CXC and CC Chemokine Receptors on Coronary and Brain Endothelia

Abstract

Chemokine receptors on leukocytes play a key role in inflammation and HIV-1 infection. Chemokine receptors on endothelia may serve an important role in HIV-1 tissue invasion and angiogenesis.
The expression of chemokine receptors in human brain microvascular endothelial cells (BMVEC) and coronary artery endothelial cells (CAEC) in vitro and cryostat sections of the heart tissue was determined by light and confocal microscopy and flow cytometry with monoclonal antibodies. Chemotaxis of endothelia by CC chemokines was evaluated in a transmigration assay.
In BMVEC, the chemokine receptors CCR3 and CXCR4 showed the strongest expression. CXCR4 was localized by confocal microscopy to both the cytoplasm and the plasma membrane of BMVEC. In CAEC, CXCR4 demonstrated a strong expression with predominantly periplasmic localization. CCR5 expression was detected both in BMVEC and CAEC but at a lower level. Human umbilical cord endothelial cells (HUVEC) expressed strongly CXCR4 but only weakly CCR3 and CCR5. Two additional CC chemokines, CCR2A and CCR4, were detected in BMVEC and CAEC by immunostaining. Immunocytochemistry of the heart tissues with monoclonal antibodies revealed a high expression of CXCR4 and CCR2A and a low expression of CCR3 and CCR5 on coronary vessel endothelia. Coronary endothelia showed in vitro a strong chemotactic response to the CC chemokines RANTES, MIP-1alpha, and MIP-1beta.
The endothelia isolated from the brain display strongly both the CCR3 and CXCR4 HIV-1 coreceptors, whereas the coronary endothelia express strongly only the CXCR4 coreceptor. CCR5 is expressed at a lower level in both endothelia. The differential display of CCR3 on the brain and coronary endothelia could be significant with respect to the differential susceptibility of the heart and the brain to HIV-1 invasion. In addition, CCR2A is strongly expressed in the heart endothelium. All of the above chemokine receptors could play a role in endothelial migration and repair.

Full text

Molecular
Medicine
5:
795-805,
1999
Molecular
Medicine
0
1999
The
Picower
Institute
Press
CXC
and
CC
Chemokme
Receptors
on
Coronary
and
Brain
Endothelia
Omri
Berger,"8
Xiaohu
Gan,2'8
Chandrasekhar
Gujuluva,"
8
Alan
R.
Burns,4
Girija
Sulur,3
Monique
Stins,'
Dennis
Way,6
Marlys
Witte,6
Martin
Weinand,6
Jonathan
Said,3
Kwang-Sik
Kim,'
Dennis
Taub,7
Michael
C.
Graves,'
and
Milan
Fiala8
Departments
of
'Neurology,
2Microbiology
and
Immunology,
and
3Pathology,
UCLA
School
of
Medicine,
Los
Angeles,
California,
U.S.A.
4Department
of
Medicine,
Section
of
Cardiovascular
Sciences
and
Leukocyte
Biology,
Baylor
College
of
Medicine,
Houston,
Texas,
U.S.A.
5Division
of
Infectious
Diseases,
Children's
Hospital
of
Los
Angeles,
Los
Angeles,
California,
U.S.A.
6Department
of
Surgery,
University
of
Arizona,
Tucson,
Arizona,
U.S.A.
7National
Institute
on
Aging,
Baltimore,
Maryland,
U.S.A.
8Department
of
Medicine,
West
Los
Angeles
VA
Medical
Center,
Los
Angeles,
California,
U.S.A.
Accepted
October
25,
1999.
Abstract
Background:
Chemokine
receptors
on
leukocytes
play
a
key
role
in
inflammation
and
HIV-
1
infection.
Chemo-
kine
receptors
on
endothelia
may
serve
an
important
role
in
HIV-
1
tissue
invasion
and
angiogenesis.
Materials
and
Methods:
The
expression
of
chemokine
receptors
in
human
brain
microvascular
endothelial
cells
(BMVEC)
and
coronary
artery
endothelial
cells
(CAEC)
in
vitro
and
cryostat
sections
of
the
heart
tissue
was
determined
by
light
and
confocal
microscopy
and
flow
cytometry
with
monodonal
antibodies.
Chemotaxis
of
endothelia
by
CC
chemokines
was
evaluated
in
a
trans-
migration
assay.
Results:
In
BMVEC,
the
chemokine
receptors
CCR3
and
CXCR4
showed
the
strongest
expression.
