD34, or PSGL-1
(9, 18, 31, 40, 54, 67, 86). The lack of effect of neuraminidase treatment on monocyte adhesion and on L-selectin/µ
binding to BAEC suggests that sialic acid residues could
not be essential for L-selectin ligand function. However,
this result must be cautiously interpreted because we cannot exclude that a subset of sialic acid residues resistant to
enzymatic cleavage could play a role in L-selectin binding.
Norgard-Sumnicht et al. (57, 58) have previously reported the presence of heparan sulfate in a calf pulmonary
artery endothelial cell line (American Type Culture Collection CCL 209). However, staining of this cell line with
an L-selectin/IgG1 heavy chain chimera revealed the presence of an intracellular pool of heparan sulfate but no significant surface expression of the ligand (57). Here, using a
decameric L-selectin chimera, we show that heparan sulfate
proteoglycans are expressed at the surface of aortic endothelial cells and play a major role in L-selectin-dependent
attachment of monocytes to TNF- The in vitro observation that heparan sulfate proteoglycans are ligands for L-selectin and mediate monocyte attachment to activated aortic endothelium needs to be extended by in vivo studies. The identification of specific
sequences responsible for the interaction of L-selectin
with sulfated glycosaminoglycans may lead to the preparation of heparan sulfate analogues with the potential of inhibiting pathological leukocyte recruitment in inflammatory diseases. The ability of some heparin oligosaccharides
to inhibit leukocyte migration at sites of inflammation suggests that this approach might have therapeutic potential
(56, 58).
, or endotoxin (12, 13, 46, 47).
4
7
integrin and L-selectin (11). CD34 is the major ligand for
L-selectin in peripheral and mesenteric lymph node high
endothelial venules as well as in human tonsil (9, 64). It is
also expressed in larger vessels (10) and on hematopoietic
cell progenitors (36). However, CD34 function in large
blood vessels has not been explored. Sialic acid, fucose,
and sulfate residues are required for the function of GlyCAM-1 and CD34 (30, 32). These residues as well as three
NH2-terminal tyrosine sulfates have also been reported to be
essential for the interaction of P-selectin glycoprotein ligand-1
(PSGL-1) with P-selectin or L-selectin (63, 68, 78, 93).
Materials and Methods
(Boehringer Mannheim,
Mannheim, Germany). After washing, cytokine-activated endothelial cells
were preincubated for 15 min with medium alone (RPMI 1640/5% FCS)
or with medium supplemented with anti-VCAM-1 mAb, L-selectin/µ, or
CD4/µ chimeric proteins. Monocytes (4 × 106 cells) were preincubated for
15 min on ice in 120 µl of medium (RPMI 1640/5% FCS) or in medium
supplemented with mAb. Endothelial cell monolayers were washed before adding monocytes. After 30 min of incubation at 4°C under rotation
at 72 rpm, nonadherent cells were discarded. Petri dishes were then placed
vertically in 2% glutaraldhehyde and fixed overnight. The number of adherent monocytes was counted in six to eight microscopic fields (0.5 mm2
per field), and the results were expressed as mean ± 1 SD.
and then treated
for 20 min with saturating levels of chimeric proteins. To determine the involvement of endothelial glycosaminoglycans, endothelial monolayers were
incubated for 45 min with heparinase I (1,200 mU/ml) or hyaluronidase
(200 mU/ml) and then extensively washed with medium. Monocytes were
pretreated for 15 min with saturating concentrations of anti-L-selectin
mAb at 4°C and then suspended in medium. Stable adhesion was determined between 10 and 12 min of monocyte perfusion by analyzing 12-14
random fields (0.14 mm2/field, ×20 objective). Monocytes were considered as adherent after 20 s of stable contact. The rate of initial attachment
was assessed by counting the number of monocytes that interacted with endothelial cell monolayers during the first 5 min of the experiments.
end of the PCR product. The PCR
product was then subcloned in a plasmid containing the CH2, CH3, and
CH4 domains of IgM heavy chain (µ) in genomic configuration (kindly
provided by A. Traunecker, Basel Institute for Immunology, Basel, Switzerland). After digestion with NotI and XhoI, the pL-selectin/µ fragment
was subcloned in the pcDNAI expression vector (Invitrogen, San Diego,
CA). A CD4/µ chimera was constructed by substituting the L-selectin
coding sequence in pcDNA I L-selectin/µ with a CD4 fragment encoding the first two NH2-terminal domains of CD4. Chimeric molecules were
produced in COS cells transiently transfected with appropriate cDNAs.
