Neutrophil adhesion to E-selectin under shear promotes the
redistribution and co-clustering of ADAM17 and its
proteolytic substrate L-selectin
Ulrich Schaff,*,1Polly E. Mattila,†,1Scott I. Simon,* and Bruce Walcheck†,2
†Department of Veterinary and Biomedical Sciences, University of Minnesota, St. Paul, Minnesota, USA; and
*Department of Biomedical Engineering, University of California, Davis, California, USA
endothelium and binds flowing neutrophils in the
blood to facilitate their recruitment into the under-
lying tissue at sites of inflammation. L-selectin on
neutrophils is engaged by E-selectin and undergoes
rapid clustering and then coalescence in the trail-
ing edge of polarizing cells. These processes are
believed to increase the valency and capacity of
L-selectin to signal CD18 integrin activity. Neutro-
phils, upon exiting the microvasculature, down-
regulate their surface L-selectin through ectodo-
main shedding by a disintegrin and metalloprotease
17 (ADAM17). We reasoned that neutrophil teth-
ering and rolling on E-selectin might initiate a co-
ordinate change in the membrane distribution of
ADAM17 as well. We found that ADAM17 indeed
underwent a dramatic cell surface redistribution to
the trailing edge of neutrophils rolling on purified
E-selectin when activated by a chemoattractant un-
der shear flow; however, its lateral migration oc-
curred at a slower rate than L-selectin. ADAM17
and L-selectin also redistributed in the same man-
ner in neutrophils attached to IL-1?-stimulated
HUVEC under shear flow. In contrast, the coales-
cence of L-selectin on the surface of neutrophils by
antibody cross-linking did not promote the redis-
tribution of ADAM17, suggesting that these mole-
cules do not constitutively associate in the plasma
membrane. Together, our findings reveal that neu-
trophil activation upon E-selectin adhesion initiates
active transport of ADAM17 and L-selectin to the
cell uropod, thus providing additional insight into
the molecular mechanisms that regulate L-selectin
during leukocyte extravasation. J. Leukoc. Biol.
83: 99–105; 2008.
E-selectin is expressed by the vascular
Key Words: ectodomain shedding ? inflammation
Neutrophils are the most prominent leukocyte component of
blood and at sites of acute inflammation following their emi-
gration through the microvasculature. The adhesion molecule
L-selectin (CD62 ligand) is constitutively expressed on neu-
trophils and is critical for directing these cells to diverse sites
of inflammation . Sialyl Lewisxon L-selectin is recognized
by the vascular adhesion molecule E-selectin . Current data
reveal that neutrophil interactions with E-selectin result in a
dramatic shift in the membrane topography of L-selectin,
which undergoes a rapid redistribution from its uniform mem-
brane dispersal to punctate clusters and eventually congregates
at the trailing edge (uropod) of neutrophils adhered to purified
E-selectin and cytokine-inflamed endothelium [3, 4]. The lat-
eral movement of L-selectin involves an active transport pro-
cess mediated by its association with the actin cytoskeleton
and membrane microdomains . The formation of L-selectin
clusters may function to increase the valency of interactions
with counter-receptors. Mathematical simulations of the impact
of L-selectin clustering on neutrophil rolling on E-selectin
predict periodic occurrences of low rolling velocity and en-
hanced adhesion, which is also observed experimentally . In
addition, artificially facilitating L-selectin clustering by anti-
body cross-linking has revealed its capacity to transduce in-
tracellular signals, which alter, among other things, the binding
activity of CD18 integrins [3, 6–13].
Another interesting feature of L-selectin is that its surface
expression is down-regulated rapidly by ectodomain shedding
following the activation of neutrophils in vitro by various
stimuli and by their extravasation into inflamed tissue [14–17].
