L-selectin binding activity for its ligand expressed by vascular endothelium is rapidly and transiently increased after leukocyte activation. To identify mechanisms for upregulation and assess how this influences leukocyte/endothelial cell interactions, cell-surface dimers of L-selectin
were induced using the coumermycin-GyrB dimerization strategy for cross-linking L-selectin
cytoplasmic domains in L-selectin cDNA-transfected lymphoblastoid cells. Coumermycin-
induced L-selectin dimerization resulted in an approximately fourfold increase in binding of
phosphomanan monoester core complex (PPME), a natural mimic of an L-selectin ligand,
comparable to that observed after leukocyte activation. Moreover, L-selectin dimerization significantly increased (by ~700%) the number of lymphocytes rolling on vascular endothelium
under a broad range of physiological shear stresses, and significantly slowed their rolling velocities. Therefore, L-selectin dimerization may explain the rapid increase in ligand binding activity that occurs after leukocyte activation and may directly influence leukocyte migration to peripheral lymphoid tissues or to sites of inflammation. Inducible oligomerization may also be a
common mechanism for rapidly upregulating the adhesive or ligand-binding function of other
cell-surface receptors.
Key words:
 |
Introduction |
Leukocyte accumulation at sites of inflammation is regulated at the level of binding to the vascular endothelium, a multistep process initiated by the selectin family of
adhesion molecules (1, 2). Although selective expression is
a prominent means of governing selectin function, other
mechanisms also regulate adhesive function. With L-selectin, lymphocyte activation through antigen receptors or
neutrophil activation by chemokines upregulates its binding affinity for ligand (3). This rapid and transient increase
in ligand binding presumably results from intracellular signals involving G proteins, since L-selectin's upregulated
binding activity correlates with rapid phosphorylation of
conserved cytoplasmic serine residues and upregulated binding activity is blocked by pertussis toxin and protein kinase
C inhibitors (4). In addition, L-selectin localization at the
tips of leukocyte microvilli (5, 6) regulates leukocyte capture since it facilitates contact with ligand-coated walls of in
vitro flow chambers (7). However, the topographical position of L-selectin does not influence rolling velocity or detachment rates once rolling is established (7). Moreover,
correct cell surface positioning is not sufficient for L-selectin-mediated adhesion, since deletion of L-selectin's cytoplasmic domain abrogates in vivo and in vitro leukocyte
rolling (8) and its interactions with the cytoskeleton, but
does not inhibit microvillus localization (9). Thus, the
L-selectin cytoplasmic domain critically regulates receptor
function. In this study, we assessed whether upregulation of
L-selectin binding activity through its cytoplasmic domain
could result from the formation of L-selectin dimers on the
cell surface and whether receptor dimerization affected receptor function and leukocyte rolling.
 |
Materials and Methods |
DNA Constructs and Cell Transfection.
A modified GyrB cDNA
fragment was generated as previously described (10) and ligated at
the 3' end of a human L-selectin cDNA (11) via an XbaI site introduced at the L-selectin translation-termination codon. All
constructs were verified by DNA sequencing, subcloned into the
pMT-2 expression vector (provided by Genetics Institute, Cambridge, MA), and used to transfect 300.19 cells (12). Transfected cells were selected in RPMI 1640 medium containing 10% calf
serum and G418 (1 mg/ml; Sigma Chemical Co., St. Louis,
MO). Multiple clones of transfected cells expressing similar cell-surface levels of wild-type L-selectin or L-selectin-GyrB fusion
proteins were identified by immunofluorescence staining with
flow cytometry analysis.
Coumermycin Treatment and Phosphomanan Monoester Core Complex Binding Assay.
Cells were washed once with RPMI 1640 medium before incubation at 37°C for 25 min (unless indicated
otherwise) in RPMI 1640 containing either 0.1% DMSO or the
indicated amounts of coumermycin and novobiocin (in 0.1%
DMSO; Sigma Chemical Co.). After washing with ice-cold PBS
without Ca2+/Mg2+, the cells were divided and incubated with
biotin-labeled PPME (5 µg/ml) in PBS containing either Ca2+/
Mg2+ or 10 mM EDTA. After a 30-min incubation on ice,
FITC-labeled avidin was added to visualize phosphomanan monoester core complex (PPME) binding as previously described (3,
4), with staining assessed immediately by flow cytometry as in
Fig. 1. Ca2+-dependent PPME-binding was calculated by subtracting the mean linear fluorescence channel number for background staining (in 10 mM EDTA) from the mean value of fluorescence staining in the presence of Ca2+. Antibiotic treatment
did not change the mean fluorescence intensity of background
staining.