Chimeras were used as concentrated COS cell conditioned media or after
purification by immunoadsorption to immobilized anti-LAM1-3 mAb (77).
The molecular characteristics of L-selectin/µ chimera were analyzed by
SDS-PAGE. In reducing conditions, purified L-selectin/µ migrated with
molecular masses ranging from 95,000 to 110,000 daltons. In nonreducing
conditions, the decameric L-selectin/µ chimera migrated as a single band
of very high molecular mass remaining at the end of the migration in the
3.75% SDS-polyacrylamide stacking gel. No additional band of lower molecular mass was observed in the 7.5% SDS-polyacrylamide running gel.
The concentration of L-selectin/µ was measured by ELISA as previously
described (75, 77). The concentration of CD4/µ chimera was determined by
ELISA using goat anti-human IgM heavy chain polyclonal antibody as capture antibody (Vector Laboratories, Inc., Burlingame, CA). The chimeric
protein was then detected with biotinylated polyclonal goat anti-human
IgM heavy chain antibody (Vector Laboratories, Inc.), avidin-HRP (Pierce,
Oud-Beijerland, The Netherlands), and O-phenylendiamine (0.125%, wt/
vol.; Sigma Chemical Co.) in 0.1 M citrate buffer, pH 4.5, as the substrate.
E- or P-selectin/µ chimeric protein concentration was determined using
purified L-selectin/µ chimera and purified human IgM as standards. Samples were run in triplicate at 1:500 to 1:5,000 dilutions. Under these conditions, a linear relationship was observed between signal intensity and protein concentration. Absorbance at 490 nm was measured using an ELISA
reader (model MR 5000; Dynatech Laboratories, Inc., Chantilly, VA).
treatment.
Results
(100 U/
ml), a significant increase in monocyte adhesion was observed (four- to ninefold, n = 6). Thus, in the experiment
illustrated in Fig. 1, the number of monocytes attached to
BAEC increased from 94 ± 10 to 425 ± 33/field upon endothelium activation with TNF-
(Fig. 1, medium). The
mechanism responsible for this observation was investigated with mAbs against L-selectin or VCAM-1. Cell binding inhibition studies revealed that monocyte adhesion to cytokine-activated BAEC monolayers was inhibited by 64 ± 18% (mean ± SD, n = 6, P < 0.005) when
monocytes were pretreated with the adhesion-blocking
mAb anti-LAM1-3 (Fig. 1) (73, 76). Cell adhesion was not
significantly inhibited in experiments with anti-LAM1-10 (not illustrated) or anti-LAM1-11 mAbs (Fig. 1), which
recognize nonfunctional domains of L-selectin. A role for
VCAM-1 in mediating monocyte attachment to activated
BAEC was demonstrated by the capacity of the anti-
VCAM-1 mAb HAE-2 to inhibit monocyte adhesion by
38 ± 6% (mean ± SD, n = 3, P < 0.01) (Fig. 1). However,
the results with anti-LAM1-3 indicate that L-selectin
plays a predominant role in monocyte attachment to cytokine-activated arterial endothelium under nonstatic conditions.
Fig. 1.
Monocyte attachment to unstimulated or TNF--activated aortic endothelium under rotation: inhibition by mAbs.
