The Center for Blood Research, and Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115
Two mechanisms have been proposed for regulating rolling velocities on selectins. These are (a) the intrinsic kinetics of bond dissociation, and (b) the reactive compliance, i.e., the susceptibility of the bond dissociation reaction to applied force. To determine which of these mechanisms explains the 7.5-11.5-fold faster rolling of leukocytes on L-selectin than on E- and P-selectins, we have compared the three selectins by examining the dissociation of transient tethers. We find that the intrinsic kinetics for tether bond dissociation are 7-10-fold more rapid for L-selectin than for E- and P-selectins, and are proportional to the rolling velocities through these selectins. The durations of pauses during rolling correspond to the duration of transient tethers on low density substrates. Moreover, applied force increases dissociation kinetics less for L-selectin than for E- and P-selectins, demonstrating that reactive compliance is not responsible for the faster rolling through L-selectin. Further measurements provide a biochemical and biophysical framework for understanding the molecular basis of rolling. Displacements of tethered cells during flow reversal, and measurements of the distance between successive pauses during rolling provide estimates of the length of a tether and the length of the adhesive contact zone, and suggest that rolling occurs with as few as two tethers per contact zone. Tether bond lifetime is an exponential function of the force on the bond, and the upper limit for the tether bond spring constant is of the same order of magnitude as the estimated elastic spring constant of the lectin-EGF unit. Shear uniquely enhances the rate of L-selectin transient tether formation, and conversion of tethers to rolling adhesions, providing further understanding of the shear threshold requirement for rolling through L-selectin.
BINDING OF SELECTINS TO CELL-SURFACE CARBOHYDRATE
LIGANDS ENABLES LEUKOCYTES THAT ARE FREE IN VASCULAR
SHEAR FLOW TO TETHER TO VESSEL WALLS AND TO ROLL IN
RESPONSE TO HYDRODYNAMIC DRAG. ROLLING IS A REMARKABLE
CLASS OF ADHESIVE INTERACTION BECAUSE THE ZONE OF ADHESIVE
CONTACT IS RAPIDLY TRANSLATED ALONG THE VESSEL WALL. ONLY
CERTAIN ADHESION MOLECULES ARE SPECIALIZED TO SUPPORT ROLLING (46). L-SELECTIN, EXPRESSED ON LEUKOCYTES, BINDS TO CARBOHYDRATE LIGANDS THAT ARE EXPRESSED ON HIGH ENDOTHELIAL
VENULES (HEVS) OF SECONDARY LYMPHOID TISSUES (5) AS WELL
AS ON CERTAIN TYPES OF LEUKOCYTES (2, 15). THE LIGAND FOR
L-SELECTIN IS A SULFATED SLEX-related carbohydrate (21, 22).
This carbohydrate ligand on HEV is expressed on a mixture of mucinlike sialoglycoproteins termed peripheral
node addressin (PNAd)1. PNAd and various components
of PNAd including CD34 have been shown to support rolling of L-selectin-expressing leukocytes in hydrodynamic
flow (6, 28, 39). The vascular selectins, P-selectin and E-selectin, can be induced on endothelium by inflammatory mediators, and both bind to carbohydrate ligands on myeloid
cells and subsets of lymphocytes (31, 42). The major P-selectin counterreceptor on human leukocytes, P-selectin glycoprotein ligand-1 (PSGL-1), is a mucin decorated with
sLex (33, 43) and with sulfated tyrosines (37, 44, 55). Both
L-selectin (36) and PSGL-1 (33) on neutrophils are concentrated on the tips of microvilli, where they are likely to
support the initial contact with the vessel wall under physiological flow.
Although bonds between selectins and their counterreceptors must be rapidly formed and broken to support
translation of the adhesive contact zone during rolling,
there are two markedly different mechanisms by which
rolling velocity can be regulated. These are (a) intrinsic
bond kinetics, i.e., kinetics in the absence of applied force;
and (b) the susceptibility of bond kinetics to applied force,
which is termed reactive compliance. Tensile force is generally expected to increase the rate of bond dissociation, but the amount of increase, i.e., reactive compliance, may
differ markedly for different types of adhesion molecules.
According to one view, rapid bond dissociation kinetics
are required for rolling (1, 25), and kinetic differences may
underlie the 7.5-11.5-fold faster rolling through L-selectin
than through E-selectin or P-selectin when ligand densities
are adjusted to give rolling adhesions of similar strength
(40). According to another view, the susceptibility of bond
kinetics to force may be the dominant factor in regulating
rolling velocity and the intrinsic bond dissociation rate
constant, i.e., koff in the absence of force, or koffo, may be
so slow that bond dissociation requires 0.5-5 h (9, 19, 49).
However, reactive compliance is predicted to be variable only within a certain range; if bonds were highly compliant,
force would shorten bond lifetime so much that tethering
and rolling could not be supported (9, 19).
In this paper, we have investigated the basis for the
markedly faster rolling through L-selectin than E- or P-selectins (40), and tested the relative contributions to rolling
velocities on selectins of intrinsic bond dissociation kinetics and the compliance of the bond dissociation reaction.
We have examined transient tethers on PNAd and E-selectin. Transient tethers have previously been studied on P-selectin (1), and occur when the densities of selectins or their
ligands on the wall of a flow chamber are too low to support rolling. Transient tethers are the smallest unit of adhesive interaction that is observable in shear flow. Transient tethers have properties that suggest but do not prove that they represent single selectin-ligand bonds. We find
that the intrinsic kinetics of dissociation of transient tethers correlates with rolling velocities for L-, E-, and P-selectins. Furthermore, we find that the L-selectin tether bonds
are less sensitive to applied force than E-selectin or P-selectin tether bonds, which is opposite to what would be expected if bond compliance dominated the differences between the selectins in rolling velocity. The exponential
constants that quantitate the effect of force on bond dissociation are interesting quantities in themselves. These constants are compared to estimates of the elastic spring constant for selectins.