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Fig. 1.
Generation of L-selectin-expressing cell lines. (A) Structure
of L-selectin and the L-selectin-GyrB (L-Gb) fusion protein containing
the entire L-selectin protein in-frame with the NH2-terminal 24-kD subdomain of the B subunit of bacterial DNA gyrase (GyrB). Domains: EGF,
epidermal growth factor-like; SCR, short consensus repeat; TM, transmembrane; Cyto, cytoplasmic. (B) Cell-surface expression of wild-type
L-selectin or L-Gb in stably-transfected 300.19 cells and wild-type
L-selectin expression by human blood lymphocytes. Cells were isolated
and stained with FITC-conjugated LAM1-116 mAb specific for L-selectin (solid line) or an isotype-matched, nonbinding control mAb (dashed
line) as previously described (16). Fluorescence histograms from flow cytometry analysis are on a three-decade log scale and are representative of
results from at least five experiments.
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|
Physiologic Shear Flow Assay.
Cells were treated with DMSO,
coumermycin, and/or novobiocin as described above. For ligation with antibody, cells (2 × 106 cells/ml in flow medium, PBS
containing Ca2+/Mg2+ and 0.5% BSA) were incubated with
LAM1-101 or LAM1-118 mAbs (10 µg/ml) at room temperature for 15 min. In vitro rolling experiments were as previously
described (13) using a transformed human umbilical vein endothelial cell (HUVEC) line (EA.hy926, provided by Dr. Cora-Jean
Edgell, University of North Carolina at Chapel Hill; reference
14) that was transfected with an
1,3fucosyltransferase-VII cDNA
(fucosyltransferase VII, provided by Dr. Brent Weston, University of North Carolina at Chapel Hill). Transfected EA.hy926 cells were grown to confluence on 25-mm circular glass coverslips and mounted in a parallel-plate flow chamber. Flow medium was
drawn through the chamber at a rate of 804 µl/min with a syringe pump (Harvard Apparatus, Natick, MA), which generates
an estimated wall shear stress of 1.85 dynes/cm2. Cells (106 cells/ml)
were perfused through the chamber for a 10-min period. Cell
rolling was observed using an inverted phase-contrast microscope (Olympus Corporation, Lake Success, NY) and videotaped using
a CCD video camera (Hitachi Denshi, Ltd., Tokyo, Japan) with a
SuperVHS video recorder (model SVO-9500MD; Sony Corporation of America, New York, NY) and an attached time-date
generator (Microimage Video Sales Co., Bechtelsville, PA). Interacting cells (tethering and rolling) were determined by analysis
of videotapes in which four fields (0.16 mm2) on a video monitor
were counted at 14 random time points throughout the flow period. For calculating velocities, the distance each cell traveled between two time points was measured, converted into actual distance, and divided by the elapsed time.
 |
Results and Discussion |
Whether the cytoplasmic domain of L-selectin enhances
ligand binding through oligomerization was tested directly
by assessing the functional activity of induced cell-surface
L-selectin dimers. Although not previously tested for inducing dimerization of transmembrane proteins, the exogenous dimeric antibiotic coumermycin can cross-link and
activate cytoplasmic Raf-1-GyrB fusion proteins by simultaneously binding two GyrB subunits (10). cDNAs encoding L-selectin and a GyrB subunit were fused (L-Gb) and
expressed in 300.19 cells (Fig. 1), an L-selectin-negative
leukemia cell line that positions L-selectin at microvillus
tips when expressed (9). The interaction of L-selectin with
its ligand was assessed using a multivalent mannose-6 phosphate-rich polysaccharide mimetic, PPME, which can be
labeled to assess L-selectin binding activity without the influence of other adhesion receptors (3, 15). Cells expressing
L-Gb or wild-type L-selectin bound PPME similarly (Fig.