Endothelial monolayers were activated for 8 h with TNF-
(100 U/ml). BAEC were preincubated with medium or anti-VCAM-1
mAb (HAE-2). Monocytes were preincubated with medium,
blocking anti-L-selectin mAb anti-LAM1-3 or control mAb
anti-LAM1-11. Adhesion assays were carried out under rotation
for 30 min at 4°C. Data are expressed as means ± SD. Results are
representative of those obtained in six experiments. *P < 0.01. **Statistically significant (P < 0.005) difference in adhesion relative to control.
[View Larger Version of this Image (34K GIF file)]
Fig. 2.
Monocyte attachment to unstimulated or TNF--activated aortic endothelium under rotation: inhibition by chimeric
proteins. Endothelial monolayers were activated for 8 h with
TNF-
(100 U/ml). Unstimulated and TNF-activated BAEC
were then preincubated with medium, L-selectin/µ, or CD4/µ.
Adhesion assays were carried out under rotation for 30 min at
4°C. Data are expressed as means ± SD. Results are representative of three experiments. **P < 0.005.
[View Larger Version of this Image (29K GIF file)]
-activated endothelium by 78 ± 12% (mean ± SD, n = 4). Thus, after
pretreatment with anti-LAM1-3 mAb, only 209 ± 44 monocytes/mm2 (mean ± SD, n = 4) interacted with activated endothelium, whereas 970 ± 84/mm2 interacting
monocytes/mm2 were observed after pretreatment with the nonblocking anti-LAM1-11 mAb (not illustrated).
Pretreatment of endothelial cells with L-selectin/µ similarly reduced monocyte primary adhesion by 65 ± 14%
(mean ± SD, n = 3). In contrast, no inhibition was observed when endothelium was pretreated with the control
chimeric protein CD4/µ. The number of stably adherent monocytes at the end of the 12-min flow experiments was
strongly reduced by pretreating monocytes with the anti-
LAM1-3 mAb (83 ± 8%; mean ± SD, n = 4) or endothelial cell monolayers with L-selectin/µ chimera (71 ± 9%;
mean ± SD, n = 3). In the experiment illustrated in Fig. 3,
97 ± 30 (mean ± SD, n = 13) adherent monocytes/mm2
were observed after monocyte preincubation with anti-
LAM1-3, and 413 ± 56 monocytes/mm2 were observed after
pretreatment with anti-LAM1-11 mAb; in the same experiment, 130 ± 31 (mean ± SD, n = 13) monocytes/mm2 adhered to activated endothelium pretreated with L-selectin/µ, whereas 380 ± 45 monocytes adhered to monolayers pretreated with CD4/µ (50 µg/ml).
Fig. 3.
Monocyte adhesion to 8-h TNF--activated aortic endothelium under flow: inhibition by anti-L-selectin mAb LAM13 or L-selectin/µ chimera. After 20 min of preincubation with
L-selectin/µ or CD4/µ chimera, BAEC monolayers were washed
and inserted into the flow chamber. Untreated monocytes or
monocytes preincubated in medium or with anti-LAM1-3 or
anti-LAM1-11 mAb at 4°C were perfused across the monolayers
at 1.8 dynes/cm2 wall shear stress. Data are expressed as means ± SD. Stably attached cells (>20 s) were counted in 13 random
fields after 10 to 12 min of perfusion (rolling and transiently interacting cells were not included). Results are representative of
four experiments. **Statistically significant (P < 0.001) difference in adhesion relative to control.
[View Larger Version of this Image (44K GIF file)]
(100 U/ml). A time-dependent increase in monocyte binding was observed up to 6 h after
the addition of TNF-
(Fig. 4, solid circles). At
2 h of activation, monocyte binding to BAEC was inhibited by 48 to 68% with anti-LAM1-3 mAb (Fig. 4, open circles). With
unstimulated BAEC, the inhibition observed with monocytes pretreated with anti-LAM1-3 did not reach statistical significance.
Fig. 4.