Rolling cells do not move smoothly, but advance in a series of jerky movements that may reflect bond dissociation
events (8, 25). New bonds to the substrate are thought to
be formed after each step forward, but forward motion is
more likely to be restrained by previously formed bonds,
which after cell movement find themselves at the rear of
the contact zone, than by newly formed bonds. We have
examined a number of parameters of cell rolling through
L-selectin that are essential to understanding its molecular
and cellular basis, and to relating transient tether dissociation to rolling velocity. Transient tethers are a uniform
population with regard to dissociation kinetics, and therefore may be considered quantal units (1). To determine
whether the same quantal tether dissociation units underlie the jerky rolling behavior of leukocytes in shear flow,
pauses in rolling were measured and were found to have
the same time scale as transient tethers. To measure biophysical parameters including the relationship between
force on a cell and force on a tether bond, the length of the
tether, and the dimension of the adhesive contact zone between the cell and the substrate, we measured the distance
that transiently tethered cells pivot when the flow direction is reversed. Furthermore, we relate this distance to
the distance moved between pauses during rolling. We
show that as few as two tether bonds to the substrate are
sufficient to support rolling.
Adhesive interactions through L-selectin have the remarkable property that they require shear above a threshold value for their promotion and maintenance (13, 29).
Rolling leukocytes accumulate on PNAd above but not
below this shear threshold of ~0.4 dyn/cm2. Furthermore,
when leukocytes rolling on PNAd or on monolayers of adherent leukocytes above the shear threshold are subjected to a rapid decrease in shear to below the threshold, in less
than 1 s they cease to interact with the substrate and move
at the hydrodynamic velocity. When shear is raised above
the threshold, leukocytes rapidly resume rolling interactions. The shear threshold phenomenon is also observed in
vivo (13), and is hypothesized to help prevent inappropriate accumulation of leukocytes in vessels with inherently
low wall shear rates and in hypoperfusion. Investigation of
the kinetics of L-selectin tethers, and of rolling on L-selectin, particularly around the shear threshold, shed further
light on this mysterious phenomenon.
Monoclonal Antibodies
mAbs used in this study as purified Igs were the anti-L-selectin mAb
DREG 56 (24), mAb MECA-79 (47), and mAb 581 to CD34 (39). Control
IgG were X63 myeloma IgG1 and the anti-CD44 mAb, A3D8 (20).
Preparation of Substrates
Human PNAd was purified from tonsil lysates by MECA-79 mAb affinity
chromatography as previously described (5, 39). PNAd aliquots (0.001-1
µg/ml in PBS/0.5% octyl glycoside) were diluted 1:10 in PBS/10 mM sodium bicarbonate, pH 8.0, and 25 µl was immediately spotted onto polystyrene dishes for 3 h at 24°C. Substrates were washed and quenched with
2% human serum albumin (HSA) (fraction V; Calbiochem-Novabiochem
Corp., La Jolla, CA). The site density of CD34 in the PNAd-coated substrates was determined by saturation binding of radiolabeled anti-CD34
mAb (39). CD34 contributes ~50% of the total L-selectin tethering and
rolling activity in human PNAd (39); therefore the densities given in the
text for PNAd are twofold that determined for CD34. For example, a final concentration of 0.45 µg/ml PNAd yielded 90 sites per µm2 of CD34 and
therefore the estimated density of PNAd was 180 sites per µm2. Site densities at low input concentrations ( Isolation of Leukocytes
Human neutrophils were isolated from citrate anticoagulated whole blood
by dextran sedimentation and density separation over Ficoll-Hypaque
(32). Leukocytes were stored in Mg2+- and Ca2+-free HBSS, containing 10 mM Hepes, pH 7.4, for up to 2 h at 24°C.
Shear Flow Assays
PNAd substrates were assembled as the lower wall in the flow chamber
and mounted on an inverted phase-contrast microscope (25, 26). Leukocytes were resuspended in binding medium (HBSS/Hepes containing 2 mM Ca2+ and 2 mg/ml HSA) and perfused through the chamber at different flow rates to obtain the indicated shear stresses at the chamber wall
(25, 29).
For inhibition studies, cells were preincubated for 5 min in binding medium with 0.3-3 µg/ml of L-selectin blocking mAb or control mAbs. The
cell suspension was perfused into the flow chamber without washing out
the antibodies. Blockade of L-selectin function was also carried out by
perfusing cells in the presence of 5 mM EDTA or 50 µg/ml fucoidan. For
comparisons of interactions in the presence or absence of inhibition, at
different shear stresses, or with different cell types, identical fields of view
were used to ensure that the results reflected uniform site density and distribution of the immobilized adhesive proteins on the substrate. Between
each comparison, any adherent cells were removed by perfusion with
binding medium supplemented with 5 mM EDTA.
Determination of Cell Displacements
Images from Nikon plan ×20 or ×40 objectives and a TEC-470 CCD
video camera (Optronics, Goleta, CA) were recorded and played back on
a Mitsubishi S-VHS (U-67) recorder. Motions of neutrophils were analyzed by determination of cell center positions in successive video frames.
Coordinates of cell centers were determined to an accuracy of ±0.68 µm
and 0.34 µm with ×20 and ×40 objectives, respectively, and the velocity
between each frame of individual cells was calculated.
Determination of Duration of Transient
Tethering Events
Transient tethers were defined as cell attachment events separated by at
least 50 µm of motion at the hydrodynamic velocity, and when no cell motion (<2 µm displacement) occurred while the cell was tethered to the immobilized ligand (1). The duration of transient tethers on PNAd was calculated by counting both the frames during which cells were motionless
and the fraction of the immediately preceding and succeeding frames for
which cells were tethered. The distance moved in these frames divided by
the distance moved between frames by a freely moving cell yielded the
fraction of time the cell was not tethered in the preceding and succeeding
frames. Using the latter technique, tethering events lasting even less than
one frame could be reliably detected, with proportionately higher accuracy at higher shear because of the greater difference between cell position in succeeding frames. The detection limit was ~0.01 s at 1.5 dyn/cm2.
The durations of transient tethers on P-selectin were measured in an integral number of frames (1). Sufficient videotape was analyzed (0.5-2 min)
to obtain 30-40 tethering events, and the natural log of the number of cells
that remained bound as a function of time after initiation of tethering was
plotted. The slope = Measurement of the Lever Arm Acting on a Tether
Neutrophils were allowed to interact in flow with P-selectin reconstituted
in a lipid bilayer on a glass slide (1, 25). Flow was controlled by switching a
pressurized air reservoir between the two ends of the flow chamber with
an air valve. Shear stress was calibrated from the hydrodynamic velocity
of nonadherent cells perfused at different flow rates in the same chamber
using a syringe pump. The lever arm is defined as half the distance moved
during flow reversal while cells were tethered to P-selectin.