CXCR4
was
localized
by
confocal
microscopy
to
both
the
cytoplasm
and
the
plasma
membrane
of
BMVEC.
In
CAEC,
CXCR4
demonstrated
a
strong
expression
with
predominantly
periplasmic
localization.
CCR5
expression
was
detected
both
in
BMVEC
and
CAEC
but
at
a
lower
level.
Human
umbilical
cord
endothelial
cells
(HUVEC)
expressed
strongly
CXCR4
but
only
weakly
CCR3
and
CCR5.
Two
additional
CC
chemokines,
CCR2A
and
CCR4,
were
de-
tected
in
BMVEC
and
CAEC
by
immunostaining.
Immu-
nocytochemistry
of
the
heart
tissues
with
monodonal
antibodies
revealed
a
high
expression
of
CXCR4
and
CCR2A
and
a
low
expression
of
CCR3
and
CCR5
on
coronary
vessel
endothelia.
Coronary
endothelia
showed
in
vitro
a
strong
chemotactic
response
to
the
CC
chemokines
RANTES,
MIP-la,
and
MIP-1,8.
Conclusions:
The
endothelia
isolated
from
the
brain
display
strongly
both
the
CCR3
and
CXCR4
HIV-
1
core-
ceptors,
whereas
the
coronary
endothelia
express
strongly
only
the
CXCR4
coreceptor.
CCR5
is
expressed
at
a
lower
level
in
both
endothelia.
The
differential
dis-
play
of
CCR3
on
the
brain
and
coronary
endothelia
could
be
significant
with
respect
to
the
differential
sus-
ceptibility
of
the
heart
and
the
brain
to
HIV-1
invasion.
In
addition,
CCR2A
is
strongly
expressed
in
the
heart
endothelium.
All
of
the
above
chemokine
receptors
could
play
a
role
in
endothelial
migration
and
repair.

796
Molecular
Medicine,
Volume
5,
Number
12,
December
1999
Introduction
Chemokines
(up
to
50)
are
members
of
the
che-
mokine
superfamily
that
were
initially
identified
as
chemotactic,
low-molecular-weight
peptides.
Chemokines
are
classified
into
CXC
(or
a),
CC
(or
(),
C,
and
CXXXC
families
(1).
Although
CXC
che-
mokines
with
the
ELR
motif
serve
as
chemoattrac-
tants
for
neutrophils
and
CC
chemokines
attract
primarily
monocytes
and
T
cells,
chemokine
dis-
tinction
based
on
leukocyte
specificity
has
become
blurred
(2).
Chemokine
receptors
belong
to
the
large
family
of
G
protein-coupled,
seven-trans-
membrane
domain
(also
called
serpentine)
recep-
tors
(3),
which
are
displayed
on
many
different
cell
types,
including
endothelia
(2).
Chemokines
or-
chestrate
monocyte
and
lymphocyte
migration
across
the
endothelial
lining
of
blood
vessels,
a
process
that
plays
a
central
role
in
pathological
states
such
as
atherosclerosis
(4),
Alzheimer's
dis-
ease
(5),
multiple
sclerosis
(6),
and
HIV-
1
enceph-
alitis
(7).
For
example,
monocyte
chemotactic
protein-
I
(MCP-
1)
is
a
pivotal
chemokine
for
monocyte
migration
in
atherosclerosis
(8),
whereas
in
multiple
sclerosis
the
chemokines
in-
terferon-y
(IFN-,y)
-inducible
protein
(IP-
10),
monokine
induced
by
IFN-y
(MIG),
and
regulated
on
activation,
normal
T
cell
expressed
and
secreted
(RANTES)
are
considered
critical
for
mononuclear
infiltration
(9).
Different
chemokines
induce
the
accumulation
of
macrophages
in
fatty
streaks
of
atherosclerosis
(10),
migration
of
monocytes
across
coronary
endothelia
in
coronary
atherosclerosis
(11,12),
and
monocyte
infiltration
of
the
brain
in
AIDS
encephalitis
(13).
In
contrast
to
the
detailed
knowledge
of
the
multiple
effects
of
chemokines
on
leukocyte
mi-
gration
and
signaling
through
chemokine
recep-
tors
(14),
the
role
of
chemokine
receptors
on
endothelia
is
just
beginning
to
be
unraveled.