2 A, top). Coumermycin treatment significantly increased
the PPME binding activity of L-Gb expressing cells by
three- to five-fold but had no effect on wild-type L-selectin bearing cells (Fig. 2 A, bottom, P < 0.01). EDTA inhibited PPME binding by all cells, consistent with the involvement of L-selectin's calcium-dependent lectin domain.
Treatment of cells with the L-selectin function-blocking mAb, LAM1-3, also blocked PPME binding (data not
shown). Coumermycin enhancement of PPME binding
by L-Gb cells was dose dependent, maximal at 0.9 µM
coumermycin, rapid, and sustained (Fig. 2, B and C). Furthermore, pretreatment of L-Gb cells with novobiocin, the
monomeric coumermycin analogue (10), completely eliminated the coumermycin-induced effect (Fig. 2 D). Coumermycin-induced dimerization did not increase L-Gb expression levels or induce L-selectin endoproteolytic release
from the cell surface (Fig. 2 E), whereas phorbol esters induced L-Gb and wild-type L-selectin endoproteolytic
release similarly (data not shown). Coumermycin-induced L-selectin dimerization in L-Gb cells did not generate
transmembrane signals leading to upregulated intercellular
adhesion over a 24-h time period. Although the molecular
explanation for why cross-linking L-selectin with some
mAbs induces potent homotypic adhesion while coumermycin does not induce homotypic adhesion is not known,
cross-linking L-Gb or wild-type L-selectin on expressing
cells with appropriate anti-L-selectin mAbs induced potent
homotypic adhesion (data not shown) as previously described (16). Therefore, coumermycin-induced dimerization of L-Gb resulted in enhanced L-selectin binding activity specific for its ligand mimetic, PPME, which provides a
mechanistic explanation for leukocyte activation rapidly
upregulating L-selectin functional activity (3).

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Fig. 2.
Coumermycin-induced changes in PPME binding activity by
L-Gb-expressing 300.19 cells. (A) Immunofluorescence analysis of PPME
binding by cells expressing wild-type L-selectin or L-Gb, before and after
treatment with 0.9 µM coumermycin. After coumermycin treatment, the
cells were incubated with PPME in the presence of either Ca2+ or EDTA,
and PPME binding was assessed by fluorescence staining and flow cytometry analysis with results shown on a three-decade log scale. These results
represent those obtained in at least five experiments with the L-Gb clone
shown (Fig. 1 B) and are representative of results obtained with two independent clones of L-Gb-transfected cells. (B) Dose-response of coumermycin-induced PPME binding in L-Gb cells. Values represent the mean
fold increase in PPME binding relative to untreated cells obtained in four
experiments. Asterisk indicates significant differences between treated and
untreated samples, P < 0.01, Student's t test. (C) Time kinetics of coumermycin-induced PPME binding by L-Gb transfectants. The cells were treated
with 0.9 µM coumermycin for the indicated amounts of time before PPME
staining. Asterisk indicates significant differences between treated and untreated samples, P < 0.05. (D) Inhibition of coumermycin-induced PPME
binding by novobiocin. Cells were first treated with medium or the indicated amounts of novobiocin at 37°C for 15 min. Coumermycin (0.9 µM
final) was then added with PPME binding assessed 25 min later. (E) Effect of
coumermycin treatment on L-selectin expression. L-Gb cells were incubated
in media containing DMSO (0.1%), 0.9 or 9.0 µM coumermycin at 37°C
for the indicated time periods. The cells were washed and L-selectin expression was assessed as in Fig. 1. Values represent mean ± SEM fluorescence
channel numbers obtained in three experiments.