Kinetics of monocyte attachment to TNF--activated
aortic endothelium under rotation. Endothelial monolayers were
stimulated with TNF-
(100 U/ml) for 0-8 h at 37°C before the
addition of monocytes. The L-selectin-dependent component of
monocyte adhesion was determined using function-blocking
mAb anti-LAM1-3. Adhesion assays were performed under rotation for 30 min at 4°C. Solid circles represent adhesion of untreated monocytes. Open circles represent adhesion of monocytes pretreated with anti-LAM1-3 mAb. Data are expressed as
means ± SD. Results are representative of two experiments.
[View Larger Version of this Image (15K GIF file)]
Fig. 5.
Interaction of L-selectin with suspended aortic endothelial cells. Unactivated or TNF--activated BAEC (8 h, 100 U/
ml) were examined by indirect immunofluorescence analysis for
the expression of L-selectin ligands. L-selectin/µ was used as the
probe (solid lines), and CD4/µ, an isotype-matched chimeric protein, was used as the control (dotted lines). The data are representative of three experiments.
[View Larger Version of this Image (30K GIF file)]
induced a progressive increase in L-selectin-dependent
monocyte adhesion (Fig. 4), L-selectin ligand expression
by BAEC was followed over a 24-h period of time. Surprisingly, unstimulated BAEC or BAEC activated by
TNF-
(100 U/ml) for 2, 4, 6, 8, or 24 h were found to bind
L-selectin/µ in a similar fashion (Fig. 6).
Fig. 6.
Time course of aortic endothelial cell activation. Endothelial cells were activated with TNF- (100 U/ml). At the indicated times, indirect immunofluorescence analysis was performed
with L-selectin/µ (solid lines) and CD4/µ (dotted lines). The data are representative of three experiments.
[View Larger Version of this Image (32K GIF file)]
(100 U/ml) (Fig.
8). Although trypsin treatment completely inhibited the
reaction (Fig. 8, bottom right), activated BAEC exposure
to heparinase I, heparitinase II, or heparitinase III only
had moderate inhibitory effects on L-selectin binding (Fig.
8, top right and middle). Thus, heparinase treatment induced a significantly higher decrease in L-selectin/µ binding to unactivated BAEC (mean percentage of decrease ± SD 42 ± 17%, n = 22) than to BAEC exposed for 8 h to
TNF-
(26 ± 14%, n = 14, P = 0.005). As observed with
unstimulated cells, hyaluronidase and chondroitinase did
not inhibit L-selectin binding to activated BAEC (bottom
left and middle right).
Fig. 7.
Interaction of L-selectin with suspended aortic endothelial cells: effect of treating unstimulated BAEC with heparinase I, heparitinase II, chondroitinase ABC, hyaluronidase, or
trypsin. Unactivated BAEC were examined by indirect immunofluorescence analysis with L-selectin/µ (solid lines) and CD4/µ
(dotted lines). Identical results were obtained by treating BAEC
with heparinase I, II, or III. The data are representative of six experiments. Percentages of BAEC that bound to L-selectin/µ were
as follows: control, 86%; heparinase I, 54%; heparitinase II, 56%;
chondroitinase, 89%; hyaluronidase, 90%; trypsin, 7%. The background staining with CD4/µ chimera was <1%.
[View Larger Version of this Image (29K GIF file)]
Fig. 8.
Interaction of L-selectin with suspended aortic endothelial cells: effect of treating TNF--activated BAEC (8 h, 100 U/ml) with heparinase I, heparitinase II, chondroitinase ABC, hyaluronidase, or trypsin. Unactivated BAEC were examined by indirect immunofluorescence analysis with L-selectin/µ (solid lines)
and CD4/µ (dotted lines). Identical results were obtained by treating
BAEC with heparinase I, II, or III. The data are representative of
six experiments. Percentages of BAEC that bound to L-selectin/
µ are as follows: control, 87%; heparinase I, 39%; heparitinase II,
47%; chondroitinase, 89%; hyaluronidase, 82%; trypsin, 4%.
[View Larger Version of this Image (29K GIF file)]
-activated BAEC cultured for 24 h in the presence of 10 mM
sodium chlorate, an inhibitor of sulfate synthesis (7). As
shown in Fig. 9, inhibition of sulfation inhibited most L-selectin binding to both unstimulated and cytokine-activated BAEC (bottom).
