Distinct kinetic and mechanical properties govern selectin-leukocyte interactions
William D. Hanley
1
Denis Wirtz
)
0
1
Konstantinos Konstantopoulos
1
0
Department of Materials Science and Engineering, The Johns Hopkins University
,
3400 N. Charles Street, Baltimore, MD 21218
,
USA
1
Department of Chemical and Biomolecular Engineering, The Johns Hopkins University
,
3400 N. Charles Street, Baltimore, MD 21218
,
USA
-
Leukocytes are recruited from the bloodstream to sites of
inflammation by the selectin family of adhesion receptors.
In vivo and in vitro studies reveal distinctive rolling
velocities of polymorphonuclear leukocytes over E-,
Pand L-selectin substrates. The kinetic and mechanical
properties of the selectin-ligand bonds responsible for these
differences at the single-molecule level are not well
understood. Using single-molecule force spectroscopy, we
probe in situ the rupture force, unstressed off-rate and
reactive compliance of single selectin receptors to single
ligands on whole human polymorphonuclear leukocytes
(PMNs) under conditions that preserve the proper
orientation and post-translational modifications of the
selectin ligands. Single L-selectin bonds to PMNs were
more labile than either E- or P-selectin in the presence of
an applied force. This outcome, along with a higher
Central to all cell-cell communications are the adhesive
interactions between biological receptors and their respective
ligands. The kinetics regulating receptor-ligand binding impart
unique properties that allow specialized cells to interact
with one other amidst the challenge of physiological and
competitive stresses. This phenomenon is perhaps best
represented by the remarkable ability of the selectins (E-,
Pand L-selectin) to mediate the initial adhesion events
during leukocyte recruitment to sites of inflammation. The
distinctiveness of selectin binding is attributed to their fast
association/dissociation rates, as well as their ability to form
high-strength tethers under rapid loading. These properties
allow polymorphonuclear leukocytes (PMNs) first to tether and
roll on activated vascular endothelium under hydrodynamic
shear, before firm adhesion and extravasation into the tissue
space (Konstantopoulos et al., 1998; McEver, 2002; Springer,
1995). In vivo (Jung et al., 1996; Kunkel and Ley, 1996) and
in vitro (Alon et al., 1997; Alon et al., 1998; Puri et al., 1997)
studies both reveal that E-, P- and L-selectin-mediated
interactions display characteristic leukocyte rolling velocities.
Structurally similar, the selectins consist of an N-terminal,
calcium-dependent lectin domain, an
epidermal-growth-factorlike (EGF) domain, a variable number of consensus repeat
units, a transmembrane domain (TM) and an intracellular
cytoplasmic tail (cyto) (Fig. 1A) (Konstantopoulos et al., 1998;
McEver et al., 1995). They also share a relatively high degree
unstressed off-rate and a higher reactive compliance,
explain the faster L-selectin-mediated rolling. By
quantifying binding frequency in the presence of a specific
blocking monoclonal antibody or following enzyme
treatment, we determined that P-selectin glycoprotein
ligand-1 is a high-affinity ligand for E-selectin on PMNs
under force. The rupture force spectra and corresponding
unstressed off-rate and reactive compliance of
selectinligand bonds provide mechanistic insights that might help
to explain the variable rolling of leukocytes over different
selectin substrates.
Supplemental figure available online
of homology within each region (McEver et al., 1995). The
primary module for adhesion is the lectin domain but other
features (including the EGF region and the number of
consensus repeat units) might confer unique biophysical and
mechanical properties on each selectin when binding to its
ligand (McEver et al., 1995). Additionally, selectin ligands
recognize a stereochemically precise arrangement of sialic acid
and fucose, termed sialyl Lewisx (McEver, 2002; Varki, 1997).
Although they share many common features, the selectins
possess differences in their expression kinetics, tissue
distributions and ability to promote leukocyte attachment,
perhaps reflecting their unique roles in diverse immunological
processes. E-selectin, expressed on the vascular endothelium,
is transcriptionally regulated and induced in response to
inflammatory stimuli (Konstantopoulos et al., 1998). P-selectin
expression on both the vascular endothelium and on platelets
is also inducible, but its storage in the Weibel-Palade bodies
of endothelial cells and the a -granules of platelets permits
rapid mobilization (within seconds to minutes) to the plasma
membrane upon cell activation (Konstantopoulos et al., 1998).
L-selectin, by contrast, is constitutively expressed on most
leukocyte subpopulations and is responsible for amplifying
the inflammatory response through leukocyte-leukocyte
interactions or so-called secondary tethers (Alon et al., 1996;
Konstantopoulos et al., 1998).
P-selectin glycoprotein ligand 1 (PSGL-1) is the
bestcharacterized ligand capable of binding to all thr (...truncated)