L-selectin shedding is mediated primarily by a metalloprotease
[18, 19]. Indeed, a disintegrin and metalloprotease 17
(ADAM17) has been shown to mediate L-selectin cleavage
directly by primary leukocytes , including mature macro-
phages and neutrophils . Studies involving radiation chi-
meric mice with leukocytes deficient in functional ADAM17
and gene-targeted mice, which express noncleavable L-selec-
tin, indicate that ectodomain shedding is important for regu-
lating the receptor’s surface density and neutrophil infiltration
into sites of inflammation [17, 21].
Currently, there is nothing known about the dynamics of
membrane redistribution of ADAM17 in relationship to L-
1These authors contributed equally to this work.
2Correspondence: University of Minnesota, 295j AS/VM Bldg., 1988 Fitch
Avenue, St. Paul, MN 55108, USA. E-mail: firstname.lastname@example.org
Received May 16, 2007; revised September 17, 2007; accepted September
0741-5400/08/0083-0099 © Society for Leukocyte Biology
Journal of Leukocyte Biology
Volume 83, January 2008
selectin during neutrophil adhesion. To begin addressing this,
we examined the diffusion of L-selectin and ADAM17 on
activated neutrophils in real-time during their adhesion to
purified E-selectin and IL-1?-activated endothelium under
shear. We show that chemoattractant stimulation of neutrophils
bound to an E-selectin substrate, as well as neutrophils at-
tached to activated HUVEC, results in the directed clustering
of L-selectin and ADAM17 to the trailing edge of cells. Of
interest is ADAM17 and L-selectin redistribute at different
rates, and the density shift by L-selectin occurs more rapidly.
Our data provide the first demonstration that ADAM17 can
redistribute and co-cluster rapidly with L-selectin by activated
neutrophils attached to E-selectin. These data thus provide
further mechanistic insight into the regulation of L-selectin
during neutrophil adhesion.
MATERIALS AND METHODS
Antibodies and other reagents
The anti-L-selectin mAb DREG-200 has been described previously [22, 23].
Humanized DREG-200 (huDREG-200) was generously provided by PDL Bio-
Pharma (Fremont, CA, USA). The L-selectin mAb FMC46 conjugated to FITC
was purchased from Dako North America (Carpenteria, CA, USA). FITC and
PE-conjugated anti-L-selectin (LAM1-116) as well as biotinylated anti-CD45
and anti-CD55 were purchased from Ancell (Bayport, MN, USA). The anti-
ADAM17 mAb M220 has been described previously [24, 25] and was conju-
gated with Alexa 546 (Molecular Probes, Eugene, OR, USA), as per the
manufacturer’s instructions. FITC-conjugated anti-CD45 was purchased from
BD Biosciences PharMingen (San Diego, CA, USA). Unconjugated and biotin-
conjugated F(ab?)2goat anti-mouse IgG, FITC-conjugated F(ab?)2goat anti-
human IgG, and Cy3-conjugated streptavidin were purchased from Jackson
ImmunoResearch (West Grove, PA, USA). QDot 655 streptavidin was pur-
chased from Quantum Dot Corp. (Hayward, CA, USA). A human E-selectin-
human IgG Fc chimeric construct (E-selectin/Fc) was purchased from R&D
Systems (Minneapolis, MN, USA). The cytokine IL-1? was purchased from
(PeproTech, Rocky Hill, NJ, USA). fMLP was purchased from Sigma-Aldrich
(St. Louis, MO, USA).
Peripheral blood was collected from healthy donors in sodium heparin in
accordance with approved protocols by the Institutional Review Board, Human
Subjects Committee at the University of Minnesota (St. Paul, MN, USA), and
at the University of California, Davis (Davis, CA, USA). Neutrophils were
isolated by Ficoll-hypaque centrifugation, and cell viabilities were assessed by
exclusion of the vital dye trypan blue, as described previously [3, 5]. All media
and buffers used for neutrophil isolation and incubations were sterile and
tested for endotoxin.