The
existence
of
chemokine
receptors
on
endothelia
has
been
obscured
by
the
presence
of
chemokine
binding
sites
that
do
not
display
the
chemokine
subclass
specificities
(
15)
and
may
represent
pro-
teoglycans
(16,17).
Specific
chemokine
binding
sites
on
human
brain
microvessels
were
visual-
ized
using
a
biotinylated
chemokine
binding
as-
say
(18).
Recently,
the
presence
of
CXCR4
on
human
umbilical
cord
endothelial
cells
(HUVEC)
was
shown
using
Northern
blotting,
immunocy-
tochemistry,
and
in
situ
hybridization
(19-21).
Some
of
these
studies
also
suggest
endothelial
expression
of
other
chemokine
receptors,
such
as
CCR3,
CCR1,
CCR2,
and
CXCR1
(20),
and
CCR1,
CCR2,
and
CCR5
(18).
Chemokines
and
chemokine
receptors
play
an
important
role
in
normal
development
and
inflammatory
and
angiogenic
responses
of
endo-
thelia.
In
the
development
of
the
vascular
sys-
tem,
CXCR4,
with
its
cognate
ligand
stromal
cell-
derived
factor-
I
(SDF-
1),
is
essential
for
vas-
cularization
of
the
gastrointestinal
tract
(22).
CXCR4
is
also
involved
in
B
cell
lymphopoiesis,
bone
marrow
myelopoiesis,
and
cardiac
ventric-
ular
septum
formation
(23).
CXC
chemokines
with
the
ELR
motif,
but
not
RANTES
or
MCP-
1,
have
a
potent
chemotactic
activity
on
bovine
adrenal
gland
endothelia
in
vitro
and
in
vivo,
although
the
binding
sites
on
endothelia
have
not
been
determined
(24).
Endothelial
CXCR4
responds
to
its
ligand,
SDF-
1,
by
mobilization
of
intracellular
calcium
and
endothelial
cell
migra-
tion
(20,19).
Endothelial
cells
from
different
sites
show
significant
heterogeneity
in
antigenic
properties
(25),
function
(26),
and
morphology
(27).
The
data
presented
by
Gupta
et
al.
(20)
and
this
com-
munication
suggest
that
endothelia
may
also
dif-
fer
in
chemokine
receptor
display.
Since
the
dis-
covery
in
HUVEC
of
a
predominant
message
for
CXCR4,
there
has
been
speculation
about
the
role of
endothelial
chemokine
receptors
and
their
modulation
by
viral
proteins
and
cytokines
in
HIV-1
invasion
into
tissues
(20,28).
CXCR4
transcription
in
endothelia
is
modulated
by
sev-
eral
cytokines,
including
IFN-,y,
interleukin-
1
3
(IL-1X3),
and
tumor
necrosis
factor-a
(TNF-a)
which
are
elevated
in
AIDS
(20,21).
Further-
more,
the
viral
protein
Tat
induces
dramatic
up-
regulation
of
CXCR4
and
CCR5
in
monocyte/
macrophages,
which
transmigrate
across
the
blood-brain
barrier
in
HIV-1
encephalitis
(29).
The
expression
of
the
HIV-
1
chemokine
corecep-
tors
on
endothelia
could
be
crucial
for
viral
in-
vasion
into
the
brain
that
may
occur
either
through
paracellular
viral
transport
(30,31)
or
by
a
transcellular
route
(C.
Gujuluva
et
al.,
unpub-
lished
results).
Furthermore,
we
speculate
that
the
receptors
could
be
involved
in
the
regulation
of
monocyte/macrophage
transmigration
across
the
blood-brain
barrier
and
the
blood-brain
bar-
rier
permeability.
In
this
work,
we
have
investigated
the
pres-
ence
of
CC
and
CXC
chemokine
receptors
in
cultured
endothelia
derived
from
the
coronary
Address
correspondence
and
reprint
requests
to:
Dr.
Milan
Fiala,
Department
of
Neurology,
UCLA
School
of
Medicine,
710
Westwood
Plaza,
Los
Angeles,
CA
90095-1769,
U.S.A.
Phone:
310-206-6392;
Fax:
310-794-5094;
E-mail:
fiala@ucla.edu

0.
Berger
et
al.:
Chemokine
Receptors
on
Endothelia
797
vessels
and
brain
microvessels,
and
in
coronary
endothelia
in
the
heart
tissues.