The Micromotion of Neutrophils During Rolling
on PNAd
To examine the jerky movements during rolling in hydrodynamic flow that are thought to occur when selectin-
ligand bonds dissociate, we studied motion on a time scale
smaller than selectin bond lifetimes, i.e., between each
0.033-s video frame (Fig. 1, A and B). Densities of PNAd
(60 sites per µm2; Fig. 1 A) and P-selectin (30 sites per
µm2; Fig. 1 B) were just above the threshold required to
support rolling, in order to minimize the number of selectin-ligand bonds between the rolling leukocyte and the substrate, and thereby maximize the effect of individual bonds
on behavior. The average rolling velocity was much faster
through L-selectin on PNAd (Fig. 1 A) than through PSGL-1
on P-selectin (Fig. 1 B). Neutrophils paused during rolling
on both substrates, but the pauses were of markedly longer duration on P-selectin than PNAd, correlating with
the slower rolling velocity on P-selectin. The five pauses
on PNAd (Fig. 1 A) were 0.073 ± 0.026 s (mean ± SD) and
the six pauses on P-selectin (Fig. 1 B) were 0.30 ± 0.14 s.
Previous studies have shown that rolling through L-selectin on PNAd requires a wall shear stress above a threshold
value of ~0.4 dyn/cm2 (13); therefore, we examined micromotion around the shear threshold. At 0.3 dyn/cm2, there
was no rolling, and only transient tethers were seen. At
0.375 dyn/cm2, a transitional type of rolling behavior was
seen. Rolling occurred in relatively short runs, a longer
representative of which is shown in Fig. 2 A. Neutrophils
rolled with pauses of 0.11 ± 0.051 s (n = 19). A remarkable feature of these pauses was their separation by leukocyte displacements that were regular in length. The modal displacement between pauses at 0.375 dyn/cm2 was 3-4 µm
(Fig. 2 D). This displacement represents the distance between successive tethers to the substrate. At 0.45 and 1.05 dyn/cm2, neutrophils rolled for substantially longer times
per run, and there was a broader distribution of displacement between pauses, including many movements of 1-2
µm (Fig. 2, B-D). The duration of pauses during rolling at
0.45 dyn/cm2 was 0.113 ± 0.077 s, and at 1.05 dyn/cm2 was
0.094 ± 0.026 s (Fig. 2, B and C). At 0.3 dyn/cm2, tethers
were separated by >10 µm, consistent with the lack of rolling (Fig. 2 D).
Transient Tethers on PNAd
At PNAd densities lower than 60 sites per µm2, rolling became faster and jerkier. At 20 sites per µm2, tethered neutrophils failed to continuously roll on PNAd (Fig. 3, cell 2),
and below this density, tethering became transient and was
separated by distances over which cells traveled at the hydrodynamic velocity (Fig. 3, cells 3 and 4). Neutrophil tethering to PNAd was highly specific, since it was completely
inhibited by pretreatment of cells with L-selectin mAb,
EDTA (Fig. 3, cell 1), soluble fucoidan, or pretreatment of
the substrate with O-sialoglycoprotease to degrade mucinlike constituents of PNAd (data not shown) (39).
The efficiency of transient tethers was studied on substrate densities below the density required to support rolling (Fig. 4 A). Tethering efficiency decreased at higher
shear stresses on both P-selectin and PNAd substrates and
declined to zero at 1.8 dyn/cm2 on P-selectin and at 2.5 dyn/cm2 on PNAd. Extraction from the substrate did not
appear to occur, because the upper shear limit for tethering was identical whether P-selectin was adsorbed to plastic or incorporated in lipid bilayers supported on glass
(Fig. 4 A). Below, we describe the kinetics of transient
tethers. At the highest shear stresses at which transient tethers occurred, their lifetimes were well within the measurable range of >0.01 s, and were 0.2 s for P-selectin and
0.04 s for L-selectin. The finding that the lifetime of transient tethers through L-selectin is much shorter than
through PSGL-1 (see below), despite the observation that
L-selectin tethers withstand higher shear stresses, argues
that the kinetics are unrelated to extraction and are due to
receptor-ligand dissociation.
We examined transient tether efficiency around the
shear threshold for rolling on L-selectin. Transient tethers
occurred at 0.30 dyn/cm2 (Fig. 2 D) and 0.35 dyn/cm2 (Fig.
4). Thus, transient tethers occur at wall shear stresses below the threshold, where rolling does not occur. However,
transient tether efficiency on PNAd at wall shear stresses
of 0.35 and 0.6 dyn/cm2 was lower than that at 0.75-1.5
dyn/cm2 (Fig. 4). This drop occurred despite correction for
the lower cell flux at lower wall shear stresses, i.e., with
tethering efficiency expressed as a function of the cell
transport distance across the substrate. By contrast, tethering efficiency on P-selectin substrates was higher at 0.3 dyn/cm2 than at 0.75 dyn/cm2 (Fig. 4). On PNAd, the percentage of tethers that resulted in rolling adhesions, as opposed to transient tethers, increased dramatically between
0.45 and 0.75 dyn/cm2 at 60 sites per µm2 (Fig. 5). At 20 and 4 PNAd sites per µm2, little or no rolling adhesions
occurred at any shear (Fig. 5). Thus, a density of PNAd
above 20 sites per µm2 and shear stress above about 0.45 dyn/cm2 are required for tethers to progress to rolling adhesions. In conclusion, shear enhances both the density of
transient tethers and the conversion of transient tethers
events on the substrate to multivalent tethers that can support rolling.
First Order Kinetics of Dissociation of Transient
Tethers on PNAd
The duration of transient tethers on PNAd was measured
for multiple cells. Synchronizing the initiation of tethering
to t = 0, transient tethers were seen to dissociate with first
order kinetics (Fig. 6 A). The data fit a single straight line
with a high correlation coefficient, and koff was independent of PNAd density (Figs. 6, A-C). L-selectin tethers
dissociated from PNAd markedly faster than P-selectin
tethers at 0.75 dyn/cm2 (Fig. 6 A). The koff on PNAd at
0.75 dyn/cm2 corresponded to a tether lifetime (the reciprocal of koff) of 0.09 s. This lifetime is comparable to the
duration of pauses seen above in rolling on PNAd. The
homogeneity in the dissociation rate constant, as shown by
fit to a straight line and lack of dependence on PNAd density at 12 sites per µm2 and below, suggested that we were
measuring the smallest detectable unit of binding between
the cell and the substrate, and that this binding unit was
homogeneous or quantal. In evidence against a contribution of multiple independent receptor-ligand interactions to transient tethers, partial saturation with an inhibitory
mAb to L-selectin, which reduced the frequency of tethers
by 70%, or inclusion of the L-selectin antagonists fucoidan
and mannose-6-phosphate, had no effect on koff (Fig. 6 B;
and data not shown). Moreover, partial desialylation of
the PNAd immobilized in the flow chamber with neuraminidase, which reduced the frequency of transient tethers by 77%, had no significant effect on koff (not shown).