To visualize the cell surface distribution pattern of L-selectin and ADAM17 on
neutrophils activated in suspension, freshly isolated neutrophils were cultured
for various lengths of time at 37°C, as detailed in the text, in RPMI plus 5 mM
HEPES (RPMI-H), in the presence or absence of fMLP (10 nM). Afterwards,
the cells were washed in cold RPMI-H, fixed with 1% paraformaldehyde, and
then treated with 1% normal goat serum in PBS to block nonspecific antibody
interactions. The neutrophils were then sequentially stained with M220 (anti-
ADAM17), biotin-conjugated F(ab?)2goat anti-mouse IgG, QDot 655 strepta-
vidin, 10% normal mouse serum, and FITC-conjugated LAM1-116 (anti-L-
Analysis of the colocalization of ADAM17, CD45, or CD55 with L-selectin
before and after its induced clustering by antibody-mediated cross-linking was
adapted from our previous studies [3, 5]. Briefly, freshly isolated neutrophils
were stained sequentially with huDREG-200 and FITC-conjugated F(ab?)2
goat anti-human IgG. The treated neutrophils were then incubated at 37°C for
30 min to facilitate L-selectin clustering. Afterwards, the neutrophils were
fixed in 1% paraformaldehyde, treated with 1% normal goat serum in PBS to
block nonspecific antibody interactions, and stained with the anti-ADAM17
mAb M220, followed by biotin-conjugated F(ab?)2goat anti-mouse IgG and
QDot 655 streptavidin, 10% normal mouse serum, and then biotinylated
anti-CD55 or biotinylated anti-CD45, followed by Cy3-conjugated streptavidin.
After the facilitation of L-selectin clustering, all antibody-staining steps
described above were performed at 4°C, and the neutrophils were washed with
RPMI-H between steps. Nonspecific antibody labeling was determined using
the appropriate isotype negative control antibodies. The appropriately labeled
cells were then applied to poly-L-lysine-coated coverslips and mounted with
Vectashield Hard-Set mounting medium (Vector Laboratories, Burlingame,
CA, USA). Fluorescence analysis was performed on an Olympus Fluoview
FV500 laser-scanning confocal microscope (Olympus, Center Valley, PA,
USA) using a 60? oil immersion objective. Images were recorded and pro-
cessed using Multi-Point Time Lapse software. Use of the confocal microscope
was made available through a National Center for Research Resources Shared
Instrumentation Grant (#1 S10 RR16851).
To assess the effects of antibody cross-linking of L-selectin on its shedding
efficiency, freshly isolated neutrophils suspended in RPMI-H were treated on
ice with the PE-conjugated anti-L-selectin mAb LAM1-116 and then with or
without F(ab?)2goat anti-mouse IgG. Cells were washed with RPMI-H between
steps. Nonspecific antibody labeling was determined using an appropriate
isotype negative control antibody. The treated neutrophils were then incubated
at 37°C for 30 min in the presence or absence of fMLP (10 nM). Afterwards,
the cells were washed in cold RPMI-H and fixed with 1% paraformaldehyde.
The antibody-labeled cells were analyzed by flow cytometry (10,000 cells/
sample) on a FACSCanto instrument (Becton Dickinson Immunocytometry
Systems, San Jose, CA, USA).
Hydrodynamic shear flow assay
Prior to their use in the shear flow assays, neutrophils were kept at 4°C in a
HEPES buffer [110 mM NaCl, 10 mM KCl, 10 mM glucose, 1 mM MgCl2, and
30 mM HEPES (pH 7.4)]. Immediately prior to the assay, CaCl2was added to
the buffer at a 1.5-mM final concentration. By this method, neutrophils were
found to remain viable and unactivated for 4 h after their separation.
Microfluidic flow chambers with a minimum feature size of 5 ? were cast
from Sylgard 184 prepolymer (Dow Corning, Midland, MI, USA) over a
patterned silicon wafer and bonded to precleaned coverslips, which were
coated with E-selectin/Fc in PBS containing 20 mM bicarbonate (1 ?g/mL) for
90 min at room temperature. To block nonspecific binding sites, 2% human
serum albumin (HSA) in PBS containing 0.05% Tween-20 was injected into
the flow channel and incubated for an additional 90 min at room temperature.