Fig. 9.
Interaction of L-selectin with suspended aortic endothelial cells: effect of treating unstimulated or TNF--activated
BAEC (6 h, 100 U/ml) with cycloheximide (10 µg/ml), V. cholerae neuraminidase (750 U/ml), or sodium chlorate (10 mM, 24 h). BAEC were examined by indirect immunofluorescence analysis with L-selectin/µ (solid lines) and CD4/µ (dotted lines). The
data are representative of six experiments. Percentages of unactivated BAEC that bound to L-selectin/µ are as follows: control,
86%; cycloheximide, 22%; V. cholerae, 91%; chlorate, 13%. Percentages of TNF-
-activated BAEC that bound to L-selectin/µ
are as follows: control, 77%; cycloheximide, 11%; V. cholerae,
89%; chlorate, 17%.
[View Larger Version of this Image (27K GIF file)]
-activated BAEC was studied by preincubating endothelial monolayers with heparinase I
before monocyte addition. Adhesion assays performed
under rotation after the addition of heparinase I indicated
that heparan sulfates support monocyte attachment to 8-h
TNF-
-activated BAEC. Monocyte adhesion to cytokineactivated aortic endothelium was reduced by 36 ± 11%
(mean ± SD, n = 4, P < 0.01) using BAEC monolayers
preexposed to heparinase I; BAEC pretreatment with
V. cholerae neuraminidase (750 mU/ml, 45 min at 37°C)
did not significantly inhibit monocyte binding (inhibition of L-selectin/µ binding
8 ± 6%, n = 3) (not illustrated).
In control experiments in which monocytes were preincubated with anti-LAM1-3 mAb, monocyte attachment to
TNF-
-activated BAEC monolayers was inhibited by 64 ± 18% (P < 0.005).
Fig. 10.
Inhibition of monocyte adhesion to 8-h TNF--activated endothelium under flow (wall shear stress estimated at 1.8 dynes/cm2). Endothelial monolayers were activated for 8 h with
TNF-
(100 U/ml), washed, and then preincubated for 45 min
at 37°C with heparinase I (1,600 mU/ml) or hyaluronidase (200 mU/ml). The adhesion assay was performed in a flow chamber,
and adherent monocytes were counted as described in the legend
to Fig. 3. Data are expressed as means ± SD. Results are representative of three experiments. **P < 0.001.
[View Larger Version of this Image (33K GIF file)]
Discussion
-activated aortic endothelial cells; and (b) heparan
sulfate proteoglycans and possibly other protein-based
ligands function as arterial counterreceptors for monocyte
L-selectin. These findings provide novel information on
the molecular mechanisms of monocyte attachment to activated arterial endothelium, a key cellular reaction in the
initial lesion of atherosclerosis.
4
1-integrin- dependent arrest (48, 49). Thus, interactions between
monocytes and venous endothelial cells seem to involve
L-selectin-dependent monocyte rolling on the endothelial
cell surface, followed by sequential involvement of
1 integrin,
2 integrin, and CD31 (PECAM-1) in subsequent
steps of monocyte migration into tissues. In this study, we
observed under rotating conditions that L-selectin plays a
major role in mediating monocyte attachment to activated
arterial endothelium. Involvement of L-selectin was demonstrated by experiments showing that adhesion-blocking
anti-L-selectin mAbs LAM1-3 and LAM1-4 had the capacity to inhibit monocyte binding to activated aortic endothelium, whereas this reaction was not inhibited by anti-LAM1-11 and anti-LAM1-10 mAbs, which recognize
domains of L-selectin not involved in cell adhesion (Fig. 1).
