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Blood First Edition Paper, prepublished online July 29, 2011; DOI 10.1182/blood-2010-11-321489
CD63 is an essential co-factor to leukocyte recruitment by
endothelial P-selectin
Emily L. Doyle1,2,3*, Victoria Ridger4*, Francesco Ferraro1,2,3, Mark Turmaine3,
Paul Saftig5, Daniel F. Cutler1,2,3
1
3
4
MRC Laboratory for Molecular Cell Biology and 2MRC Cell biology unit and
Department of Cell and Developmental Biology, UCL, London, WC1E 6BT, UK.
Department of Cardiovascular Science, Faculty of Medicine, Dentistry and Health,
University of Sheffield, S10 2JF, UK
5
Biochemisches Institut, Christian-Albrechts-Universität Kiel, D-24098 Kiel
Germany
Correspondance:
Daniel
F.
Cutler,
[email protected]
T:00442076797808;
F:00442076797805
* Denotes equal contribution.
Short title: Function of Endothelial CD63 in Inflammation
1
Copyright © 2011 American Society of Hematology
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Abstract
The activation of endothelial cells is critical to initiating an inflammatory response.
Activation induces the fusion of Weibel-Palade Bodies with the plasma membrane,
thus
transferring
recruitment
of
P-selectin
leukocytes
and
and
VWF
to
platelets
the
cell
surface,
respectively.
where
CD63
has
they
act
long
in
the
been
an
established component of WPB, but the functional significance of its presence within
an organelle that acts in inflammation and haemostasis was unknown. We find that
ablating CD63 expression leads to a loss of P-selectin-dependent function: CD63–
deficient HUVECs fail to recruit leukocytes, CD63-deficient mice exhibit a significant
reduction
in
leukocyte
extravasation
phenotype
to
both
loss
leukocyte
of
in
a
rolling
and
peritonitis
P-selectin
itself,
recruitment
model.
thus
selectin.
2
CD63
and
Loss
is
an
of
we
show
CD63
essential
a
has
failure
a
of
similar
co-factor
to
P-
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Introduction
Endothelial cells contain specialised regulated secretory organelles, Weibel-Palade
1
bodies (WPB) , which play an important role in inflammation and haemostasis. The
best-known components of WPB are the major cargo protein von Willebrand Factor
2
3
4
(VWF) , P-selectin , and CD63 . Upon WPB fusion the VWF tubules unfurl into long
5
platelet-catching strings at a site of injury , playing a major role in haemostasis.
VWF also has an important function in inflammation, since activated platelets make
a
major
contribution
leukocyte
receptor
to
the
PSGL-1
inflammatory
and
integrin
6
response .
αMβ 27
Further,
implicating
a
VWF
binds
the
more direct role
in
leukocyte recruitment to the endothelium.
The integral membrane protein leukocyte receptor P-selectin is stored within the
membrane
surface
of
WPB
within
of
endothelial
minutes
following
cells
3,8
from
where
it
secretagogue-triggered
is
delivered
to
9
exocytosis .
the
cell
P-selectin
plays a key early role in the inflammatory trafficking of leukocytes, being the first
receptor involved in recruiting leukocytes from flowing plasma to the endothelial
surface
CD63
12
10,11.
4
is the third long-established component of WPB , but this protein has no
identified endothelial-specific function in either haemostasis or inflammation. This
universally expressed membrane protein of the tetraspanin family is best known as
a
marker
phenotype
of
of
the
intra-lumenal
CD63
knockout
vesicles
mice
within
suggests
multi-vesicular
a
redundant
endosomes.
role
for
The
CD63
in
development and distribution of immune system cells, very mild effects on platelet
adhesion, and a role in kidney physiology
13.
It has been reported that CD63 might modulate the trafficking of other membrane
proteins, including altering internalisation from the plasma membrane, and most
recently, in targeting
plasma
membrane
synaptotagmin VII to the lysosome, thus facilitating lysosome-
fusion
12.
Whether
CD63
3
is
involved
in
the
haemostatic
or
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inflammatory roles of WPB is unknown, but if so, it might operate by modulating
trafficking events that underpin WPB formation and function. CD63 is found only on
mature
WPB
14.
It
is
therefore
unlikely
to
be
directly
involved
in
initial
WPB
formation at the TGN, but could influence events late in WPB biogenesis including
maturation, late recruitment of a critical modulator of WPB function, exocytosis, or
post-exocytic events at the plasma membrane.
We report that siRNA depletion of CD63 from human umbilical vein endothelial cells
(HUVEC) results in loss of P-selectin dependent recruitment of THP-1 monocytic
cells under flow in vitro. CD63 knockout mice showed both a loss of P-selectin
dependent leukocyte rolling and a failure of leukocyte extravasation in a peritonitis
model.
These
are
very
similar
to
observations
made
in
the
P-selectin-deficient
mouse itself, thus demonstrating a critical inflammatory role for CD63. Scanning
electron microscopy revealed that CD63 co-clusters with P-selectin on the plasma
membrane
of
activated
endothelial
cells,
and
this
close
co-localisation
was
confirmed by an in situ proximity ligation assay (PLA). Ablation of CD63 expression
by siRNA in HUVECs leads to reduced P-selectin on the cell surface, plus a reduction
in
the
number
and
size
of
remaining
clusters
seen
by
EM.
We
have
therefore
identified an endothelial role for this ubiquitous and poorly understood tetraspanin:
it acts to cluster and maintain P-selectin at the plasma membrane of endothelial
cells and is thus an essential co-factor to P-selectin in the initiation of inflammation.
4
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Material and Methods
Cell Culture and Transfection
HUVECs (TCS-Cellworks) HEK293 and THP-1 cells were cultured and Nucleofected
(Nucleofector II, Amaxa Biosystems, Gaithersburg, MD) as previously described
15
30, and see supplementary methods. HUVECs between passage 3-4 were used for all
experiments.
elsewhere
Wild-type
and
tail-deficient
P-selectin
constructs
are
described
14.
Immunofluorescence
Immunoflurescence staining was carried out as described previously
imaged
through
40X
or
63X
oil-immersion
lenses
on
a
Leica
TCS
15.
Slides were
SPE
confocal
system (Leica, Wetzlar, Germany). Adobe Photoshop CS2 and Illustrator CS2 were
used to generate figures from the digital images (Adobe, San Jose, CA). Contrast
adjustments were carried out to improve clarity of images but did not alter overall
appearance. This was done equally in all channels for all images in the same figure.
siRNA Knockdown of CD63 in HUVECs
All
siRNA
duplexes
used
were
sequences
(Applied
Biosciences,
Ambion
Foster
Silencer
City,
®
CA,
Select
pre-designed
USA).
See
siRNA
supplementary
experimental procedures for sequences. Cells were nucleofected with 100 pmol of
CD63-targeting or non-targeting control siRNA (see supplementary Materials and
Methods for sequences) and then a second time 48-72h later.
confluent
15cm
petri
dish
was
used
for
4
reactions.
Typically a 70-80%
Cells
were
used
for
experimentation 48-72 hours after the second round of nucleofection.