The dissociation rate constant for the L-selectin-PNAd
tether increased as shear was increased (Figs. 6 C and 7 A).
For comparison, we examined transient tethers of neutrophils on E-selectin at seven sites per µm2. Transient
tethers required Ca2+ and were sensitive to neuraminidase
treatment of neutrophils. Like L-selectin tethers, dissociation of E-selectin tethers showed first order kinetics with a
single rate constant, which was independent of density
when E-selectin was present at 10 sites per µm2 or below.
As with other selectins, the koff of transient E-selectin tethers increased as shear was increased (Fig. 7 B). E-selectin tethers had a longer lifetime than L-selectin tethers.
Parameters of the Adhesive Contact Zone
An understanding of the mechanics of transient tethers
and rolling requires knowledge of where the cell is tethered to the substrate (Fig. 8, A and B). Therefore, the direction of flow was reversed every few seconds during
transient tethering experiments. Measurements were made
on P-selectin substrates, since the short lifetime made
measurements on PNAd impractical; however, tether parameters are expected to be similar or identical for L-selectin because of its colocalization with the P-selectin ligand
PSGL-1 on microvilli tips (33, 36). Thus, tethers to both
PNAd and P-selectin are predicted to be through microvilli tips. When, by chance, flow reversal occurred while
a cell was transiently tethered, the distance moved by the
cell was measured (Fig. 8 C). Cells that had transiently
tethered as shown by lack of movement between one or
more successive video frames (Fig. 8 C, 0.333-0.733 s)
were observed to move during flow reversal, to then again
remain stationary for one or more successive video frames
(Fig. 8 C, 0.933-1.633 s), and to subsequently dissociate
and move at the hydrodynamic velocity (Fig. 8 C, 1.833-
2.000 s). The lever arm is the distance between (a) the
point on the substrate where the adhesion molecule is attached to which the cell is tethered, and (b) the projection
of the center of the cell on the substrate, and is equal to
half the distance the cell moves during flow reversal (Fig. 8
A). Measurements on 27 tether events that coincided with flow reversal (Fig. 8 D) showed a mean ± SD of 3.06 ± 0.53 µm for the length of the lever arm. This length was independent of shear from 0.3 to 0.8 dyn/cm2 (Fig. 8 D). No
change in cell dimension in any direction within the plane
of focus was visible microscopically during tethering or
flow reversal. The distance moved during flow reversal defines the maximal length of the adhesive contact zone, because a cell would be predicted to be capable of interacting with the substrate between the tether point and the
projection of the center of the cell on the substrate, and at
most for an equal distance on the other side of the projection of the center of the cell (Fig. 8 A). Using the lever arm
length and the model of a spherical nondeformable cell
(8.5 µm in diam) with a tether that is flexible where it joins
the cell body, and balancing the forces and torques on a tethered cell in shear flow, the length of the tether (d) can be estimated to be 1.00 ± 0.32 µm (Fig. 8 B). This length
may include microvillus and adhesion molecule lengths, as
well as deviations of the cell from its idealization as a nondeformable sphere.
Both tether koff and the time required for a cell to move
between successive tethers, i.e., to pivot about a tether,
will affect rolling velocity. Therefore, we determined the
time required for transiently tethered cells to move the
distance of 2 l (l is the distance between the tether point
and the projection of the center of the neutrophil on the
substrate) after flow reversal (Fig. 8 E). This took approximately twice as long as for untethered cells in the same
field of view and focal plane, perhaps because slippage and
rotation of tethered cells relative to the substrate in shear
(17) is retarded by the tether.
The Intrinsic koff and Effect of Force on koff
for Selectins
The lever arm measurement allows the relationship between wall shear stress and force on the tether bond to be
estimated. Given the lever arm length, the force on the
tether bond (Fb), can be calculated to be 2.15 ± 0.47 times
the hydrodynamic drag force on the cell (Fs) (Fig. 8 B).
Using the solution for Fs for a sphere near a wall in shear
flow (17), the relationship between wall shear stress and Fb
can be estimated (Fig. 8, legend). Thus, data on koff for
L-selectin and E-selectin determined here and for P-selectin (1) can be plotted as a function of wall shear stress and
the estimated force on the tether bond (Fig. 9). There are
several important conclusions: first, the koff for L-selectin is markedly faster than that for the vascular E- and P-selectins at all forces examined, and when extrapolated to zero
shear stress to yield koff° (unstressed koff; Fig. 9). Therefore, the faster koff of L-selectin will make an important
contribution to its faster rolling velocity. Second, the increase in koff with increased force, i.e., the mechanical
property of reactive compliance, is not related to differences in rolling velocity between selectins. To quantitate
reactive compliance, all data were fit to Bell's equation, in
which the potential energy stored in the bond increases linearly with the applied force and is proportional to
To understand the molecular basis of selectin-mediated
rolling, we have examined the kinetic and mechanical
properties of the transient tether, and its relationship to
properties of rolling adhesions including velocity, pause
duration, and step length. We have focused on L-selectin
because rolling through L-selectin is faster in velocity than
through the vascular selectins and exhibits a shear threshold dependence (13, 29). We have compared the intrinsic
koff and mechanical properties of L-, P-, and E-selectins by
studying the dissociation of transient tethers. In all cases, dissociation of transient tethers followed first order kinetics and was independent of ligand density over a wide
range of densities below the threshold required to support
rolling. These properties suggest but do not prove that
transient tethers represent the formation and dissociation
of single receptor-ligand bonds. Elsewhere, we have directly compared rolling velocities on PNAd, P-selectin, and
E-selectin at densities on the substrate that were similar, and that gave rolling adhesions of equal strength, as shown
by resistance to detachment by increased shear. Over a
wide range of wall shear stresses, rolling through L-selectin
was 7.5-9-fold faster than on P-selectin, and 8-11.5-fold
faster than on E-selectin (40). An important question addressed in the current study was the relative importance of
the intrinsic bond dissociation rate constant, koff°, and the
susceptibility of koff to applied force, i.e., reactive compliance, in determining the more rapid rolling velocity through
the leukocyte L-selectin molecule than through the vascular E- and P-selectin molecules.