Both
coronary
artery
endothelial
cells
(CAEC)
and
brain
micro-
vascular
endothelial
cells
(BMVEC)
display
strongly
CXCR4,
as
described
by
others,
but
BM-
VEC
differ
from
CAEC
in
a
strong
expression
of
CCR3.
CCR5
is
expressed
at
a
lower
level
in
both
endothelia.
As
shown
by
confocal
microscopy,
CCR5
and
CXCR4
are
localized
both
on
the
plasma
membrane
and
in
the
cytoplasm
but
the
cytoplasmic
localization
is
stronger
in
BMVEC
than
in
CAEC.
CAEC
migrate
in
response
to
the
CC
chemokines
RANTES,
macrophage
inflam-
matory
protein-I
a
(MIP-
1
a),
and
macrophage
inflammatory
protein-
1/3
(MIP-
1,B).
Materials
and
Methods
Cells
CAEC
(Clonetics/BioWhittaker,
San
Diego,
CA)
were
isolated
by
the
producer
from normal
hu-
man
heart
tissues
by
dissection
of
coronary
ar-
teries
and
enzymatic
digestion
and
were
propa-
gated
in
75
cm2
T.C.
flasks
(Falcon
Plastics/
Becton
Dickinson
Labware,
Franklin
Lakes,
NJ)
using
EGMR-2-MV
Bullet
Kit
medium
(Clonet-
ics/BioWhittaker).
Adult
BMVEC
were
prepared
from
surgical
brain
tissues
obtained
during
removal
of
epilep-
togenic
foci
that
were
dissociated
and
cultured
by
the
techniques
described
previously
(32).
The
outgrowing
cells
were
purified
by
fluorescence-
activated
cell
sorting
(FACS)
using
uptake
of
acetylated
low-density
lipoprotein
labeled
with
1,1'
-dioctadecyl-
3,3,3',3
'tetramethylindocarbo-
cyanine
perchlorate
(DiI-Ac-LDL)
(Biomedical
Technologies,
Stoughton,
MA)
as
described
pre-
viously
(32).
BMVEC
were
propagated
in
75
cm2
T.C.
flasks
using
RPMI
1640
medium
(Irvine
Sci-
entific,
Santa
Ana,
CA)
with
10%
fetal
bovine
serum
(FBS),
10%
NuSerum
(Collaborative
Re-
search,
Bedford,
MA),
sodium
pyruvate,
and
modified
Eagle's
medium
(MEM)
nonessential
amino
acids
and
vitamins.
Three
different
strains
(two
from
adult
brain
and
one
from
a
pediatric
brain)
were
examined
for
chemokine
receptors.
HUVEC
were
prepared
by
harvesting
umbil-
ical
veins
by
collagenase
perfusion
(33),
and
were
pooled
and
plated
in
75
cm2
T.C.
flasks
pretreated
with
0.2%
gelatin
(Difco,
Detroit,
MI).
HUVEC
were
cultured
in
a
1:1
mixture
of
M199
(Gibco
BRL)
and
astrocyte-conditioned
medium
was
supplemented
with
10%
FBS
and
10%
bovine
calf
serum
(BCS)
(Hyclone
Labora-
tories),
1%
penicillin-streptomycin
(Gibco
BRL),
1
%
fungizone
(Gibco
BRL),
1
%
HEPES
buffer
(Gibco
BRL),
1
mg/ml
heparin
(Sigma),
and
50
mg/ml
endothelial
cell
growth
supplement
(Col-
laborative
Biomedical
Products,
Bedford,
MA)
(34).
The
endothelia
were
removed
from
flasks
using
0.05%
trypsin/5
mM
EDTA
(Gibco
BRL),
seeded
onto
glutaraldehyde-cross-linked,
gela-
tin-coated
coverslips
(34)
in
24-well
plates,
and
were
grown
to
confluence
in
the
medium
appro-
priate
for
each
cell
strain.
CAEC,
BMVEC,
and
HUVEC
were
stained
100%
positively
with
the
antibody
to
von
Willebrand
factor
and
vimentin
and
were
unstained
with
antibody
to
a-smooth
muscle
actin
(all
from
DAKO,
Carpinteria,
CA).
Antibodies
Monoclonal
antibody
(MAb)
anti-CXCR4
(clones
44708.111,
44716.111,
44717.111)
(R&D
Sys-
tems),
MAb
anti-CCR3
(7B
11)
(LeukoSite),
MAb
anti-CCR5
(clones,
45502.111,
45523.111,
45549.111,
and
45531.111)
(R&D
Systems),
MAb
anti-CXCRI
(also
designated
as
IL-8RA),
and
MAb
anti-CXCR2
(also
designated
IL-8RB)
were
from
Pharmingen
International
(La
Jolla,
CA).