HUVEC were obtained from Cascade Biologics (Portland, OR, USA) and grown
in Media 200 containing low-serum growth supplement (Cascade Biologics). At
passages 4–5, HUVEC were layered onto the glass coverslips coated with
cross-linked gelatin, grown to confluence, and then stimulated with 5 ng/mL
IL-1? for 4 h, as described previously . Finally, each flow channel was
washed three times with PBS and connected to polyethylene tubing in prep-
aration for shear flow experiments. To impose a specific shear stress, fluid was
withdrawn from a reservoir through the flow chamber via a syringe pump
(Harvard Apparatus, Holliston, MA, USA).
To label live neutrophils, the cells were incubated for 10 min at 4°C with 10
?g/mL of the L-selectin mAb FMC46-FITC (nonfunction blocking) or anti-
CD45-FITC and the ADAM17 mAb M220 conjugated to Alexa 546 in HEPES
buffer containing 1% HSA to block nonspecific antibody interactions. Follow-
ing labeling, neutrophils were spun down and resuspended to 1 ? 106/mL in
HEPES buffer and then introduced into the microfluidic flow chamber. The
average shear stress at the flow channel floor was set at 1 dyne/cm2by
adjusting the flow rate according to the Couette approximation (T?6 Q?/wh2)
. To activate neutrophils rolling on E-selectin/Fc, the inlet reservoir fluid
was replaced with 10 nM fMLP at a defined time-point. For all experiments,
one fluorescent image was captured every 5 s by a Cascade 512B camera
(Roper Scientific, Duluth, GA, USA) after sequential exposure using a filter
wheel/shutter system on an inverted Nikon 1200 microscope (Nikon, Melville,
NY, USA). A 50-frame sequence of three-color (Mean Red, Mean Green, and
Mean Blue) images was reconstructed from each set of 488 nm, 546 nm, and
100 Journal of Leukocyte Biology
Volume 83, January 2008
brightfield channel data and saved for analysis. Image sequences were ana-
lyzed for the position of fluorescent clusters. Based on a histogram of all pixel
intensities within an image region bounded by a neutrophil, clusters of
ADAM17, L-selectin, or CD45 staining were defined as regions with pixel
intensity, 2 SD greater than the mean neutrophil intensity. Using custom macros
written in Image Pro 5.1 (Mediacybernetics, Silver Spring, MD, USA), these
regions of dense protein were analyzed for percent area overlap in the red and
green channels and cluster position.
Data analysis was performed using GraphPad Prism Version 4.0 software
(GraphPad Software, San Diego, CA, USA). All data are reported as mean ?
SD. Gaissian-distributed mean values were analyzed by Student’s t-test. Group
comparisons were deemed significant for P values below 0.05.
Distribution pattern of L-selectin and ADAM17
on the surface of activated neutrophils
L-selectin is distributed primarily on the tips of microvillous
surface projections of resting leukocytes in suspension .