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The physiological importance of L-selectin dimerization
for leukocyte/endothelial interactions was assessed using in
vitro flow chambers that mimic vascular flow conditions in
vivo. L-selectin supports leukocyte rolling on a monolayer
of transformed HUVEC that express L-selectin ligand(s)
(14, 17). Both L-Gb and wild-type L-selectin mediated a
basal level of 300.19 cell rolling (Fig. 3, A and C), but neither cell type arrested on these HUVEC monolayers due to
the absence of other operable adhesion molecules expressed
by this cell line (data not shown). L-Gb-mediated rolling of cells on HUVEC monolayers was significantly enhanced
(by >700%, P < 0.002, n = 3 experiments) by coumermycin treatment, which was competitively inhibited by
novobiocin pretreatment (Fig. 3 A). All rolling on HUVEC
monolayers was completely blocked by the LAM1-3 mAb
(Fig. 3, A and C). Remarkably, coumermycin treatment also significantly lowered rolling cell velocities by 35%,
from a median (±SEM) rolling velocity of 272 ± 9 to 175 ± 12 µm/s (P < 0.001; n = 3, Fig. 3 B). Therefore,
L-selectin dimerization markedly increased the number of
leukocytes rolling under conditions of physiologic shear
stress.

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Fig. 3.
L-selectin dimerization enhances leukocyte rolling on endothelial cells under physiologic flow. (A) Effect of coumermycin and novobiocin treatments on the number of L-Gb cells rolling on a HUVEC
monolayer in an in vitro flow chamber assay. Values represent the number of L-Gb cells interacting with HUVEC monolayers in a 0.16-mm2
field. Asterisk indicates significant differences from all other groups, P < 0.01. (B) Effect of coumermycin (0.9 µM) on rolling velocities of L-Gb
cells interacting with a HUVEC monolayer. Each symbol represents the
velocity of an individual cell plotted in rank order with median (50%) velocities indicated by horizontal and vertical lines. (C) Effect of anti-
L-selectin mAbs on the number of wild-type or L-Gb cells rolling on
HUVEC monolayers. (D) Effect of anti-L-selectin mAbs on the rolling
velocities of L-Gb cells interacting with HUVEC monolayers. A and C
values are mean ± SEM of results obtained in three experiments, and B
and D values are representative of results obtained in three experiments.
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|
To further verify that L-selectin dimerization enhances
its functional activity, a panel of IgG mAbs was screened to
identify those that cross-linked L-selectin extracellular domains in a functionally appropriate configuration while not
blocking ligand binding or inducing transmembrane signals
(16). Of 32 mAbs screened, the LAM1-118 mAb reactive
with the short consensus repeat domains of L-selectin fit
these criteria. LAM1-118 mAb treatment of wild-type L-selectin or L-Gb-bearing cells significantly increased the
frequency (310 and 350% increase, respectively; P < 0.01)
of cells interacting with HUVEC monolayers under physiologic flow conditions (Fig. 3 C). This was consistent with
an increase in high endothelial venule binding by lymphocytes pretreated with this mAb (16). As with coumermycin
treatment of L-Gb cells, LAM1-118 mAb treatment significantly lowered rolling cell velocities by 35%, from a median (±SEM) of 266 ± 3 to 172 ± 6 µm/s (P < 0.001, n = 3; Fig. 3 D). LAM1-118 mAb treatment of wild-type
L-selectin-bearing cells lowered rolling cell velocities similarly (data not shown). LAM1-118 mAb treatment of L-Gb
or wild-type L-selectin transfected cells did not increase or
decrease receptor expression levels (data not shown), and
did not induce homotypic adhesion of 300.19 cells (16).
Furthermore, treatment of wild-type or L-Gb L-selectin- bearing cells with LAM1-101, an isotype-matched mAb
that binds the epidermal growth factor-like/short consensus repeat domains of L-selectin, had no significant effect
on cell attachment or rolling velocities (Fig. 3, C and D).
Neither 300.19 cells alone nor wild-type L-selectin-
expressing cells treated with the LAM1-3 mAb showed any
detectable interaction with the HUVEC monolayers (Fig.
3 C). Therefore, coumermycin-mediated cross-linking of
L-selectin cytoplasmic domains and mAb-mediated cross-linking of the L-selectin extracellular domains both generated identical results under physiological flow conditions.