THP-1 Adhesive Interaction Assays
Briefly (for details see supplementary methods) control or CD63-deficient HUVECs
treated with or without IL-4 were seeded into
before
analysis
on
a
microscope
perfused in media (HBSS + Ca
2+
stage
+ Mg
2+
μ-slides
maintained
at
and grown to confluence,
o
37 C
HUVECs
were
+ 0.2% BSA) with or without PMA
5
then
for 5
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6
minutes under flow, then THP-1 cells (10 /ml) were added to the media +/- PMA
(100ng/ml)
to
stimulate
or
mock
stimulate
HUVECs.
For
the
antibody
blocking
experiments sheep polyclonal anti-human P-selectin (R&D Systems, Minneapolis,
MN, USA) or IgG from sheep serum (Sigma-Aldrich, St Louis, MO) were incubated at
o
37 C with the HUVECs for 30 minutes prior to experimentation at a concentration of
25
μg/ml.
Antibody
was
added
to the
perfusion media
for
the
remainder
of
the
experiment at the same concentration. Movies were recorded to observe THP-1
adhesive interactions with the monolayer.
Scanning Electron Microscopy and Immuno-labelling
HUVECs were treated with IL-4 (0.02
experimentation
stimulated
μg/ml,
to increase total cell
with
PMA
Sigma-Aldrich) for 48 hours prior to
levels of
(100ng/ml)
for
10
P-selectin expression.
minutes
and
fixed
Cells
with
were
2%
paraformaldehyde/2% gluteraldehyde in 0.1M cacodylate for 30 minutes, Samples
were then immuno-gold labelled and processed for analysis by scanning electron
microscopy (supplementary Materials and Methods).
Duolink
®
II In situ PLA
®
(Proximity Ligation Assay)
HUVECs grown on glass coverslips were stimulated with or without 100ng/ml PMA
o
(Sigma-Aldrich) for 10 minutes at 37 C. HEK293 cells were transfected 24 hours
before experimentation with wild-type or the tail-deficient mutant of P-selectin or
left untransfected. Coverslips were fixed with 3% (w/v) paraformaldehyde in PBS
for 15 minutes at room temperature. Cells were then either permeabilised or left
4
intact and incubated with 50mM NH Cl in PBS to quench the fixative. Following the
manufacturers
Sweden)
through
Wetzlar,
was
a
instructions,
used
40X
to
a
detect
oil-immersion
Germany)
and
Duolink
®
II
Probemaker
kit
P-selectin-CD63
interactions.
lens
TCS
Z-stacks
on
a
Leica
acquired.
procedures for more detailed methods.
6
See
SPE
(Olink
Slides
confocal
supplementary
®
Bioscience,
were
system
imaged
(Leica,
experimental
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Intravital Microscopy
Male
CD63-/-
Germany.
All
description
and
mouse
of
Observations
leukocytes
mice
the
of
within
littermate
studies
controls
were
generation
of
approved
these
leukocyte-endothelial
the
mouse
were
obtained
by
mice
the
is
venules
UK
Kiel
Home
University,
Office.
given
in
Schroder
and
of
the
interactions
cremaster
from
were
as
A
et
full
al.
behaviour
previously
13.
of
described
16
Venules were observed 10-30 min after surgical stimulation of cremaster, when
leukocyte
rolling
is
exclusively
P-selectin-dependent
17.
Venules
were
observed
using a Nikon E600 FN microscope (Nikon U.K.) equipped with a water immersion
objective (20x/0.5W). Leukocyte rolling was observed using brightfield illumination
and centreline velocities of the observed venules were measured in real time using a
dual
photodiode
velocimeter
and
on-line
digital
cross-correlation
program
(Microvessel Velocity OD-RT System, Circusoft Instrumentation LLC, Hockessin, DE).
Leukocyte
rolling
fluxes
before
and
after
anti-P-selectin
antibody
(RB40.34,
30mg/mouse i.v.) were normalized and expressed as % control rolling.
Thioglycollate-Induced Peritonitis
Peritoneal neutrophil influx after thioglycollate
previously described
18
administration was measured as
.
Subcellular Fractionation of WPB
Two days post-nucleofection, medium was aspirated from HUVECs that were then
washed
with
PBS
then
homogenisation
protease inhibitors (Sigma).
by
centrifugation,
and
cell
buffer
(HB)
at
o
4 C
supplemented
with
Cells were scraped into a small volume of HB, collected
pellets
resuspended
passes through a ball-bearing homogenizer (10
in
μm
HB
then
homogenized
by
12
clearance). Homogenates were
o
centrifuged at 1000 g for 5 min (4 C) and supernatants retained. The pellets were
then resuspended, homogenized and centrifuged as described. The supernatants
from the second and first centrifugations were ten pooled, diluted to 3 ml with HB,
loaded on discontinuous sucrose gradients (1.0 M, 2 ml; 1.1 M, 2 ml; 1.3 M, 2 ml; 1.6
7
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M, 1.5 ml; and 1.8 M, 1.5 ml) and centrifuged at 36000 rpm (SW40ti) for 90 min
o
(4 C). Fractions were collected from the top and probed for their VWF content by
ELISA, and SDS-PAGE and western blotting carried out to analyse P-selectin levels in
the WPB peak, quantified by densitometric analysis using a Molecular Imager GS-
800
calibrated
densitometer
and
ImageJ
and
normalised
to
levels
of
VWF.
See
supplementary experimental procedures for a full description.
Surface Biotinylation Assay
Mock
treated
or
CD63
siRNA
treated
HUVECs
were
incubated
with
serum-free
media alone or containing PMA (100ng/ml) for 5, 15, 30, 45 and 60 minutes. Cells
were transferred to ice and washed with PBS,
incubated with EZ-Link
® Sulfo-NHS-
LC-Biotin (Thermo Scientific, MA, USA) (0.2mg/ml in ice-cold PBS) for 30 minutes,
rinsed twice with ice-cold PBS and residual biotin quenched with ice-cold 50mM
Tris-HCL pH 7.5 in PBS for 5 minutes. Cells were then lysed with a RIPA buffer-
protease
inhibitor
cocktail
(Sigma)).
Lysates
were
incubated
with
NeutrAvidin
o
agarose beads (Thermo Scientific, MA, USA) for 1 hour rotating at 4 C. Protein was
eluted
from
the
beads
by
addition
of
20
μl
Laemmli
sample
buffer
followed
by
o
incubation at 95 C for 5 minutes. Samples were analysed by SDS-PAGE and Western
blotting
to
determine
P-selectin
expression.
procedures for a more detailed description.
8
See
supplementary
experimental
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Results
Loss of CD63 does not affect WPB formation, exocytosis or maturation
Efficient knockdown of CD63 was achieved with two rounds of nucleofection using a
pool of two siRNA oligonucleotides. CD63 is clearly absent from HUVECs as shown
by immunofluorescence staining (Figure 1A) where it has disappeared from WPB
(labelled by antibodies to their major content protein, von Willebrand factor), and
late endosomes (the large peri-nuclear punctae present in control cells, clearly seen
as red punctae in the merged image). Cell numbers, morphology, and the formation
and
cellular
distribution
of
WPBs
remained
indistinguishable
from
control
cells
(Figure 1A). siRNA depletion of CD63 typically reduced protein levels by at least
95% as compared to cells transfected with two rounds of control siRNA as shown by
western blotting (Figure 1B).