Upon cell activation, the distribution pattern of L-selectin on
suspended cells remains essentially unchanged, but the num-
ber of surface molecules decreases rapidly as a result of their
shedding . Using confocal fluorescence microscopy, we
examined the surface-staining pattern of anti-L-selectin and
anti-ADAM17 mAb simultaneously by neutrophils activated in
suspension with fMLP. L-selectin on unstimulated neutrophils
demonstrated punctate but uniform staining (Fig. 1), as re-
ported previously [3, 5]. Upon cell activation, essentially the
same distribution pattern for L-selectin was noted, but its
staining level decreased to that of background within a few
minutes (Fig. 1). This did not occur in the absence of fMLP
within the same time-frame (data not shown). ADAM17 also
exhibited a punctate but uniform staining pattern on unstimu-
lated and activated neutrophils, but unlike L-selectin, its stain-
ing level remained essentially unchanged following neutrophil
activation (Fig. 1). These findings indicate that ADAM17 and
its substrate L-selectin do not undergo a striking topographical
redistribution on the surface of neutrophils when activated in
Spatial and temporal redistribution of L-selectin
and ADAM17 in activated neutrophils during
tethering on an E-selectin substrate
under shear flow
Neutrophil attachment to E-selectin induces a rapid clustering
of L-selectin and its eventual migration to the trailing edge of
polarizing cells [3–5]. Using two-color, live cell immunofluo-
rescence, we examined the cell surface distribution of L-
selectin and ADAM17 simultaneously by fMLP-activated neu-
trophils adhered to E-selectin under shear flow. As described
previously , L-selectin underwent a rapid redistribution to
the trailing edge of E-selectin-bound neutrophils during their
arrest and shape polarization (Fig. 2A). The membrane distri-
bution of ADAM17 changed as well, and its staining pattern
shifted toward the trailing edge of E-selectin-attached neutro-
phils and began to occupy the L-selectin cluster (Fig. 2, A and
B). In previous studies, we have found that a control surface
receptor CD45 did not redistribute in the plasma membrane of
neutrophils as efficiently as L-selectin upon E-selectin binding
[3, 5]. In the current study, we observed that CD45 underwent
significantly less co-clustering with ADAM17 than did L-
selectin during the same time-frame of E-selectin tethering
(Fig. 2B). Of particular interest was that the rates of L-selectin
and ADAM17 redistribution to the uropod of polarizing neu-
trophils were significantly different, and the retrograde motion
of L-selectin was more rapid (Fig. 2C).
Neutrophils attached to stimulated endothelial monolayers
undergo clustering of L-selectin as well . IL-1?-stimulated
Fig. 1. Cell surface distribution pattern of
ADAM17 and L-selectin on neutrophils ac-
tivated under static conditions. Freshly iso-
lated human neutrophils under static condi-
tions were treated with 10 nM fMLP for the
indicated time-points. After which, the cells
ADAM17, as described in Materials and
Methods. Confocal micrographs are repre-
sentative of three independent experiments.
Schaff et al.
L-selectin and ADAM17 co-clustering by adhered neutrophils101
HUVEC express high levels of E-selectin and chemokines [26,
30], and we measured the redistribution and colocalization of
L-selectin and ADAM17 on adhered neutrophils under shear
flow. We observed that during the process of firm attachment
and transmigration beneath the endothelial monolayer,
ADAM17 and L-selectin clustered at the trailing edge of
neutrophils, and the relocation of L-selectin occurred at a
faster rate than ADAM17 (Fig. 3, A and B). Altogether, these
data demonstrate that in contrast to neutrophils activated in
suspension, L-selectin and ADAM17 undergo a coordinated
lateral movement and focal coalescence in activated neutro-
phils rolling on E-selectin under shear flow, and the redistri-
bution of L-selectin was more rapid than ADAM17.
Effects of facilitated L-selectin clustering on the
cell surface distribution of ADAM17
We next examined whether the facilitated clustering of L-
selectin on the surface of resting neutrophils resulted in a
redistribution of ADAM17. Antibody cross-linking has been
used extensively for specific and precise regulation of the
extent of L-selectin clustering [5, 6, 12, 31–36]. Using this
approach, we examined whether ADAM17 co-clustered with
Fig. 2. ADAM17 and L-selectin undergo redistribution following the activa-
tion of neutrophils rolling on E-selectin. Freshly isolated human neutrophils
were immunofluorescently labeled with nonfunction blocking, anti-L-selectin-
FITC or anti-CD45-FITC, and anti-ADAM17-Alexa 546 mAb, as described in
Materials and Methods. The labeled cells were perfused into a flow chamber
and sheared over adsorbed E-selectin. After a constant shear stress for ?1
min, fMLP (10 nM) was added to the inlet reservoir fluid, and the cells were
monitored by immunofluorescence microscopy for the indicated times. (A)
Image sequences indicate the position of fluorescent staining by L-selectin
(green) and ADAM17 (red) on an individual adhered neutrophil, as indicated.