Biologically, L-selectin appears specialized to mediate
fast interactions that generally represent the initial event
in adhesion cascades. L-selectin nucleates leukocyte-leukocyte interactions (2) that subsequently lead to slower
rolling on E- and P-selectins (1, 53) and firm adhesion
through leukocyte integrins (41). Similarly, rolling interactions through L-selectin appear to precede slower rolling
through mucosal cell adhesion molecule-1 (MAdCAM-1) in Peyer's patches (3). Fast rolling through L-selectin occurs across species including rabbit (50), mouse (6), cow
(2), and human (28); thus it appears from its evolutionary
conservation to be biologically important. We have not examined the association rate constant, kon, because this requires knowledge of the concentration and diffusion of the
selectin and the ligand in the adhesive contact zone, and
may also be affected by transport of the cell by shear flow
and the shear threshold phenomenon. Maintenance of stable rolling requires that the average number of bonds that
are formed and broken be equal. The faster rate of tether
dissociation through L-selectin than through E- and P-selectins requires an equally faster overall rate of L-selectin
tether bond formation, and if the effective concentrations
of reactants were identical, this would require a faster kon.
Thus, while our investigations have focused on koff , they
have implications for the overall rate of bond formation,
and for understanding the basis for the fast interactions
through L-selectin that are important in initiating adhesion cascades in the vasculature.
Both koff° and mechanical properties must be important
for selectin-mediated rolling. Reactive compliance has
been predicted to be low to obtain conditions that are permissive for rolling (9, 19). Reactive compliance in the
Hookean spring model is inversely related to the exponential constant By contrast with koff°, we found that mechanical compliance, i.e., We have recently measured the L-selectin-carbohydrate interaction in the other direction, i.e., with L-selectin
adsorbed to the substrate interacting with a carbohydrate
ligand for L-selectin that is expressed on the surface of
neutrophils (Alon, R., S. Chen, R. Fuhlbrigge, K.D. Puri,
and T.A. Springer, manuscript in preparation). Despite
the difference in direction and nature of the carbohydrate
ligand, the kinetics (koff° = 7.0 ± 0.5 s To demonstrate a relationship between tether koff and
rolling velocity, it was important to examine the kinetics of
rolling on a time scale shorter than the tether lifetime. The
duration of pauses during rolling was measured on low
densities of PNAd and P-selectin, and near the shear threshold on PNAd, to minimize the number of receptor-ligand
bonds and hence to accentuate the importance of dissociation of individual tethers. At a wall shear stress of 1.5-1.8
dyn/cm2, the duration of pauses on P-selectin of 0.30 ± 0.14 s was longer than on PNAd of 0.073 ± 0.026 s, correlating with the longer lifetime of P-selectin than PNAd
tethers. On PNAd at 1.5 dyn/cm2, pause duration during
rolling was 0.073 ± 0.026 s compared to a transient tether
lifetime of 0.061 s. On PNAd at 0.375 and 0.45 dyn/cm2,
pause durations were 0.11 ± 0.051 and 0.113 ± 0.077 s
compared to tether lifetimes of 0.121 and 0.116 s at these
shear stresses, respectively. This close agreement between
transient tether lifetime and rolling pause duration provides strong evidence that pauses during rolling through
L-selectin on PNAd at the tested shear stresses correspond to the time required for the dissociation of tether
bonds, and that movements during rolling reflect dissociation of tether bonds. Further support for this comes from a
comparison of the estimate for the koff of E-selectin of 0.5 s Measurement of the distance that transiently tethered
cells moved during flow reversal yielded information on the
geometry of the contact region between the cell and the
substrate. The lever arm distance of 3.06 ± 0.53 µm yields
an estimate of 1.0 ± 0.32 µm for the length of the tether
between the cell and the substrate, including both adhesion molecule and microvillus lengths and the effects of cell
roughness and deformation. This is in reasonable agreement with recent estimates by a different measurement technique (45). Furthermore, the dimension of the adhesive
contact zone in the direction parallel to flow can be estimated to be twice the lever arm distance, 6.12 ± 1.06 µm.
On PNAd at 0.375 dyn/cm2, near the shear threshold
where the number of tethers formed to the substrate is
predicted to be the minimum required to support rolling,
the step distance was found to be unimodal, with a peak at
3-4 µm. The close correspondence between the step distance of 3-4 µm and the lever arm distance suggests that
when a cell is restrained by a tether, the most favored location for the formation of the next tether is under the center of the cell. Dividing the dimension of the contact zone
of 6 µm by the step distance of 3 µm during rolling on low
densities of PNAd near the shear threshold yields an estimate of two tethers per contact zone. Two tethers is the
minimum required to support rolling; one tether would
give a transient tether. The minimum number of tethers
was expected under these conditions, near the transition
between rolling and transient tethering. The results support the idea that rolling can be supported by a small number of bonds between the cell and the substrate (25). Several models assume a large number of receptor-ligand
bonds (9, 49), whereas one finds that the number of bonds
never exceeds 100 (19).
Our data show a significantly better fit to two different
models of exponential relationships between Fb and koff
than to a linear relationship. Thus, it is valid to consider
the meaning of the exponential constants, Using the highest Fb at which we consistently saw transient tethers, 230 pN, and the range of estimates for the
elastic spring constant, the lectin-EGF unit is predicted to
be stretched by 0.38-1.15 Å; i.e., the strain (percentage increase in the rest length) would be 0.76-2.3%. This is less
than the critical strain of 3-5% observed for globular proteins, i.e., the strain at which denaturation occurs (35). The
EGF-lectin domain pair would also be assumed to denature at these strains, and this could contribute to the upper
shear threshold for transient tethers.
L-selectin is remarkable for its requirement for shear
above a threshold value for adhesive interactions (13).
Tensile force could theoretically increase bond lifetime as
in a Chinese finger prison (9). Such "catch-bonds" might
be theorized to account for the shear threshold phenomenon. However, our data show instead a decrease in bond
lifetime at higher shear forces, and therefore rule out this
explanation. Several other observations are relevant to the
shear threshold phenomenon. We found that transient
tethers occur below the shear threshold, and that conversion of transient tethers to rolling adhesions is sharply dependent on shear, with a 10-fold increase in the probability
of converting a transient tether to a rolling adhesion between 0.45 and 0.6 dyn/cm2. The frequency of transient
tethers also increased from 0.4-0.7 dyn/cm2, by contrast to
results on P-selectin, where tethering frequency decreased
with increasing shear. Thus, shear appears to promote formation of tether bonds with the substrate, both in the case of the initial transient tether, and more so in the formation of subsequent tethers so that rolling can be supported.