One possible function of CD63 might be in WPB exocytosis: WPB are lysosome-
related organelles
19
and CD63 can influence lysosome/plasma membrane fusion
20,
We therefore measured amounts of VWF secreted from control and CD63 depleted
HUVECs. No significant difference in basal or PMA stimulated VWF release was seen
(Figure 1C). We also assessed the maturity of the WPBs that are released from
CD63-depleted versus control cells through analyses of the multimerisation state
and functionality of secreted VWF. Since multimerisation of VWF continues after the
initial formation of WPB
21,
the state of VWF at exocytosis is an effective measure of
granule maturity. We observed that CD63-depleted cells produce normal multimers
and release VWF strings
5
(that function in platelet-catching) of normal length and
quantity (Figure S1), suggesting that WPB are fully mature when released. Loss of
CD63
therefore
maturation
of
has
no
WPBs.
demonstrable
We
hypothesised
impact
that
it
on
the
might
formation,
instead
be
exocytosis
involved
in
or
a
subsequent inflammatory function of endothelial cells, i.e. after WPB exocytosis.
Loss of CD63 leads to a failure of P-selectin-dependent rolling by monocyte-like
THP-1 cells on HUVECs
9
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To
determine
a
post-exocytic
role
for
CD63
in
the
inflammatory
function
of
endothelial cells, we analysed recruitment under flow of the PSGL-1-positive human
monocytic
cell
line
THP-1
to
HUVECs
in
vitro.
We
performed
experiments
on
undifferentiated cells (THP-1 and HUVEC) and HUVEC were stimulated only briefly
before
THP-1
addition,
ensuring
a
small
repertoire
present. HUVECs were grown to confluence in
μ-slide
to a pump to generate a shear stress of 0.7 dyn/cm
2
of
adhesion
molecules
are
chambers (Ibidi), connected
and THP-1 cells then perfused
over control and CD63-ablated HUVECs briefly activated with PMA. The data show a
set of adhesive interactions (Materials and Methods) between the THP-1 cells and
HUVECs that are PMA and P-selectin dependent (as blocked by a specific antibody),
and that disappear when the endothelial cells lack CD63 (Figure 2A, and Video 1, 2
and 3,
supplementary data). These interactions were not inhibited by a non-specific
antibody (data not shown). Numbers of firmly adherent cells were also determined
(Figure. 2B) and showed the same pattern of PMA-, P-selectin- and CD63-dependent
adherence of THP-1 cells. We then treated HUVECs with IL-4 for 24h before PMA
stimulation,
to
integrin levels
22
determine
whether
upregulated
P-selectin
and
pro-inflamatory
affect the pattern of adherence. IL-4 treatment does indeed increase
the number of firmly adherent cells, and again this recruitment is dependent on P-
selectin and CD63 (Figure 2C).
These data strongly suggest a post-exocytic change
in the ability of endothelial P-selectin to function in the absence of CD63.
CD63-deficient mice show defects in leukocyte rolling and extravasa tion.
The physiological importance in vivo of CD63 to P-selectin function was determined
by examining leukocyte recruitment to the endothelium in CD63 -/- mice, firstly
confirming
15,
that loss of CD63 has no effect on leukocyte numbers (Figure S2).
Intravital microscopy of CD63 deficient mice
13
showed that rolling flux of leukocytes
was significantly reduced compared to WT mice (Figure 3A and Supplementary
Movies 4 and 5). To confirm the role of P-selectin in these experiments, P-selectin
blocking
antibody
was
injected
(RB40.34,
30
μg
intravenous
(i.v.))
into
WT
and
CD63 -/- mice (Figure 3A) within 30 minutes of surgery. Anti-P-selectin significantly
10
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(p<0.001) inhibited leukocyte rolling in WT mice whereas the minor residual rolling
observed in CD63 -/- mice was not significantly altered implying that the rolling
seen in CD63 -/- mice is not P-selectin dependent. Analysis of the rolling velocities
in WT versus CD63 -/- mice was also carried out, indicating that cells rolled at a
lower (but not significantly so) median velocity in CD63 -/- mice (Supplementary
Figure 2B). Thus altogether, the CD63-/- mice have fewer rolling cells, and those
that do roll are not doing so with the same characteristics as do those in wild-type
mice.
The loss of P-selectin-dependent leukocyte recruitment to the endothelium when
CD63
is
absent
in
vivo
strongly
suggests
that
P-selectin
is
unable
to
function
normally. If this is the case, a patho-physiological inflammatory phenotype should
be seen in CD63 -/- mice comparable to that of P-selectin -/- mice; i.e. a significant
delay in recruitment of neutrophils in a peritonitis model
the
peritoneal
ml/mouse,
i.p.)
neutrophil
in
CD63
influx
-/-
and
after
WT
23.
thioglycollate
mice
observing
We therefore measured
administration
compromised
(3%,
2
leukocyte
extravasation in CD63-/- as compared to WT mice (Figure 3B), closely mirroring the
data obtained from the P-selectin deficient mouse
23.
Taken together, the in vivo
data gives a strong indication that the presence of CD63 is essential for P-selectin to
effectively recruit leukocytes to the endothelium.
CD63 and P-selectin co-cluster at the endothelial surface.
We hypothesised that as a tetraspanin - a class of proteins that co-cluster with
associated proteins within the membrane bilayer
12,24
CD63 might affect P-selectin
function via an ability to cluster the leukocyte receptor. Loss of CD63 could reduce
clustering of P-selectin, reducing its avidity, thus explaining the loss of leukocyte
recruitment to the endothelium. To determine if this is the case we examined the
distribution of CD63
resolution
using
and
P-selectin
scanning
on
electron
the
activated
microscopy
endothelial
(SEM).
surface
HUVECs
at
were
high
PMA-
stimulated, fixed, immuno-gold labelled for P-selectin and CD63 and samples then
11
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processed for SEM (Materials and Methods). Although the labelling density is quite
variable,
presumably
reflecting
variation
of
stimulation-response
and
P-selectin
expression at the level of individual cells, P-selectin and CD63 do co-cluster, with
some
clusters
shown
in
containing
iv-viii).
As
up
to
negative
25
gold
particles
controls
primary
(Figure
and
4Ai-iii,
bridging
magnifications
antibodies
were
omitted, resulting in very few gold particles. Quantification of CD63 and P-selectin
co-clustering
(Figure
4B)
shows
that
65.3%
of
clusters
contain
both
proteins
compared to just 11.5% and 23.2% containing only P-selectin or CD63 respectively.
The clusters are a variety of shapes as exemplified in the enlarged images in Figure
4A. The commonest form has gold particles on top of a membrane structure (Figure
4A
compare
iv-viii
upper
and
lower
panels),
but
some
clusters
have
the
gold
particles arranged around an outer perimeter, as shown in Figure 4A viii.
To obtain an independent confirmation of this associative behaviour between P-
selectin
proteins
and
CD63,
within
we
used
a
proximity
20-30nm of each other.
ligation
Their
assay
for
heterotypic
proximity allows
pairs
of
hybridisation
of
complementary oligonucleotides attached to their cognate antibodies, which then
supports a PCR-driven
fluorescent readout. To ensure that our PLA reflects the
relative localisation of CD63 and P-selectin, we assayed their behaviour in an easily
manipulated non-endothelial model system. HEK-293 cells, which express CD63 but
not P-selectin, were transfected so as to express either wild-type P-selectin, which
travels to the cell surface and then internalises and is targeted to the lysosome, or a
variant form lacking a cytoplasmic tail that accumulates on the plasma membrane
19.