One fluorescent image was captured every 5 s. (B) Cell micrographs were
analyzed for the percentage of L-selectin or CD45 pixels overlapped by
ADAM17 pixels (i.e., yellow pixels were divided by green and yellow pixels
and then multiplied by 100). *, P ? 0.005, versus (CD45 vs. ADAM17). (C)
Cell micrographs were analyzed for L-selectin and ADAM17 staining concen-
trations in the trailing edge of activated neutrophils during their arrest, spread,
and polarization. Micrograph pixel intensity for each labeled mAb was defined
as 2.5 SD above average cellular fluorescence. *, P ? 0.001; **, P ? 0.008,
versus ADAM17. (B and C) Data are given as mean ? SE for ?15 cells per
time-point for five independent experiments.
Fig. 3. ADAM17 and L-selectin colocalize upon neutrophil transmigration
through activated HUVEC. Freshly isolated human neutrophils were labeled
immunofluorescently with nonfunction-blocking anti-L-selectin-FITC or anti-
CD45-FITC and anti-ADAM17-Alexa 546 mAb, perfused into a flow chamber,
and sheared over HUVEC monolayers, as described in Materials and Methods.
(A) Image sequences indicate the position of fluorescent staining of L-selectin
(green) and ADAM17 (red), as indicated on an individual transmigrating
neutrophil, starting from the moment a subendothelial pseudopod is visible by
brightfield microscopy. Upper panels represent the phase-contrast image of an
attached neutrophil with overlaid fluorescence, and the lower panels display
fluorescence overlap occurring in the L-selectin cluster in a pixilated manner.
Values indicated in the lower panels represent the percentage of L-selectin
pixels overlapped with ADAM17 pixels (i.e., yellow pixels were divided by
green and yellow pixels and then multiplied by 100). (B) Cell micrographs were
analyzed for the percentage of L-selectin or CD45 pixels overlapped by
ADAM17 pixels. Micrograph pixel intensity for each labeled mAb was defined
as 2.5 SD above average cellular fluorescence. Data are given as mean ? SE for
?15 cells per time-point for five independent experiments. *, P ? 0.008,
versus (CD45 vs. ADAM17).
102 Journal of Leukocyte Biology
Volume 83, January 2008
antibody cross-linked L-selectin on resting neutrophils using
confocal fluorescence microscopy. As we have reported previ-
ously , antibody cross-linking resulted in L-selectin patch-
ing on the neutrophil surface, which in turn, promoted appre-
ciable clustering of CD55 (Fig. 4A, h), a GPI-anchored pro-
tein resident in leukocyte lipid rafts [37–40], but not CD45
(data not shown; ref. ). Moreover, the vast majority of the
CD55 clusters colocalized with L-selectin (Fig. 4A, i). This
degree of clustering or increased colocalization was not ob-
served for ADAM17 upon antibody cross-linking of L-selectin
(Fig. 4A, e and f). It is interesting that antibody cross-linking
of L-selectin greatly diminished its down-regulation in surface
expression upon neutrophil activation with fMLP compared
with noncross-linked L-selectin on stimulated neutrophils (Fig.
4B), further indicating that L-selectin clustering in this manner
sequestered it from ADAM17. The above findings thus suggest
that ADAM17 and L-selectin are not associated in a physical
manner in unactivated neutrophils, nor do they occupy a
common membrane microdomain.