Further support for the notion that monocytes are attached to arterial endothelium via L-selectin was provided
by experiments showing the capacity of L-selectin/µ to inhibit monocyte-endothelial interactions (Fig. 2). Equivalent inhibitions were obtained by preincubating activated aortic cell monolayers with L-selectin/µ or by treating monocytes with mAb LAM1-3, indicating that L-selectin/µ had
the capacity to completely inhibit L-selectin-dependent
cell adhesion. Under the same conditions, CD4/µ had no
inhibitory effect on monocyte binding to activated aortic
endothelium (Fig. 2).
-activated aortic endothelium primarily through L-selectin. mAb blockade of L-selectin inhibited by ~80% monocyte attachment to TNF-
-activated endothelium (Fig. 3).
These observations are consistent with those describing,
under flow, monocyte interactions with 6-h TNF-
-activated human umbilical vein endothelial cells (49), and
they are the first to show that L-selectin mediates monocyte attachment to activated aortic endothelium. Because
recent studies reported that neutrophils can roll on already adherent neutrophils (8, 22, 78), L-selectin-mediated
monocyte primary adhesion to activated endothelium was
examined during the first 5 min of each experiment, when
the number of stably adherent monocytes is low. Careful
analysis of the video records showed that flowing monocytes occasionally slowed down and arrested on adherent
monocytes, facilitating their attachment to activated endothelium. These interactions were discarded for quantitative analysis. Only single-cell interactions with activated
endothelium were taken into consideration. The strong inhibition of monocyte attachment to activated endothelium induced by the pretreatment of endothelium with L-selectin/µ further indicated that L-selectin interaction with endothelial ligand(s) is an important mechanism of monocyte
recruitment at the vascular endothelial cell surface.
observed here is consistent with results from earlier studies reporting expression of that receptor on aortic endothelium in acute rejection of rabbit cardiac allograft
(82) or after balloon injury of the aorta (81). In addition,
several studies have reported that VCAM-1 is expressed
on atherosclerotic lesions, suggesting that VCAM-1 could
play a critical role in regulating monocyte entry into the
arterial wall (20, 59). Because this study has identified
L-selectin as a major mediator of monocyte attachment to
cytokine-activated arterial endothelium, it will be important
to assess in subsequent work the extent to which L-selectin
is also involved in regulating monocyte entry into atherosclerotic lesions. Clearly, a detailed elucidation of the molecular mechanisms involved in monocyte attachment to the arterial wall will be required to understand how atherosclerotic plaques are formed and to generate drugs that
may have the capacity to inhibit the formation of these lesions.
(34, 48, 49, 76). Other investigators have reported that additional endothelia can also express cytokine-inducible ligands (14). Here, the progressive increase
in L-selectin-mediated monocyte adhesion observed after
activation of endothelial cells with TNF-
suggested again
that inducible ligands for L-selectin are expressed on activated aortic endothelial cells (Fig. 4). The nature of these
ligands was probed by experiments examining the binding of soluble recombinant L-selectin/µ to live endothelial
cells. Considering that multivalency could be an important
factor in selectin function (65), we used a decameric form
of L-selectin instead of a dimeric chimera to improve the
detection of L-selectin ligands. Surprisingly, soluble L-selectin/µ was also found to bind to unactivated aortic endothelial cells (Fig. 5). This result was unexpected because unactivated endothelium supported only little monocyte
binding (Figs. 1-3). The specificity of L-selectin/µ binding
to aortic cells was established using EDTA or functionblocking mAb anti-LAM1-3 or anti-LAM1-4, which completely inhibited L-selectin binding, whereas control anti-
L-selectin mAbs had no effect on this reaction. Activation
of endothelial cells by TNF-
had little influence on L-selectin/µ binding (Figs. 5 and 6). Endothelial cell treatment
with various glycosaminoglycan-cleaving enzymes demonstrated that ligands expressed on both unactivated and activated aortic endothelium were sensitive to heparinase I
and heparitinase II (Figs. 7 and 8). In addition, binding of
L-selectin to aortic endothelium was completely abolished
by trypsin, which indicates that L-selectin binds to heparan
sulfate chains attached to protein in the form of proteoglycans. Importantly, the reactivity of L-selectin with cytokineactivated aortic cells was only partially susceptible to heparinase I and heparitinase II digestion. This latter observation suggests that cytokine activation could induce the expression of additional ligands, distinct from heparan sulfate
proteoglycans that interact with L-selectin to support monocyte adhesion. Alternatively, TNF-
could increase monocyte adhesion to endothelium by modifying heparan sulfate
proteoglycan glycosylation or sulfation. This mechanism
could induce expression of L-selectin-binding sequences responsible for high affinity interactions between L-selectin and cytokine-activated aortic cells; these sequences
would not be expressed on unstimulated arterial endothelium. An additional option is that the arterial endothelial
response observed after TNF-
activation occurs with preexisting L-selectin ligands. In this scenario, unstimulated
aortic endothelial cells have dispersed L-selectin ligands
on their surface, which can bind L-selectin/µ but cannot
support monocyte adhesion; in contrast, on the surface of
activated endothelial cells, L-selectin ligands could form patches capable of both L-selectin/µ and monocyte binding. Further studies will be needed to investigate these
possibilities.