The variant P-selectin accumulating at the plasma membrane should give rise to a
higher number of PLA signals at the surface than the wild-type receptor for a similar
level of protein expression. This was indeed the case, thus even at the high levels of
expression found in HEK293 cells, and when normalised to the level of P-selectin in
each
individual
cell,
a
significantly
higher
number
of
signals
were
found
at
the
surface of HEK-293 cells transfected to express the tail-deficient receptor (tail-)
than
those
expressing
the
wild-type
P-selectin
(Figure
5A,
quantified
in
B).
Conversely, there are fewer internal signals from the mutant receptor (Figure 5A,
12
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quantified in B). Thus the location of P-selectin relative to CD63 controls the PLA
signal.
In
addition,
no
PLA
signals
are
observed
when
one
probe
demonstrating the signals are interaction-dependent (data not shown).
is
omitted
In activated
HUVECs an average of 7.9 signals per cell is seen at the surface but this decreases to
only 1.7 signals per cell on average when CD63 is depleted, with a maximum of 20.7
versus
3.2
signals
per
cell
observed
in
mock
transfected
versus
CD63
depleted
samples respectively (Figure 5C, quantified in D). This data fits well with the direct
EM observation of these two proteins clustering together at the endothelial surface.
CD63 depletion leads to de-clustering and loss of surface P-selectin.
We determined whether CD63 is required for P-selectin clustering. The distribution
of P-selectin analysed by SEM in control and CD63 siRNA treated cells showed that
in
CD63
depleted
cells
reduced
P-selectin
was
found
in
clusters
(Figure
6A
quantified in B and C) compared to mock transfected cells, and the mean cluster size
was reduced in CD63 depleted cells with a higher frequency of smaller clusters
compared to mock transfected HUVECs. We found a 50% fall in clusters containing 4
or more P-selectin-associated gold particles in CD63-deficient cells. Finally, the total
level of P-selectin-associated gold particles is reduced by an average of 25% (114
particles vs 84 particles over the same 95 square microns of plasma membrane).
These data suggest that CD63 is indeed required for efficient clustering of P-selectin
at the surface of activated HUVECs and that in the absence of CD63 reduced levels of
P-selectin are located in smaller clusters on the plasma membrane.
These
changes
targeting,
in
12,20(and
P-selectin
distribution
could
also
reflect
changes
in
its
references within). We therefore determined total, cell surface,
and WPB levels of P-selectin in both control and CD63-deficient HUVECs. We found
that total P-selectin expression is unchanged in CD63 depleted cells compared to
control cells (Figure 7A), implying that loss of CD63 does not divert P-selectin to
lysosomes for degradation. However, P-selectin expression at the surface of resting
and PMA-stimulated CD63-deficient HUVECs is lower (in agreement with our EM
and PLA data) compared to controls over a 60 minutes time-course, in Figure 7B. An
13
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approximate four-fold increase in surface P-selectin expression is seen 15 minutes
after activation in PMA-stimulated mock-transfected HUVECs compared to resting
cells. This response to PMA is reduced by around 30% in CD63 depleted HUVECs,
and this significant differential is maintained over 60 minutes of post-exocytosis
analysis. In the relatively slow and continuous exocytic response shown by PMA-
stimulated
relatively
HUVECs
25,
we
synchronous
do
not
see
disappearance
a
clear
as
appearance
compensatory
phase
followed
endocytosis
by
a
commences
(Figure 7B). The difference in surface levels during this assay could thus reflect an
increase in internalisation of P-selectin or a lower delivery to the plasma membrane,
or both. We therefore analysed the levels of P-selectin within WPB to determine
whether loss of CD63 reduces P-selectin available for delivery to the surface. Sub-
cellular fractionation on a discontinuous sucrose gradient resolves VWF into one
major and three minor peaks, and the distribution of over-expressed Rab27a-GFP, a
marker
for
mature
WPB,
confirms
that
peak
3
contains
the
bulk
of
secretory
organelles, with a smaller fraction being within peak 4 (Figure 7C). In addition,
following
60 minutes of
PMA stimulation
VWF levels in
peak 3 are reduced
by
approximately 40% due to fusion of WPB at the plasma membrane, confirming that
this peak contains the majority of functionally active WPB (data not shown). Loss of
CD63 has no effect on the numbers of WPB (measured by total VWF content) within
HUVECs,
quantitatively
confirming
the
impression
from
immunofluorescence
(Figure 1A) and consistent with the unchanged release of VWF (Figure 1C). Western
blotting followed by
densitometric
quantification
shows
that
levels of
P-selectin
within the WPB, when normalised to levels of VWF, are unchanged by loss of CD63
(Figure 7D). The likeliest explanation of the observed fall in levels and clustering of
P-selectin at the cell surface is therefore of an increase in re-internalisation of P-
selectin.
14
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Discussion.
We report here that P-selectin cannot effectively recruit leukocytes to endothelial
cells lacking CD63. CD63 acts as an essential co-factor; its loss is as deleterious for
leukocyte
recruitment
in
vitro,
or
reported for loss of P-selectin itself
in
an
inflammatory
response
in
vivo,
as
that
23.
Our investigation shows that levels of P-selectin at the plasma membrane are clearly
reduced, by 25% as measured by numbers of P-selectin-associated Gold particles, or
by 30% as measured biochemically by surface biotinylation and western blotting.
Delivery of P-selectin to the cell surface, however, appears unaffected. The best
explanation for a change in plasma membrane P-selectin levels is therefore that
internalisation of P-selectin increases. Since CD63 is known to regulate the surface
levels
of
membrane
proteins,
function of this tetraspanin
Oligomerisation
of
12,20
this
finding
is
in
line
with
current
views
on
the
leukocytes
26-28.
(and references within).
P-selectin
enhances
its
ability
to
bind
Dimerisation of P-selectin occurs during biosynthesis of the receptor, is maintained
at
the
plasma-membrane
increased shear stress
suggested
as
a
26.
strategy
28,
and
is
Clustering
to
important
of
achieve
in
maintaining
P-selectin into
increased
even
avidity
29.
tethering
larger groups
Certainly
receptor, PSGL-1, is found clustered within membrane extensions
30.
the
under
is also
counter-
It is likely that
multimerisation of leukocyte receptors will increase their avidity, and this may be
particularly
important
for
P-selectin
as
it
forms
bonds
to
decelerate
leukocytes
under flow.
Despite
the
importance
of
the
oligomeric
state
of
P-selectin
at
the
plasma
membrane, the micro-distribution of P-selectin has not previously been established
by high resolution EM on the surface of endothelial cells. We used scanning EM to
find that that clusters of up to 200
seen,
analogous
to
those
nm that contain both P-selectin and CD63 can be
involving
other
adhesion/tetraspanin
complexes
31.
Interestingly, the P-selectin clusters are similar in size to the clusters of its counter-
15
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receptor PSGL-1 found on the membrane protrusions of leukocytes
30.