Goat
polyclonal
antibodies
(PAb),
anti-
CCR1,
anti-CCR2A,
anti-CCR2B,
anti-CCR3,
an-
ti-CCR4,
anti-CCR5,
anti-CXCR3,
and
anti-
CXCR-4
(also
designated
anti-fusin)
were
from
Santa
Cruz
Biotechnology
(Santa
Cruz,
CA).
The
Santa
Cruz
goat
antibodies
were
prepared
with
the
following
peptides:
LERVSSTSPSTGEHELSA-
GF
(CCR1),
KSIGRAPEASLQDKEGA
(CCR2A),
DGVTSTNTPSTGEQEVSAGL
(CCR2B),
LERTSS-
VSPSTAEPELSIVF
(CCR3),
DTPSSSYTQSTMDH-
DLHDAL
(CCR4),
ERASSVYTRSTGEQEISVGL
(CCR5),
SSSRRDSSWSETSEASYSGL
(CXCR3),
and
ALTSVSRGSSLKILSKG
(CXCR4).
CCR2A
staining
of
the
coronary
vessels
was
inhibited
with
the
peptide
KSIGRAPEASLQDKEGA.
The
antibodies
produced
by
R&D
Systems
and
LeukoSite
were
obtained
through
the
Na-
tional
Institutes
of
Health
(NIH)
AIDS
Research
and
Reference
Reagent
Program.
Immunofluorescence
and
Immunocytochemistry
The
30,000
cells
were
grown
to
confluence
for
4
days
on
gelatin-coated,
glutaraldehyde
cross-
linked
coverslips
(34).
After
fixation
for
30
min
with
3
%
paraformaldehyde
at
40C,
the
cells
were
permeabilized
with
0.1%
Triton,
incubated
for
1
hr
with
a
primary
antibody
(MAb
or
PAb
at
5

798
Molecular
Medicine,
Volume
5,
Number
12,
December
1999
,ug/ml),
washed,
and
then
incubated
with
FITC-
conjugated
secondary
antibody.
For
dual
stain-
ing,
two
primary
mouse
and
rabbit
antibodies
and
two
secondary
FITC-
or
Texas
Red-conju-
gated
antibodies
specific
for
mouse
or
goat
im-
munoglobulin
G
(IgG)
were
used.
Immunofluo-
rescent
preparations
were
viewed
using
either
an
Olympus
Research
microscope
with
a
mercury
lamp
or a
Zeiss
LSM
310
confocal
laser
micro-
scope.
Immunocytochemical
staining
of
the
en-
dothelia
was
done
using
the
DAKO
LSAB+
Per-
oxidase
kit
(DAKO)
as
described
previously
(35).
Flow-Cytometric
Analysis
of
Chemokine
Receptors
The
endothelial
cells
were
removed
from
the
flask
using
the
Cell
Dissociation
Buffer
Enzyme-
Free
(Life
Technologies,
Gaithersburg,
MD).
All
staining
steps
were
performed
on
ice.
The
endo-
thelial
cells
were
washed
with
phosphate-buff-
ered
saline
(PBS)/1%
bovine
serum
albumin
(BSA)
by
centrifugation.
The
cells
were
then
re-
suspended
in
PBS/1%
BSA
and
stained
with
2
,ug/ml
of
each
primary
antibody
(CXCR4
MAb,
CCR5
MAb,
CXCR4
MAb,
or
IgG
isotype
control)
for
30
min.
After
two
washes
with
PBS/1
%
BSA,
rabbit
anti-mouse
FITC-antibody
was
added
for
30
min.
The
cells
were
washed
two
times
and
resuspended
in
PBS/1%
BSA.
Finally,
the
cells
were
fixed
with
2%
paraformaldehyde.
Receptor
expression
on
the
endothelia
was
analyzed
by
EPICS
XL-MCO
(Coulter).
Histochemistry
Fresh
myocardium
from
an
explanted
heart
of
two
patients
with
dilated
cardiomyopathy
was
obtained
from
the
Surgical
Pathology
Division
of
the
UCLA
Department
of
Pathology.
Specimens
were
embedded
in
O.C.T.
embedding
medium
from
Sakura
Finetek
U.S.A.