Several factors might contribute to the shear threshold.
The force on the tether bond can be resolved into two
components, one equal to Fs, and another at a right angle
to this, which will push the cell onto the substrate at the
point of contact below the cell's center of mass (Fig. 8 B).
This may flatten the cell at the area of contact, and hence
enlarge the adhesive zone and promote bond formation
(14). Furthermore, shear flow can elongate mucin molecules (30), and this might better expose the carbohydrates
that they bear for recognition by selectins.
In conclusion, we have found that L-selectin has a fast
koff, and that this is related to its ability to support fast rolling. Rolling through L-selectin is faster than through any
other known adhesion molecule. The fast rate of bond dissociation must be compensated by a correspondingly fast
overall rate of bond formation. These rapid kinetics may
be biologically important for L-selectin's function in the
earlier steps of adhesion cascades. We have examined two
distinct mechanisms that could account for differences in rolling velocity, and have found that differences between
selectins are explained by intrinsic bond kinetics, and not
by the mechanical property of reactive compliance. Indeed, koff increases less for L-selectin than for other selectins with increasing shear; this may be important for L-selectin's function in initiating adhesion cascades, and may help
compensate for its fast intrinsic koff°. Our measurements
on cell-substrate contact parameters have elucidated a
number of remarkable features of selectin-mediated rolling, including its ability to be supported by as few as two
tether bonds in the zone of adhesive contact.
Materials and Methods
0.15 µg/ml) were below the detection
limit and were extrapolated from higher site density determinations; since
the concentration of detergent in the adsorption media was kept constant,
the site density of adsorbed PNAd was assumed proportional to PNAd
concentration. Proportionality has previously been demonstrated in the
range of 90-290 sites per µm2 (39). Purified E-selectin (26) was reconstituted in glass-supported lipid bilayers as described for P-selectin (1).
koff.
Results
Fig. 1.
The microkinetics of rolling of individual neutrophils
interacting with the lowest densities of purified PNAd or P-selectin that can support rolling. (A) Neutrophils were perfused in a
parallel wall flow chamber on PNAd at 60 sites per µm2 at a wall
shear stress of 1.5 dyn/cm2. (B) Neutrophils were perfused on
P-selectin at 30 sites per µm2 at 1.8 dyn/cm2. Velocities of cells
free in flow adjacent to the wall are shown for comparison. Coordinates of cell centers were determined within ±0.68 µm and the
velocity between each frame was calculated for individual cells.
[View Larger Version of this Image (31K GIF file)]
Fig. 2.
The microkinetics
of neutrophil rolling near the
shear threshold. (A) The position of a neutrophil selected for continued interaction with a 100 sites per µm2
PNAd substrate at 0.375 dyn/
cm2 was determined to ±0.34
µm accuracy in each video
frame and interframe velocities were calculated. (B) As
in A, at 0.45 dyn/cm2. (C) As
in A, at 1.05 dyn/cm2. Cells
rolled for shorter distances at
0.375 dyn/cm2 than at 0.45 and 1.05 dyn/cm2; the cell in
A rolled for 3.2 s. (D) The
distance between pauses during rolling is modal near the
shear threshold. The distance traveled between each pause
(velocity = 0) was determined at each shear stress for
two representative neutrophils that showed substantial interaction with the substrate
after tethering within the
field of view, in the same experiment as in A-C. Note
that fewer events were seen
at 0.3 dyn/cm2 because only
transient tethers occurred.
[View Larger Version of this Image (47K GIF file)]
Fig. 3.
The microkinetics of transient tethers on PNAd. Neutrophils were perfused at 1.5 dyn/cm2 on substrates with PNAd at
the indicated density. Velocities of representative cells on each
substrate are shown. Cell 1 in EDTA is shown to demonstrate the
hydrodynamic velocity. Coordinates of cell centers were determined within one pixel accuracy (±0.9 µm) and the velocity between each frame was calculated for individual cells.
[View Larger Version of this Image (31K GIF file)]
Fig. 4.
L-selectin forms
tethers at higher shear forces
than PSGL-1. The number of
transient tethers was counted
and divided by the distance
cells were transported by
flow across the field of view and by the number of cells
that flowed across the field
that were within the focal
plane of the substrate. This
yields the density of tethers. L-selectin blocking mAb DREG-56
added to neutrophils in the binding medium at 3 µg/ml, shortly
before perfusion on the substrate, inhibited 95% of tethering
events on PNAd. The density of leukocyte tethering to control
substrates coated with HSA was <0.005 events cell1 · mm
1. No
tethering events were observed in the presence of 5 mM EDTA.
[View Larger Version of this Image (30K GIF file)]
Fig. 5.
Conversion of tethering events to rolling is a
function of wall shear stress
and site density of PNAd.
Tethers were considered to
result in rolling when neutrophils moved for at least 2 s at a mean velocity of at least
fivefold lower than the mean
hydrodynamic velocity of untethered cells at a given shear
stress.
[View Larger Version of this Image (24K GIF file)]
Fig. 6.
The kinetics of dissociation of transiently tethered neutrophils from PNAd and its independence from PNAd or L-selectin densities. The koff values equal the negative slope of the lines
through the dissociation data. (A) Kinetics at a wall shear stress
of 0.75 dyn/cm2 on different low densities of PNAd. Data at the
same wall shear stress on P-selectin (1) are shown for comparison. (B) Kinetics at a wall shear stress of 0.75 dyn/cm2 on higher
density PNAd (60 sites per µm2) in the presence of subsaturating
concentrations of control mAb AD38 to CD44 or mAb DREG-56 to L-selectin at 0.3 µg/ml. The L-selectin mAb reduced tethering frequency by 70% and abolished all rolling adhesions. (C)
Dissociation rate constants at different wall shear stresses, as a
function of PNAd site density. At low shear stresses, when both
transiently tethered and continuously rolling cells could be observed, the few transient events that occurred had the same dissociation kinetics as those derived at low densities.
[View Larger Version of this Image (23K GIF file)]
Fig. 7.
Effect of wall shear stress on the kinetics of neutrophil
dissociation from PNAd and E-selectin. (A) PNAd at four sites
per µm2. (B) E-selectin at seven sites per µm2.
[View Larger Version of this Image (28K GIF file)]
Fig. 8.