L-selectin binds its ligands with rapid association and dissociation rates, which results in a minimal shear stress
threshold requirement for the initiation and maintenance
of leukocyte rolling (18). Despite the coumermycin-
induced increase in L-selectin binding activity, both coumermycin-treated and -untreated L-Gb cells required shear
stress for the promotion of L-selectin-dependent interactions since no tethering or rolling was observed below wall
shear stresses of 0.75 dynes/cm2 (Fig. 4). Nonetheless,
coumermycin-treated L-Gb cells interacted with HUVEC
monolayers at a significantly greater frequency compared with untreated L-Gb cells at shear stresses between 0.75 and 3.0 dynes/cm2 (Fig. 4). Thus, L-selectin dimerization
influences rolling velocity and receptor detachment rates as
opposed to selectin localization to the tips of microvilli,
which appears to promote cell capture (5). L-selectin
dimerization may retard the dissociation of selectin bonds,
which would enhance the lifetime of L-selectin binding to
its endothelial ligand(s) and promote leukocyte tethering to
endothelium during rolling.

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Fig. 4.
L-selectin dimerization enhances leukocyte attachment and rolling on endothelial
cells under physiologic flow.
Wall shear stress was varied at
1-min intervals by changing the
flow rate through the flow
chamber. Asterisk indicates coumermycin-treated cells that were
significantly different from untreated cells, P < 0.05. Values
represent means ± SEM of results from three experiments.
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|
That inducing L-selectin dimerization by either coumermycin or mAbs can enhance L-selectin adhesion is consistent with a model in which the transient activation-
enhanced adhesive function of L-selectin results from receptor
dimerization. However, these results do not exclude other
mechanisms for transiently enhancing the adhesive function
of L-selectin and do not necessarily prove that L-selectin
dimerization is a physiologic process. Nonetheless, the formation of cell-surface dimers may also provide a mechanistic explanation of why L-selectin-mediated tethers operate
at high shear forces (22). Thus, the rapid upregulation of
L-selectin binding activity after leukocyte activation and
L-selectin dimerization may stabilize L-selectin bonds under shear force, which facilitates the formation of a second
receptor/ligand bond before the first one breaks during
rolling. Multivalent L-selectin binding would also distribute the tensile force applied on each tether among several
L-selectin/ligand bonds. Therefore, L-selectin's cytoplasmic domain and cytoskeletal associations may be required
for its oligomerization within the cell membrane, providing it with strong resistance to shear stresses. Whether
dimerization also regulates P- and E-selectin function is
unknown, although P-selectin isolated from activated
platelets is found in a tetrameric configuration, which facilitates its binding activity in vitro (23).
Since leukocyte rolling may involve multivalent binding
(22), rapid oligomerization of L-selectin would favor the
formation of multivalent bonds with its low affinity endothelial cell ligands (24). Consistent with this notion, L-selectin ligands consist of multimeric sialylated and sulfated oligosaccharides appropriately presented by mucin scaffolds
(1). As such, oligomerized L-selectin molecules may interact cooperatively with ligands presenting multiple low affinity oligosaccharide binding sites that are optimally stabilized by multivalent bonding. This is consistent with the many animal lectins that dramatically increase their affinity for carbohydrate ligands by combining multiple oligosaccharide binding sites in each lectin polypeptide (25). However, in the case of L-selectin the generation of multimeric
binding by receptor oligomerization may provide a rapid
means for upregulating adhesion receptor function with
leukocyte activation. In addition, dimerization may be particularly important when L-selectin or its ligands are expressed at low site densities (26). Therefore, this study supports the notion that selectin oligomerization is of primary physiologic significance and is likely to directly influence
leukocyte migration and entry into sites of inflammation.
Moreover, the coumermycin-GyrB dimerization strategy is
likely to be useful for studying other transmembrane proteins
and adhesion molecules that share the property of being
functionally upregulated in response to cellular activation.
Address correspondence to Thomas Tedder, Box 3010, Department of Immunology, Rm. 353 Jones Bldg.,
Research Dr., Duke University Medical Center, Durham, NC 27710. Phone: 919-684-3578; Fax: 919-684-8982; E-mail: tedde003{at}mc.duke.edu
We thank Drs. M.D. Delahunty, S.D. Rosen, F.W. Luscinskas, L. Robinson, M. Inaoki, and X.-Q. Zhang
for reagents and help with these experiments.
This work was supported by National Institutes of Health grants AI-26872, CA-54464, and HL-50985.
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[Abstract/Free Full Text].
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