The exact
numbers of molecules within P-selectin clusters is unclear, since the relationship
between numbers of gold particles and the numbers of either CD63 or P-selectin
molecules that each particle represents is unknown. Each gold particle is likely to be
coated with about 10 IgG molecules (manufacturers data), which implies perhaps a
maximum of 5 receptors per particle could be engaged (only up to half of a spherical
particle could be facing the cell).
Our EM data suggests that a fall of only 20% in P-selectin within clusters is sufficient
to cause a near-total loss of receptor function, perhaps because the contribution of
the larger clusters is disproportionately important; Clusters containing 4 or more
gold particles which make up around only 25% of all clusters (using a baseline of
those observed in mock treated HUVECs) were reduced by 53% in CD63 depleted
cells, as against a reduction of only 20% of all clusters. These numbers suggest that
these
larger
clusters
are
more
important
to
the
capture
of
THP-1
cells
to
roll.
Further, the EM analyses are likely to underestimate the size of the larger clusters
since steric hindrance will have the biggest effect on gold-binding to the largest
clusters.
The
actual
cluster
size
for
P-selectin
molecules
may
therefore
be
significantly larger than suggested by simply multiplying numbers of gold particles
by how many receptors each could potentially engage.
CD63
TEM
cells
12
33)
has been found in the tetraspanin microdomains (known as TERM
32;
or
that generally act to cluster a variety of proteins within a bilayer in different
to
promote
different
functions.
These
properties,
and
the
very
close
association with P-selectin that we see by SEM and by in situ PLA (Duo-link II)
analysis, strongly argue that P-selectin and CD63 do indeed interact. One question is
where they might come together. We have not directly addressed this, but we note
that when the trafficking adaptor AP-3 is ablated
14,
CD63 traffics to the cell surface
directly, not via WPB, yet leukocyte rolling is unaffected. Thus P-selectin and CD63
may associate into functional structures at the plasma membrane, although this
does not rule out the possibility that they normally assemble within the WPB.
16
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Our data suggest a model in which CD63 acts to cluster P-selectin, retaining it at the
plasma membrane. How might this occur? One simple explanation would be that
association of these two molecules in itself inhibits the internalisation of P-selectin
from the plasma membrane. In principle, clustering P-selectin into larger structures
might slow internalisation. However, both partners have cytoplasmic motifs that
can bind AP-2
34-36
and thus support clathrin and accessory protein recruitment to
drive endocytosis; clustering into a pre-sorted patch of AP-2 –binding membrane
proteins is therefore arguably more likely to drive surface down-regulation of P-
selectin, especially since many clusters are potentially small enough to fit into a
clathrin-coated vesicle
Alternatively, clustering of P-selectin/CD63 into the tetraspanin adhesion domains
reported by Barreiro et al.
31
might sequester P-selectin away from internalisation.
We regard this as possible since CD63 co-clusters with CD9 and CD81 in HeLa cells
and CD9 and CD81 cluster with ICAM and VCAM in endothelial cells
37.
33
Importantly,
incorporation into such tetraspanin domains affects surface residence for otherwise
itinerant receptors
includes
adhesion
38.
In addition, if P-selectin was thereby present in a domain that
molecules
acting
later
within
the
adhesion
cascade
then
the
handover of decelerated leukocytes to proteins such as ICAM or VCAM would be
facilitated.
If P-selectin is either incorporated into large stable clusters via its association with
CD63,
or
is
internalised
after
its
rapid
appearance
presumably when not functionally engaged
39
at
the
plasma
membrane
–
- its residence at the surface (i.e. its
recruitment to/disengagement from the tetraspanin domain via its binding to CD63)
could
be
regulated.
This
might
be
by
Phosphorylation of P-selectin has been reported
influencing
association
with
tetraspanin
post-translational
40-42
modification.
43
as has S-acylation
microdomains
for
other
(reportedly
proteins
24).
Alternatively, since internalisation of CD63 can be controlled by an association with
17
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syntenin
44,
or L6
45,
this in turn may control the trafficking of P-selectin together
with CD63.
It
has
been
reported
that
the
rate
of
internalisation
of
P-selectin
and
its
co-
localisation with the endocytic clathrin adaptor AP-2 correlate with its ability to
support leukocyte rolling
might
concentrate
46.
(14). These findings suggested that the clathrin lattice
P-selectin
molecules
into
clusters,
thus
increasing
its
avidity.
Similar findings have been reported for the transcriptionally-upregulated leukocyte
receptor E-selectin
47.
This concentration of the selectins into patches presumably
increases the number of bonds formed within a tether, lengthen the lifetime of the
interaction and enhance rolling stability. How the counter-intuitive demonstration
of a reduction in clathrin-mediated internalisation efficiency, (i.e. an increase in
surface
residency)
of
P-selectin
correlates
with
reduced
leukocyte
recruitment
relates to our own data is unclear. EM analyses showing tetraspanin microdomains
adjacent to clathrin-coated domains or pits
33
potentially explains the apparent co-
localisation of P-selectin with the endocytic clathrin adaptor AP-2 seen by the lower
resolution of diffraction limited light microscopy
46.
However, our data showing that
reduced levels of P-selectin at the cell surface correlate with reduced leukocyte
recruitment does provide a simpler explanation of the relationship between surface
behaviour and functioning of P-selectin.
Several
previous
leukocyte
major
investigations
adhesion
cell-type
and
involved,
have
migration
platelets,
suggested
within
have
the
not
a
role
vascular
yet
been
for
leukocyte
system
48,49,50.
CD63
The
investigated e.g.
are
in
other
their
alpha-granules also affected by loss of CD63? However, leaving aside speculations
for a yet wider role, already the current existing data plus our own experiments
together suggest that CD63 is actually a major player in the inflammatory adhesion
cascade, acting at multiple points not only within leukocytes but now also- and
essentially- the endothelium, to ensure efficient leukocyte migration from plasma to
tissue.
18
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Acknowledgements
This
work
was
supported
by
the
UK
Medical
Research
Council
(grant
U12260000200001) to DFC and by the Deutsche Forschungsgemeinschaft and the Center
of Excellence “Inflammation at Interfaces” to P.S.
The authors thank
Marie O’Connor
for helpful comments and statistical expertise, Ian White, Jemima Burden and Andrew
Vaughan
for
their
assistance
with
scanning
electron
and
light
microscopy,
all
members of the Cutler lab for their valuable comments, Keith Norman for early
suggestive experiments, Dr Mark Ariaans for technical assistance, Bernd Schröder
for assistance with the mice, and Mark Marsh for anti-CD63 and THP-1 cells.
Author Contributions
E.D. performed research on HUVECs, data analysis, and co-wrote the manuscript.
V.R. performed research on mice, data analysis and co-wrote the manuscript. F.F.
and
M.T.
performed
research
on
HUVECs.
P.S.
analysed
data
and
co-wrote
the
manuscript. DC conceived of, managed the project, analysed data and co-wrote the
manuscript.
Conflict of interest disclosure: The authors disclose no conflicts.
19
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References
1.
Weibel ER, Palade GE. New Cytoplasmic Components in Arterial Endothelia.
Cell Biol.
2.
J
1964;23:101-112.
Wagner DD, Olmsted JB, Marder VJ. Immunolocalization of von Willebrand
protein
in
Weibel-Palade
bodies
of
human
endothelial
J
cells.
Cell
Biol.
1982;95(1):355-360.
3.