activation does not
preclude the existence of inducible ligands for L-selectin
(Figs. 5 and 6). Thus, the increase in L-selectin-dependent
binding of monocytes observed after BAEC activation
could be mediated by ligands that react with high affinity
with L-selectin but are expressed at low density at the cell
surface. One can speculate that heparan sulfate proteoglycans function as low affinity L-selectin ligands that attract
monocytes at the vascular cell surface (coreceptor function) and direct them to less abundant high affinity receptors. This process could be analogous to the one that regulates the presentation of FGF by multimeric heparan
sulfate proteoglycans to high affinity FGF receptor (69).
In addition, the capacity of heparan sulfate proteoglycans
to present cytokines to attracted monocytes may provide
adhesion-inducing signals that regulate subsequent steps of
adhesion (66, 79, 80). As discussed above, the partial susceptibility of L-selectin ligands to heparin lyases suggests
that additional ligands for L-selectin could be expressed
by TNF-
-activated endothelium. Heparan sulfate proteoglycans could have an important role in supporting
monocyte rolling along endothelium, whereas less abundant
high affinity ligands could be required to allow monocyte
arrest. The increase in L-selectin-dependent monocyte adhesion observed after activation of BAEC with TNF-
(Fig. 4) could be explained by the expression of ligands not
present on unactivated BAEC.
-activated aortic endothelium (Fig. 10). Moreover, endothelial monolayer treatment with heparinase I inhibited monocyte adhesion to activated endothelial monolayers. Future studies will be aimed
at identifying and characterizing heparan sulfate proteoglycans involved in L-selectin endothelial cell interactions and the additional ligand(s) that may cooperate with
heparan sulfate proteoglycans to support monocyte adhesion. It is possible that heparan sulfate expressed by arterial endothelium has L-selectin-specific recognition sequences that are not present on heparan sulfate extracted
from bovine intestinal mucosa. Indeed, Diamond et al.
(21), using a flow system, did not observe interactions between L-selectin and bovine intestinal mucosa heparan
sulfate.
Received for publication 4 June 1996 and in revised form 15 October 1996.
Please address all correspondence to Dr. Olivier Spertini, Division of Hematology, University of Lausanne, 1011-CHUV Lausanne, Switzerland.The authors are grateful to Dr. Philippe Schneider, Dr. Jean-Daniel Tissot, and the staff of the Centre de Transfusion Sanguine at Lausanne for providing buffy coats. We thank Dr. Jacques-Antoine Haeffliger for providing BAEC, and Drs. André Traunecker and Klaus Karjalainen for providing CD4 and IgM heavy chain DNAs.
This work was supported by grant 31-43235.95 from the Swiss National Foundation for Scientific Research, the Marie Heim-Vögtlin Foundation, and the Emma Muschamp Foundation.
BAEC, bovine aortic endothelial cells; PSGL-1, P-selectin glycoprotein ligand-1; TNF, tumor necrosis factor.