The lever arm acting on a transient tether, the
dimension of the adhesive
contact zone, and the force
on the tether bond. (A)
Scheme for measurement of
the lever arm by reversing
the direction of flow. The lever arm, l, is the distance between the tether point and
the projection of the center
of the neutrophil on the substrate. (B) Estimation of
forces on a neutrophil tethered in shear flow (drawn to
scale). The force balance
equations are Fb cos = Fs
and Fblsin
=
s + RFs,
where Fb is the force on the tether bond, Fs is the force on
the cell, and
s is the torque on the cell. The exact solution for Fs for a motionless
hard sphere in shear flow
near a wall is Fs = 6
·viscosity·R·h·shear·C where R is sphere radius, h is the distance from the center of the cell to the wall, and C is a numerical factor determined
by integration that depends on h/R and ranges from 1 to 1.7 (17). With R = 4.25 µm for a neutrophil (48), variation of h-R from 0 to 0.5 µm has little effect on Fs (±3%), and we have assumed h = R for a tethered cell. The assumption of hardness is good because no neutrophil deformation is visible microscopically within the range of shear used here. Roughness such as from microvilli is thought to increase Fs only modestly; for a rough object, Fs is intermediate between Fs on a sphere contained in the object and Fs on a sphere containing the object. We estimate uncertainty in R to measurement variation (48) and its effective increase by microvilli as ±0.25 µm. The
calculated value of Fs and its uncertainty are 59.8 ± 6.9 pN per dyn/cm2 wall shear stress. Both the uncertainty in R and in the lever arm
measurement l = 3.06 ± 0.53 µm introduce uncertainties into the other calculated values. The uncertainties stemming from R and l
were estimated separately for these values and their variances were added. We calculate
= 62.3 ± 4.2°, d = 1.0 ± 0.32 µm, Fb/Fs = 2.15 ± 0.32, and Fb = 124.4 ± 26.1 pN per dyn/cm2 wall shear stress. (C) Movement of a representative transiently tethered neutrophil on
P-selectin at five sites per µm2 during reversal of flow at 0.3 dyn/cm2. The vertical dashed line marks the left boundary of the tethered cell before flow reversal. (D) Measurement of the lever arm. Each point is a measurement on an individual cell; the lever arm is defined
as half the distance moved during flow reversal while cells were tethered to the indicated density of P-selectin. Shear stress was calibrated from the hydrodynamic velocity of cells in the same chamber using a syringe pump. (E) Velocities after flow reversal of untethered neutrophils and of neutrophils transiently tethered to P-selectin (five sites per µm2). The velocity of tethered cells was derived
from a frame by frame analysis of the number of video frames required for a transiently tethered cell to move the distance of 2 l after
flow reversal. Values are the average of two velocity determinations at each wall shear stress. Velocities of cells free in flow (i.e., untethered, traveling at the hydrodynamic velocity) were determined in the same video segments. Velocities of cells free in flow are the mean
of four determinations. Similar velocity determination of cells free in flow was performed on an identical field using a syringe pump generating known wall shear stresses, and the shear stress was calibrated from these velocities. The movement of indicator nontethered
cells in the same field of view was observed to mark flow reversal, and time was not counted until indicator cells reached full velocity
(dead time was about one frame at the lowest shears).
[View Larger Versions of these Images (19 + 22 + 20 + 56K GIF file)]
, the "bond interaction distance" (Fig. 9). The value of
can
quantitatively represent reactive compliance. There is less
variation in
than in koff° for selectins; furthermore, L-selectin has the lowest reactive compliance. Therefore, differences in mechanical properties cannot explain faster rolling through L-selectin than through E- and P-selectins
because greater, not lesser, compliance would lead to
faster rolling. Third, the most robust data set, that on L-selectin off rates, supports an exponential relationship between
Fb and koff. In addition to Bell's equation, these data were
fit to a Hookean spring model in which koff increases exponentially with the second power of Fb, and a straight line
model in which koff increases linearly with Fb. The experimental data on L-selectin show a good fit to the Bell expression (
2 = 0.305 with 9 degrees of freedom), and a
somewhat better fit to the Hookean spring model (
2 = 0.213, with 9 degrees of freedom). Both exponential equations appear to be adequate approximations to the data;
neither fit the data significantly better than the other. By
contrast, the straight line model yields
2 = 0.848, with 9 degrees of freedom. The Hookean spring and Bell expressions both fit the data significantly better than the linear relationship as shown with an F-test of the
2 values (10)
(P = 0.014 and 0.03, respectively), providing support for
an exponential dependence of koff on Fb.
Fig. 9.
Fit to theoretical predictions of the effect of Fb on koff.
koff was measured on PNAd at 4 and 20 sites per µm2 ( and
,
respectively) or E-selectin (
); data on P-selectin (
) (1) are
plotted against Fb using the lever arm measured in the current paper. Fb was calculated as described in the legend to Fig. 7 B. The
thin lines are fit of all three selectin interactions to Bell's expression (4) koff = koff° exp (
Fb/kT) where koff° is the unstressed koff,
is the separation between receptor and ligand that weakens the
bond, k is Boltzmann's constant, and T is the absolute temperature. The fits yield for PNAd: L-selectin koff° = 6.8 ± 0.2 s
1,
= 0.20 ± 0.01 Å; for P-selectin koff° = 0.93 ± 0.15 s
1,
= 0.40 ± 0.08 Å; for E-selectin koff° = 0.7 ± 0.05 s
1,
= 0.31 ± 0.02 Å.
The thick line is fit of the Hookean spring model, koff = koff° exp
(f
Fb2/2
kT), where
is the spring constant for the bond; in the
formalism of Dembo et al. (9) and Hammer and Apte (19), f
is the fraction of the bond spring constant that is applied to bond dissociation; the remainder is applied to bond association. In other words, f
is the fraction of bond strain that is devoted to
bond dissociation, and is also known as the fractional spring slippage (9, 19). The fit yields for the PNAd: L-selectin koff° = 8.8 ± 0.2 s
1 and
/f
= 7.1 ± 0.4 N/m. Data was fit using the program Igor (WaveMetrics, Inc., Lake Oswego, OR). Values for
and
koff° shown in the figure are from the fit to Bell's equation.
[View Larger Version of this Image (35K GIF file)]
Discussion
/f
, i.e., the tether bond spring constant divided by the fraction of the bond spring constant that is
applied to bond dissociation; the range of permissive values has been predicted to correspond to values of
/f
in
Newtons (N)/m that are between 0.1 and infinity (19).