L-selectin undergoes rapid clustering on the surface of neutro-
phils upon its engagement and rolling on a cell substrate
expressing E-selectin [3, 4]. L-selectin is also down-regulated
promptly in expression by the metalloprotease ADAM17 fol-
lowing overt activation of neutrophils by various stimuli and
during their extravasation into inflamed tissues [14–17]. At
this time, the distribution pattern of ADAM17 in relationship
to L-selectin, while the adhesion molecule undergoes cluster-
ing during neutrophil tethering on E-selectin, has not been
determined. We simultaneously imaged the distribution of
L-selectin and ADAM17 on live human neutrophils upon their
rolling and activation on purified E-selectin and inflamed
vascular endothelial cells under shear flow. Of interest is that
ADAM17 also underwent membrane redistribution and pro-
gressed to polarized clusters at the trailing edge of neutrophils,
although at an apparently slower rate than L-selectin. Such
striking focal colocalization of L-selectin and ADAM17 was not
apparent on neutrophils activated in suspension. These find-
ings reveal the involvement of additional mechanisms in the
regulation of L-selectin activity and its shedding by adherent
leukocytes under shear flow conditions.
The clustering of L-selectin increases its valency [4, 41, 42]
and transduces cellular signals from the outside in, resulting in
various post-L-selectin adhesion events, including oxidative
burst, degranulation, cytokine expression, actin polymeriza-
tion, and CD18 integrin activation [3, 6, 8, 12, 31–36, 43].
Hence, L-selectin clustering appears to promote various leu-
kocyte effector activities prior to its down-regulation by ectodo-
main shedding. L-selectin clustering may also be important in
Fig. 4. Antibody-mediated cross-linking of L-selectin in resting
neutrophils does not facilitate the clustering of ADAM17. (A)
Freshly isolated neutrophils were treated with an L-selectin mAb
and FITC-conjugated secondary antibody and incubated on ice
(a–c) or at 37°C for 30 min (d–i) to coalesce L-selectin (L-selectin-
XL). After which, the neutrophils were stained for CD55 or
ADAM17, as indicated. Neutrophils incubated on ice demonstrated
uniform, punctate staining of L-selectin (a). Neutrophils incubated
at 37°C demonstrated increased patching of L-selectin (d and g). A
merged image of L-selectin and CD55 (i) shows several yellow
areas (some are indicated by arrowheads). Values indicated in the
merged panels represent the percentage of ADAM17 or CD55
pixels overlapped with L-selectin pixels (i.e., yellow pixels were
divided by red and yellow pixels and then multiplied by 100).
Confocal micrographs are representative of 25 cells from three
independent experiments. (B) Freshly isolated neutrophils were
treated with a fluorochrome-conjugated L-selectin mAb, plus or
minus F(ab?)2goat anti-mouse IgG, and then incubated for 15 min at 37°C [only the former treatment promoted L-selectin clustering (data not shown)], with or
without 10 mM fMLP, as indicated. Neutrophils treated with a fluorochrome-conjugated L-selectin mAb, plus or minus F(ab?)2goat anti-mouse IgG at 37°C in the
absence of fMLP, demonstrated equivalent mean fluorescent cell-staining levels (data not shown). Cell staining was examined by flow cytometry, and 10,000 cells
were examined per sample. The dashed line indicates cell staining by a fluorochrome-conjugated, isotype-matched, negative control mAb. Data are representative
of three independent experiments.
Schaff et al.