(Torrance
CA)
and
snap
frozen
in
liquid
nitrogen
and
dry
ice.
Cry-
ostat
sections
were
fixed
in
4%
paraformalde-
hyde
in
0.1
M
PBS
for
30
min
at
40C.
After
washing
in
PBS
the
slides
were
incubated
with
blocking
buffer
containing
0.5%
BSA
and
then
with
the
primary
antibody
diluted
1:200
in
PBS
with
0.1%
Triton
X-100
(Sigma)
for
12-18
hr
at
4°C.
As
the
primary
antibody,
mouse
MAb
to
CCR3,
CXCR4,
or
CCR5
(R&D),
or
goat
PAb
to
CCR1,
CCR2A,
CCR2B,
CCR3,
CCR4,
or
CCR5
(Santa
Cruz
Biotechnology)
was
used.
The
slides
were
washed
in
PBS
containing
Tween
20
(PB
ST)
(Sigma)
for
5
min
and
incubated
sequen-
tially
with
biotinylated
rabbit
anti-mouse
and
biotinylated
swine
anti-rabbit
antisera
(both
from
DAKO)
for
30
min
at
room
temperature.
Slides
were
then
washed
in
PBST
and
incubated
with
streptavidin-horseradish
peroxidase
(DAKO)
for
30
min.
Following
the
diaminobenzidine
reaction,
the
slides
were
counterstained
with
hematoxylin.
In
the
case
of
the
goat
PAb,
the
second
step
con-
sisted
of
biotin-conjugated
anti-goat
antibody.
This
was
followed
by
avidin
and
biotinylated
horserad-
ish
peroxidase
(ABC
Staining
System,
Santa
Cruz
Biotechnology).
Endothelial
Chemotaxis
Assay
Chemotaxis
of
endothelia
by
CC
chemokines
was
tested
in
96-well
chemotaxis
chambers
(NeuroProbe,
Gaithersburg,
MD),
and
50
,ul
of
EGMR-2-MV
Bullet
Kit
medium
(Clonetics/Bio-
Whittaker)
with
RANTES,
MIP-l1a,
or
MIP-1,B
at
the
indicated
concentration
was
placed
in
the
lower
chamber
and
1000
endothelial
cells
in
20
,ul
medium
was
introduced
in
the
upper
well
above
the
plate
(with
8-,um
pores)
separating
the
chambers.
The
chamber
was
incubated
at
370C
for
48
hr,
then
the
plate
was
removed
and
the
cells
on
the
upper
surface
were
removed
using
a
scraper.
The
lower
surface
of
the
plate
was
stained
using
0.1%
crystal
violet
and
the
cells
were
enumerated
using
10OX
magnification
in
a
horizontal
strip
across
each
well.
The
number
of
cells
was
multiplied
by
4
to
obtain
the
total
num-
ber
of
transmigrated
cells.
Results
Coronary
and
Brain
Endothelia
Display
CCR3,
CXCR4,
and
CCR5
In
Vitro
We
investigated
the
expression
of
chemokine
re-
ceptors
in
cultured
endothelia
using
immunofluo-
rescence
and
immunoperoxidase
techniques
with
monodonal
antibody
(MAb)
(CXCR1,
CXCR2,
CXCR4,
CCR3,
and
CCR5)
and/or
polydonal
anti-
body
(PAb)
(CXCR3,
CXCR4,
CCR1,
CCR2A,
CCR2B,
CCR3,
CCR4,
and
CCR5).
The
specificity
of
each
reaction
was
shown
by
the
absence
of
staining
when
the
primary
antibody
to
a
chemo-
kine
receptor
was
omitted
or
substituted
with
an
isotype-matched
IgG
(Fig.
1J-L).
CAEC
and
BM-
VEC
showed
strongly
positive
staining
by
the
im-
munofluorescence
(Fig.
IA-I)
and
the
immuno-
peroxidase
technique
(not
shown)
with
anti-
CCR3,
-CXCR4,
and
-CCR5.
The
staining
of
BMVEC
and
CAEC
was
consistently
greater
when
the
cells
were
fixed
before
the
staining
than
when

0.
Berger
et
al.:
Chemokine
Receptors
on
Endothelia
I
Fig.
1.
Immunofluorescent
stainiing
of
CCR3,
CCR5,
and
CXCR4
chemokine
receptors
in
BMVEC
and
CAEC.