Bonfanti
component
R,
of
Furie
BC,
Furie
Weibel-Palade
B,
Wagner
bodies
of
DD.
PADGEM
human
(GMP140)
endothelial
cells.
is
a
Blood.
1989;73(5):1109-1112.
4.
Vischer UM, Wagner DD. CD63 is a component of Weibel-Palade bodies of
Blood.
human endothelial cells.
5.
Dong
secreted
JF,
Moake
ultralarge
JL,
von
Willebrand
under flowing conditions.
6.
Semple
JW,
1993;82(4):1184-1191.
Nolasco
Blood.
Freedman
L,
et
al.
factor
ADAMTS-13
multimers
on
rapidly
the
cleaves
endothelial
newly
surface
2002;100(12):4033-4039.
J.
Platelets
and
innate
immunity.
Cell Mol Life Sci.
2010;67(4):499-511.
7.
and
Pendu R, Terraube V, Christophe OD, et al. P-selectin glycoprotein ligand 1
beta2-integrins
cooperate
in
the
adhesion
of
leukocytes
to
von
Willebrand
factor.
Blood.
8.
McEver RP, Beckstead JH, Moore KL, Marshall-Carlson L, Bainton DF. GMP-
2006;108(12):3746-3752.
140, a platelet alpha-granule membrane protein, is also synthesized by vascular
endothelial
cells
and
is
localized
in
Weibel-Palade
bodies.
J
Clin
Invest.
1989;84(1):92-99.
9.
McEver RP. Selectins: lectins that initiate cell adhesion under flow.
Cell Biol.
10.
2002;14(5):581-586.
McEver RP, Martin MN. A monoclonal antibody to a membrane gl ycoprotein
binds only to activated platelets.
11.
the
Curr Opin
J Biol Chem.
1984;259(15):9799-9804.
Larsen E, Celi A, Gilbert GE, et al. PADGEM protein: a receptor that mediates
interaction
of
activated
platelets
with
neutrophils
and
monocytes.
Cell.
1989;59(2):305-312.
12.
Pols MS, Klumperman J. Trafficking and function of the tetraspanin CD63.
Cell Res.
13.
Exp
2009;315(9):1584-1592.
Schroder
J,
Lullmann-Rauch
R,
Himmerkus
N,
et
al.
Deficiency
of
the
tetraspanin CD63 associated with kidney pathology but normal lysosomal function.
Mol Cell Biol.
14.
2009;29(4):1083-1094.
Harrison-Lavoie
KJ, Michaux
G,
Hewlett
L,
et al.
P-selectin
different mechanisms for delivery to Weibel-Palade bodies.
Traffic.
and
CD63
use
2006;7(6):647-
662.
15.
Michaux G, Abbitt KB, Collinson LM, Haberichter SL, Norman KE, Cutler DF.
The physiological function of von Willebrand's factor depends on its tubular storage
in endothelial Weibel-Palade bodies.
16.
Ridger
support
VC,
Hellewell
leukocyte
rolling
PG,
in
Dev Cell.
Norman
vivo
2006;10(2):223-232.
KE.
when
glycoprotein ligand-1 interaction is inhibited.
20
L-
and
P-selectins
high-affinity
Am J Pathol.
collaborate
to
P-selectin-P-selectin
2005;166(3):945-952.
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
17.
Ley K, Bullard DC, Arbones ML, et al. Sequential contribution of L- and P-
selectin to leukocyte rolling in vivo.
18.
J Exp Med.
1995;181(2):669-675.
Hicks AE, Abbitt KB, Dodd P, Ridger VC, Hellewell PG, Norman KE. The anti-
inflammatory effects of a selectin ligand mimetic, TBC-1269, are not a result of
J Leukoc Biol.
competitive inhibition of leukocyte rolling in vivo.
19.
2005;77(1):59-66.
Hannah MJ, Williams R, Kaur J, Hewlett LJ, Cutler DF. Biogenesis of Weibel-
Semin Cell Dev Biol.
Palade bodies.
20.
2002;13(4):313-324.
Flannery AR, Czibener C, Andrews NW. Palmitoylation-dependent association
with
CD63
targets
the
Ca2+
sensor
synaptotagmin
VII
to
J Cell Biol.
lysosomes.
2010;191(3):599-613.
21.
Wagner DD, Marder VJ. Biosynthesis of von Willebrand protein by human
J Cell Biol.
endothelial cells: processing steps and their intracellular localization.
1984;99(6):2123-2130.
22.
Yao L, Pan J, Setiadi H, Patel KD, McEver RP. Interleukin 4 or oncostatin M
induces a prolonged increase in P-selectin mRNA and protein in human endothelial
cells.
J Exp Med.
23.
1996;184(1):81-92.
Mayadas
TN,
Johnson
RC,
Rayburn
H,
Hynes
RO,
Wagner
DD.
Leukocyte
rolling and extravasation are severely compromised in P selectin-deficient mice.
Cell.
1993;74(3):541-554.
24.
Yanez-Mo M, Barreiro O, Gordon-Alonso M, Sala-Valdes M, Sanchez-Madrid F.
Tetraspanin-enriched microdomains: a functional unit in cell plasma membranes.
Trends Cell Biol.
25.
2009;19(9):434-446.
Michaux G, Hewlett LJ, Messenger SL, et al. Analysis of intracellular storage
and
regulated
secretion
Blood.
Willebrand factor.
26.
of
3
von
Willebrand
disease-causing
variants
of
von
2003;102(7):2452-2458.
Ramachandran V, Yago T, Epperson TK, et al. Dimerization of a selectin and
its ligand stabilizes cell rolling and enhances tether strength in shear flow.
Acad Sci U S A.
27.
Proc Natl
2001;98(18):10166-10171.
Ushiyama S, Laue TM, Moore KL, Erickson HP, McEver RP. Structural and
functional characterization of monomeric soluble P-selectin and comparison with
membrane P-selectin.
J Biol Chem.
28.
Barkalow
Barkalow
FJ,
platelets and endothelial cells.
29.
at
1993;268(20):15229-15237.
KL,
Blood.
Mayadas
TN.
Dimerization
of
P-selectin
in
2000;96(9):3070-3077.
Zhu C, Long M, Chesla SE, Bongrand P. Measuring receptor/ligand interaction
the
single-bond
level:
Biomedical Engineering .
30.
Moore
KL,
Experimental
and
interpretative
issues.
Annals
of
2002;30(3):305-314.
Patel
KD,
Bruehl
RE,
et
al.
P-selectin
mediates rolling of human neutrophils on P-selectin.
glycoprotein
J Cell Biol.
ligand-1
1995;128(4):661-
671. Prepublished on 1995/02/01 as DOI.
31.
Barreiro O, Zamai M, Yanez-Mo M, et al. Endothelial adhesion receptors are
recruited
to
nanoplatforms.
32.
adherent
leukocytes
J Cell Biol.
by
inclusion
in
preformed
tetraspanin
2008;183(3):527-542.
Kurita-Taniguchi M, Hazeki K, Murabayashi N, et al. Molecular assembly of
CD46 with CD9, alpha3-beta1 integrin and protein tyrosine phosphatase SHP-1 in
human
macrophages
through
differentiation
2002;38(9):689-700.
21
by
GM-CSF.
Mol
Immunol.