L-selectin and ADAM17 co-clustering by adhered neutrophils 103
enhancing its proteolytic turnover by increasing the proximity
of L-selectin molecules to facilitate more efficient shedding. A
limitation of the fluorescent imaging approaches used in our
study is a spatial resolution cutoff of approximately 0.02 ?m to
assess molecular scale interactions between L-selectin and
ADAM17. However, approaches such as electron microscopy,
which can assess L-selectin and ADAM17 intermixing at the
ultrastructural level, or fluorescence resonance energy transfer,
which detects signal emissions dependent on intermolecular
proximity, present various technical challenges when real-time
imaging live cells in our microfluidic flow chamber. Although
not measured directly, L-selectin shedding by adherent neu-
trophils may begin prior to its redistribution. In consideration
of this, it is tempting to speculate that the observed redistri-
bution of L-selectin molecules ahead of ADAM17 in tethered
neutrophils may delay their shedding upon clustering in the
uropod by altering the stoichiometry of the proteolytic reaction
or temporarily sequestering L-selectin from its sheddase. Con-
sistent with the latter assumption, we observed that antibody-
mediated L-selectin clustering greatly reduced the level of
L-selectin down-regulation upon neutrophil activation with
fMLP when compared with neutrophils that were subjected to
It has been reported that L-selectin clustering in neutrophils
upon their binding to E-selectin involves an active transport
process . Indeed, the lateral mobility of L-selectin appears to
be regulated by dynamic associations with membrane domains
and the actin cytoskeleton [5, 44, 45]. The topographical
redistribution of ADAM17 during this process may also involve
an active transport process. This assumption is based on sev-
eral findings. ADAM17 and L-selectin appear not to associate
in a physical manner or partition in a common membrane
domain, either of which might facilitate passive diffusion by
ADAM17 upon the coalescence of L-selectin, as appears to
occur for CD55 . L-selectin and ADAM17 progressed to the
trailing edge of activated neutrophils during adhesion and
transmigration at different rates under shear flow conditions.
Lastly, under static conditions, ADAM17, but not L-selectin,
demonstrated little redistribution to the uropod of neutrophils
undergoing transmigration through activated HUVEC (data not
shown). These findings together indicate that L-selectin and
ADAM17 are not constitutively associated and exhibit distinct
membrane transport properties. Canault et al.  reported
that four-and-a-half LIM domain 2 protein, which is involved
in various protein-binding interactions, associates with the
cytoplasmic region of ADAM17 and the actin cytoskeleton.
This and other intermolecular interactions may facilitate cy-
toskeletal linkages and lateral movement by ADAM17. It will
be important to gain a better understanding of the mechanisms
that direct the lateral movement of surface ADAM17, as this
may provide for additional means to manipulate the activity of
ADAM17 during leukocyte extravasation into sites of inflam-
In conclusion, when neutrophils bind inflamed vascular
endothelial cells, they become polarized, and L-selectin con-
gregates at their trailing edge. We show that this directed
clustering occurs for ADAM17 as well. When adhered to
E-selectin, activated neutrophils redistribute first L-selectin
and then ADAM17 to the uropod in a time course consistent
with the onset of L-selectin shedding. Such redistribution may
facilitate transendothelial migration in several ways. First, the
redistribution of L-selectin may decrease adhesion events at
the neutrophil’s leading edge. Second, L-selectin clustering by
attached neutrophils provides potent inside-out signaling, re-
sulting in a conformational shift in the CD18 integrins, which
leads to neutrophil deceleration and arrest [3, 9, 12]. Finally,
ADAM17 redistribution to the uropod may provide a means for
extinguishing L-selectin adhesion events and outside-in sig-
naling, facilitating a smoother transition from surface adhesion
to interstitial migration. In support of this theory, Venturi et al.
 reported that neutrophils in gene-targeted mice expressing
noncleavable L-selectin were impaired in their transendothe-
lial migration across postcapillary venules during keratinocyte-
derived cytokine-induced inflammation. We thus conclude that
a proteolytic step supplied by ADAM17 redistribution in rela-
tion to L-selectin aids in the precision of the process of rolling,
activation, arrest, and transmigration by neutrophils.
This study was supported by grants HL61613 (B. W.) and
AI47294 (S. I. S.) from the National Institutes of Health. We
thank Drs. Roy Black and Jacques Peschon for providing the
anti-human ADAM17 mAb M220 and Drs. Zhenya Ni and Josh
Mattila for their technical assistance with Photoshop.
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L-selectin and ADAM17 co-clustering by adhered neutrophils 105