Variation within this wide range would have a dramatic effect on koff in the presence of force, and hence on rolling
velocity. Therefore, we have sought to determine whether koff° or reactive compliance is varied biologically to obtain the considerably faster rolling through L-selectin than
through the vascular selectins. The comparisons between
selectins on koff° and the reactive compliance for bond dissociation were clear cut. The intrinsic L-selectin koff in the
absence of force, koff°, is sevenfold faster than for P-selectin and 9.4-fold faster than for E-selectin. This is in excellent agreement with the 7.5-9-fold faster rolling through
L-selectin than through P-selectin and 8-11.5-fold faster
rolling than through E-selectin, on substrates of equal adhesive strength (40). The koff° are 6.8, 0.93, and 0.7 s
1 for
L-, P-, and E-selectins, respectively, including our previous estimate for P-selectin (1); a previous estimate of a
stressed koff for E-selectin obtained by measuring pauses
during rolling on endothelium is 0.5 s
1 (23). By contrast,
current biophysical models assume koff° of 5 × 10
4-5 × 10
5 s
1, which corresponds to unstressed bond lifetimes of
0.5-5 h (19, 49). Reactive compliance must increase the
unstressed koff° by >1,000-fold to obtain a stressed koff that
can support rolling in these models. Thus bond compliance
has a major role in determining rolling velocity in current
biophysical models, whereas the effect of short intrinsic
bond lifetimes has not been modeled. The difference between the koff°'s measured here and koff°'s assumed in
these models range between 2 × 103 and 106-fold, and
could impact model predictions.
, varied only twofold among selectins, and did
not correlate with rolling velocity. Greater compliance
means a greater increase in koff with applied force, thus
giving rise to faster rolling velocity. However, L-selectin
has lower reactive compliance than the vascular selectins,
and therefore this does not contribute to its faster rolling
velocity. To the contrary, the lower reactive compliance of
L-selectin may help compensate for its faster koff, so that
its already short bond lifetime is not excessively diminished by the hydrodynamic drag forces experienced by adherent leukocytes in postcapillary venules.
1) and reactive compliance (
= 0.24 ± 0.02 Å) are quite similar to those reported here for the L-selectin-PNAd interaction (koff° = 6.8 ± 0.2 s
1 and
= 0.20 ± 0.01 Å). We have also found
that rolling on L-selectin is faster than on E- and P-selectins when substrate densities are adjusted to give rolling
adhesions of similar strength, further supporting the generality of the findings reported here. It has been suggested
that proteolytic shedding of L-selectin may contribute to
the rapidity with which it supports rolling (52). The equivalent kinetics of rolling on PNAd and L-selectin argue
against this, because the protease is cell associated and
cannot function in trans as would be required to shed L-selectin from a substrate (12, 38).
1 derived from analysis of neutrophil pauses during rolling on stimulated endothelium (23) and our measurement
of koff° of 0.7 s
1 from transient tether kinetics. Thus, the
agreement between tether lifetime and pause duration
during rolling provides an explicit link between koff and
rolling velocity.
and
/f
.
There are two caveats to the measurements of these constants. First, neither exponential model fits significantly
better than the other, and thus each should be viewed as
no more than an approximation to the data. Second, although measurements of selectin bond mechanical properties with leukocytes is clearly of the highest biological significance, it is not clear whether leukocytes are accurate biophysical measuring devices; we cannot rule out some
systematic error in the estimate of Fb because leukocytes
are not rigid as modeled, but are viscoelastic. Nonetheless,
we have estimated the error in the calculation of Fb as
±21% (Fig. 8, legend) and the estimates of the constants
may be viewed at least as useful first approximations. The
absolute values of these constants might differ between adhesion molecules and molecules that do not have to function while withstanding force.
was proposed as the increase in distance between the receptor and ligand that leads to
an increased rate of bond dissociation (4); note that
is
not only distinct from, but also less than the unbinding distance and that separation by larger distances than
would
be required to abolish interaction.
was found to be 0.20 ± 0.01 Å. The constant
/f
is the tether bond spring constant divided by the fraction of tether bond stress devoted
to bond dissociation (9), and it was found to be 7.1 ± 0.4 N/m. Note that membrane and cytoskeletal elements of
the microvillus that bear L-selectin (36) may also contribute to elasticity; we acknowledge this with the designation
"tether bond". Tether bond stress is modeled as decreasing Keq; i.e., a portion increases koff and the remainder decreases kon. Since f
1, the value for
/f
derived here
places an upper limit on the tether bond spring constant,
,
of 7.1 N/m. This may be compared to estimates of the elastic spring constant for the EGF-lectin domain unit, since
the elastic spring constant for the binding domain will be
linked by the binding site dislocation that accompanies domain elongation to the spring constant for the bond dissociation reaction. Domains that fold up independently of
one another act independently in elasticity (11). The elastic unit thus is the lectin and EGF domain pair, because
they can be expressed independently of the short consensus repeat domains (7, 54). This unit as shown for E-selectin by x-ray crystallography is a cylinder ~3 nm in diam
and 5.2 nm long (18). Because of the high homology to
P-and L-selectins, essentially identical dimensions can be
safely predicted. Ligand binding to the lectin domain and
connection of the EGF domain to the more membrane-proximal short consensus repeats occur on opposite ends of the
cylinder, and stretching is therefore along its axis. Using
this model, the elastic spring constant can be calculated
from the Young's moduli for globular and structural proteins, which are in the range of 1.4-4 × 109 N/m2 (16, 34,
51), to be 2-6 N/m. This estimated elastic spring constant is
thus of the same order of magnitude as the upper limit for
the tether bond spring constant for L-selectin of 7.1 N/m.
Received for publication 11 October 1996 and in revised form 30 June 1997.
Please address all correspondence to Timothy A. Springer, The Center for Blood Research and Department of Pathology, Harvard Medical School, 200 Longwood Avenue, Room 251, Boston, MA 02115. Tel.: (617) 278-3200. Fax: (617) 278-3232.We would like to thank U. von Andrian for providing purified PNAd used in preliminary investigations, R. McEver for purified P-selectin and mAb to PSGL-1, and D. Hammer, M. Dembo, H. Brenner, and A. Nadim for helpful discussion.
This work was supported by a National Institutes of Health grant (HL 48675).
HSA, human serum albumin; PNAd, peripheral node addressin; PSGL-1, P-selectin glycoprotein ligand-1.
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