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
33.
Nydegger
S,
Khurana
S,
Krementsov
DN,
Foti
M,
Thali
M.
Mapping
J Cell
tetraspanin-enriched microdomains that can function as gateways for HIV-1.
Biol.
of
2006;173(5):795-807.
34.
Owen
DJ,
Setiadi
H,
Evans
PR,
McEver
RP,
Green
SA.
A
third
specificity-
determining site in mu 2 adaptin for sequences upstream of Yxx phi sorting motifs.
Traffic.
2001;2(2):105-110.
35.
op den Camp RG, Przybyla D, Ochsenbein C, et al. Rapid induction of distinct
stress
responses
after
the
release
of
singlet
oxygen
in
Plant Cell.
Arabidopsis.
2003;15(10):2320-2332.
36.
Janvier K, Bonifacino JS. Role of the endocytic machinery in the sorting of
Mol Biol Cell.
lysosome-associated membrane proteins.
37.
Barreiro
O,
microdomains
Yanez-Mo
regulate
M,
Sala-Valdes
leukocyte
firm
M,
2005;16(9):4231-4242.
et
adhesion
al.
Endothelial
during
tetraspanin
Blood.
extravasation.
2005;105(7):2852-2861. Prepublished on 2004/12/14 as DOI 2004-09-3606 [pii]
10.1182/blood-2004-09-3606.
38.
Shoham T, Rajapaksa R, Boucheix C, et al. The tetraspanin CD81 regulates the
expression
of
compartment.
39.
CD19
McEver
metastasis.
40.
during
J Immunol.
RP.
Crovello
cell
development
Selectin-carbohydrate
Glycoconj J.
CS,
dephosphorylation
B
in
a
postendoplasmic
reticulum
2003;171(8):4062-4072.
interactions
during
inflammation
and
1997;14(5):585-591.
Furie
of
BC,
Furie
P-selectin
B.
Rapid
accompanies
phosphorylation
platelet
activation.
and
J
selective
Biol
Chem.
1993;268(20):14590-14593.
41.
Fujimoto
T,
McEver
RP.
The
cytoplasmic
Blood.
phosphorylated on serine and threonine residues.
42.
domain
of
P-selectin
is
1993;82(6):1758-1766.
Crovello CS, Furie BC, Furie B. Histidine phosphorylation of P-selectin upon
stimulation of human platelets: a novel pathway for activation-dependent signal
transduction.
43.
Cell.
1995;82(2):279-286.
Blagoveshchenskaya AD, Hewitt EW, Cutler DF. A balance of opposing signals
within
Chem.
44.
the
cytoplasmic
tail
controls
the
lysosomal
targeting
of
P-selectin.
J Biol
1998;273(43):27896-27903.
Latysheva N, Muratov G, Rajesh S, et al. Syntenin-1 is a new component of
tetraspanin-enriched
microdomains:
interaction of syntenin-1 with CD63.
45.
Lekishvili
associated
T,
antigen
Fromm
L6
E,
mechanisms
Mol Cell Biol.
Mujoomdar
(L6-Ag)
is
and
consequences
of
the
2006;26(20):7707-7718.
M,
Berditchevski
recruited
microdomains: implication for tumour cell motility.
to
the
J Cell Sci.
F.
The
tumour-
tetraspanin-enriched
2008;121(Pt 5):685-
694.
46.
in
Setiadi H, McEver RP. Signal-dependent distribution of cell surface P-selectin
clathrin-coated
pits
affects
leukocyte
rolling
under
flow.
J
Cell
Biol.
2003;163(6):1385-1395.
47.
Setiadi H, McEver RP. Clustering endothelial E-selectin in clathrin-coated pits
and lipid rafts enhances leukocyte adhesion under flow.
1998.
22
Blood.
2008;111(4):1989-
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
48.
Skubitz KM, Campbell KD, Iida J, Skubitz AP. CD63 associates with tyrosine
kinase activity and CD11/CD18, and transmits an activation signal in neutrophils.
Immunol.
49.
J
1996;157(8):3617-3626.
Radford
KJ,
Thorne
RF,
Hersey
P.
Regulation
migration by CD63 in a human melanoma cell line.
of
tumor
J Immunol.
cell
motility
and
1997;158(7):3353-
3358.
50.
Mantegazza AR, Barrio MM, Moutel S, et al. CD63 tetraspanin slows down cell
migration
and
translocates
to
the
endosomal-lysosomal-MIICs
extracellular stimuli in human immature dendritic cells.
1190.
23
Blood.
route
after
2004;104(4):1183-
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
Figure Legends
Figure
1.
Loss
maturation.
of
CD63
HUVECs
oligonucleotides,
does
were
not
treated
individually
or
affect
for
pooled,
WPB
two
formation,
rounds
directed
with
against
2
exocytosis
different
CD63
or
a
or
siRNA
control
oligonucleotide. (A) Representative maximum intensity projection confocal images
of immunofluorescence staining of CD63 and VWF and P-selectin in control and
CD63 pooled siRNA treated cells showing depletion of CD63. Merged panel shows
CD63 in red, VWF in blue and P-selectin in green. Cell morphology, WPB formation
and P-selectin recruitment to WPBs remain unaffected by transfection of siRNA.
Scale bars represent 25μm. (B) Western blot analysis of cell lysates for control and
two separate or pooled CD63 targeted siRNA treated cells showing down-regulation
of
CD63
using
mouse-anti-CD63,
with
β-tubulin
as
a
loading
control.
(C)
Representative secretion assay data for mock-transfected and CD63 depleted cells,
where cells were incubated with serum free release media (basal) or release media
containing PMA (100 ng/ml) (stimulated) for 45 minutes. Results represent % total
VWF secreted in basal and stimulated conditions. Data are shown as mean ± SEM
(n=3).
Figure 2. Loss of CD63 results in a failure of P-selectin dependent rolling and
adhesion of THP-1 cells on HUVECs. HUVECs were treated with two rounds of 2
different siRNA oligonucleotides directed against CD63 or control siRNA. HUVECs
treated with IL-4 or culture media alone were transferred to
μ-slides 24 hrs before
2
flow experiment. Slides were placed under flow at 0.7 dyne/cm , and incubated with
sheep
polyclonal
anti-human
P-selectin
(25ug/ml)
or
media
alone
followed
by
perfusion for 5 mins to ensure the monolayer was intact. HUVECs were treated with
PMA
(100ng/ml)
6
(10 /ml)
were
or
then
perfusion
added
to
media
the
alone
for
perfusion
5
mins
media
under
with
or
flow.
THP-1
without
PMA
cells
and
antibody and allowed to flow across the monolayer for 5 mins. The entire movie was
recorded for each condition. (A) Quantification of THP-1 adhesive interactions on
HUVECs
showing
a
significant
loss
of
interactions
24
in
CD63
depleted
cells.
(B)
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Quantification of THP-1 firm adhesion to HUVEC monolayers. The number of THP-1
cells firmly adhered to the monolayer after the 5 mins of perfusion was recorded for
at least 5 fields of view for each condition. (C) Quantification of THP-1 firm adhesion
to HUVEC monolayers treated with IL-4. ***P<0.001, **P<0.01 by 2 way ANOVA
comparing between PMA stimulated control-siRNA treated cells and CD63 siRNA-
treated
cells
and
1
way
ANOVA
comparing
between
control-siRNA
groups
with
Bonferroni post tests (n=3). Error bars represent standard deviation of the mean.
Figure
3.
The
effect
of
CD63
deficiency
on
P-selectin-dependent
leukocyte
rolling and neutrophil migration in vivo. Surgically-induced leukocyte rolling flux
was measured in WT littermate control (open bars) and CD63
-/-
(closed bars) mice
before and after administration of P-selectin antibody (RB40.34, 30 µg/mouse) (A).
Results are presented as mean ± SEM for n=9-12 venules from at least 4 mice per
group. *** = P<0.001 compared to wild type rolling prior to anti-P-selectin antibody
administration; NS = not significantly different. Peritoneal neutrophil influx in CD63
/-
(
) or WT mice (
-
) after thioglycollate (3 %, 2 ml/mouse, i.p.) was also measured
(B). Peritoneal lavage was performed at the times indicated and the number of
neutrophils present in the lavage fluid was determined from total and differential
cell counts. Two mice of each genotype were used for 0h time point and six mice of
each used for all other time points. Data are shown as mean ± SEM. *** P<0.001
compared to wild type control at the same time point.
Figure 4. Cell surface clustering of P-selectin and CD63 in stimulated HUVECs.
(A) IL4 treated HUVECs were grown to a confluent monolayer and stimulated with
PMA
followed
by
fixation.
Coverslips
were
labelled
with
primary
antibodies
followed by secondaries conjugated to colloidal gold particles. Samples were then
processed for scanning EM and distribution of gold particles analysed. Labelling for
P-selectin (10nm gold) and CD63 (15nm gold) shows a tendency of the two proteins
to co-cluster. Panels i-iii show 3 example backscatter images of the gold labelling of
P-selectin and CD63. Magnifications of example clusters are shown in iv-viii with
gold
particles
shown
in
backscatter
images
25
(compo)
and
corresponding
surface
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structures
200nm.
shown
(B)
in
secondary
Quantification
of
electron
scanning
images
EM
(SEI).
clusters,
All
scale
showing
bars
the
represent
percentage
of
clusters observed containing P-selectin, CD63 or both molecules together. Data was
obtained
from
5-10
separate
low
power
images
of
equal
magnification
from
4
independent experiments. A total of 538 and 994 gold particles were counted for P-
selectin and CD63 labelling respectively. Data are shown as mean ± SD.
Figure 5: Proximity ligation assay (PLA) demonstrates P-selectin and CD63
interactions. (A) Following fixation and blocking, HEK 293 cells transfected with
wild-type P-selectin, tail-deficient mutant P-selectin or non-transfected (NT) cells
were
assayed
Duolink
and
®
for
CD63-P-selectin
interactions
(surface
and
total)
using
the
II Probemaker kit following the manufacturers instructions. PLA signals
P-selectin
expression
levels
were
then
detected
using
confocal
microscopy.
Images show representative confocal maximal projections of surface PLA signals
and P-selectin. Scale bars represent 25
μm (B) The mean number of surface and total
PLA signals per cell were quantified using Volocity Software
and this value was
normalised according to the level of P-selectin expression in each cell.
A total of 8-
10 cells were analysed for each condition. (C) CD63 siRNA or mock treated HUVECs
were PMA-stimulated followed by fixation and blocking. Endothelial surface CD63 -
P-selectin interactions were assayed the same way. Images show representative
confocal maximal projections with PLA signals in red and nuclei in blue.
represent 25
Scale bars
μm. (D) Quantification of (C). Images were quantified by counting total
number of PLA signals in each image and dividing by the number of nuclei in field of
view. A total of 286 and 471 individual cells were counted for mock and CD63 KD
samples
respectively
taken
from
3
independent
experiments.
***P<0.001
by
Student t-test. Data are shown as mean ± SD.
Figure
6.
Cell
surface
P-Selectin
clustering
is
reduced
in
CD63-depleted
HUVECs. HUVECS, mock-treated or treated with two rounds of siRNA against CD63,
were grown to a confluent monolayer. Cells were PMA-stimulated and fixed then
coverslips
labelled
with
primary
antibodies
26
against
P-selectin
followed
by
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secondary
antibodies
conjugated
to
colloidal
gold
particles.
Samples
were
then
processed for scanning EM and distribution of gold particles then quantified. Scale
bars represent 200nm. (A) General views of cell surfaces indicating distribution of
P-selectin.
reveals
a
Large
inserts
reduction
in
are
the
magnifications
percentage
of
of
the
smaller
P-selectin
boxes.
found
in
Quantification
clusters
(B)
and
a
reduction in the mean cluster size (C) following loss of CD63. Data was obtained
from 5-10 separate low power
images
for each condition
across 4
independent
experiments. (B,C) ***P<0.001 by Student t test. Data are shown as mean ± SD.
Figure
7.
P-Selectin
cell
surface
delivery
and
expression
levels
in
CD63-
depleted HUVECs. Western blot analysis of whole cell lysates for mock transfected
and CD63 depleted cells. Blots were probed with sheep polyclonal-anti- human P-
selectin
and
mouse
anti-
β-tubulin
as
a
loading
control.
The
blot
is
one
representative result from 3 independent experiments. (B) Biotinylation assay to
determine surface levels of P-selectin in mock transfected and CD63 depleted cells.
IL-4
treated
mock-transfected
or
CD63
siRNA
transfected
HUVECs
were
PMA-
stimulated for 0-60 minutes followed by incubation with non-cell permeable biotin
on ice for 30 minutes to label surface proteins. Cells were subsequently lysed and
biotinylated proteins pulled down using Neutravidin agarose beads. Protein was
eluted by boiling with sample buffer and P-selectin levels analysed by SDS-PAGE
followed by immuno-blotting for P-selectin using sheep polyclonal anti-human P-
selectin. Quantification by densitometry analysis (ImageJ) of the immuno-blots are
shown.
Relative
band
intensity
is
shown,
normalised
to
P-selectin
surface
expression in non-stimulated mock HUVECs. ***P<0.001 by two-way ANOVA. Data
are shown as mean ± SD (n=3-10 for each time point). (C) Sub-cellular fractionation
of HUVECs treated with control or CD63-targeted siRNA was performed using a
sucrose
step-gradient
percentage total
for
two
VWF across
independent
the gradient
experients.
The
for both conditions.
graph
shows
the
The major peaks
referred to in the text are labelled 1-4. In addition, a western blot below shows the
distribution of over-expressed Rab27a-GFP, a marker for WPB. (D) Western blotting
27
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of the WPB peak fractions for mock-transfected and CD63 siRNA treated cells was
performed to analyse levels of P-selectin.
Densitometric analysis of the immuno-
blots was carried out to quantify protein expression levels, normalising to VWF
expression.
The graph shows the change in P-selectin expression in CD63 depleted
cells compared to mock treated, which are set at 1. Two separate biological repeats
were performed for each condition.
28
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Prepublished online July 29, 2011;
doi:10.1182/blood-2010-11-321489
CD63 is an essential co-factor to leukocyte recruitment by endothelial
P-selectin
Emily L. Doyle, Victoria Ridger, Francesco Ferraro, Mark Turmaine, Paul Saftig and Daniel F. Cutler
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