Human
brain
(A,
D,
G,
J)
and
coronary
(B,
C,
E,
F,
H,
I,
K,
L)
endothelia
were
grown
on
coverslips
coated
with
glutaraldehyde-
fixed
gelatin
(34).
Confluent
monolayers
were
stained
by
the
immunofluorescent
technique
using
MAb
against
CCR3
(IgG2A
isotype)
(A-C),
CCR5
(IgG2B
isotype)
(D-F),
CXCR4
(IgG2A
isotype)
(G-
I),
or
an
IgG
mouse
isotype
antibody,
IgG2A
(J,
K),
or
IgG2B
(L).
The
cells
were
either
permeabilized
first
with
Triton
(A,
B,
D,
E,
G,
H,
J,
K),
or
they
were
stained
without
permeabilization
(C,
F,
I,
1).
The
IgG
isotype-stained
samples
were
photographed
using
a
six
times
longer
exposure
(compared
to
MAb-stained
preparations)
to
demonstrate
the
back-
ground
autofluorescence
staining
of
the
endothelia.
they
were
stained
without
fixation
(compare
Fig.
lB
with
Fig.
IC,
Fig.
IE
with
Fig.
IF,
and
Fig.
IH
with
Fig.
11).
In
addition,
two
CC
chemo-
kine
receptors
(CCR2A
and
CCR4)
were
positively
stained
by
inmmunocytochemistry
in
both
CAEC
and
BMVEC,
and
two
CXC
chemokine
receptors
(CXCR1
and
CXCR3)
were
stained
in
BMVEC
but
not
in
CAEC
(data
not
shown).
CXCR2
was
nega-
tive
by
immunocytochemical
staining
in
CAEC
and
BMVEC
(data
not
shown).
Laser
confocal
microscopic
examination
of
CAEC
revealed
CXCR4
(Fig.
2A)
and
CCR5
(Fig.
2B)
(green)
in
a
patchy
distribution
along
the
plasma
membrane
and
von
Willebrand
factor
(VWF)
(red)
in
a
granular-to-homogeneous
pat-
tern
in
the
perinuclear
location.
In
contrast
to
this
pattern
seen
in
CAEC,
in
BMVEC
both
CCR5
and
CXCR4
were
expressed
in
the
cytoplasm
and
on
the
plasma
membrane.
CXCR4
(Fig.
3B)
was
strongly
expressed
and
showed
substantial
colo-
calization
with
VWF
in
the
cytoplasm.
CCR5
ex-
pression
was
weak
(Fig.
3A).
799

800
Molecular
Medicine,
Volume
5,
Number
12,
December
1999
Fig.
3.
Confocal
microscopy
of
the
subcellular
localization
of
CCR5
(A)
and
CXCR4
(B)
in
BM-
VEC.
BMVEC
were
grown
on
coverslips
and
stained
as
described
in
Figure
2.
Fig.
2.
Confocal
microscopy
of
the
subcellular
localization
of
CXCR4
(A)
and
CCR5
(B)
in
CAEC.
CAEC
were
grown
on
coverslips
coated
with
glutaraldehyde-fixed
gelatin
(34).
The
cells
were
double
stained
using,
as
the
primary
antibody,
MAb
to
CXCR4
or
CCR5
followed
by
the
rabbit
antibody
to
von
Willebrand
factor
and,
as
the
secondary
anti-
body,
mouse
or
rabbit
IgG
labeled
with
FITC
or
Texas
Red,
respectively.
Chemokine
receptors
CCR5
and
CXCR4
appear
as
green
and
von
Willebrand
fac-
tor
as
red.
CCR3,
CXCR4,
and
CCR5
Receptor
Density
in
CAEC,
BMVEC,
and
HUVEC
The
expression
of
chemokine
receptors
in
BMVEC,
CAEC,
and
HUVEC
was
quantified
us-
ing
flow
cytometry
performed
with
the
cells
stained
without
prior
fixation.
In
the
case
of
BMVEC,
the
receptor
density
(%
staining
by
the
specific
antibody
-
%
staining
by
the
IgG
isotype
control)
was
determined
in
two
experiments
as
follows:
CCR3,
45%
and
77%;
CXCR4,
41%
and
51%;
and
CCR5,
4%
and
5%.
In
one
additional
experiment,
CXCR4
was
40%
and
CCR5
was
27%.
In
CAEC,
the
density
was
measured
in
two
experiments
with
the
following
results:
CCR3,
11°%
and
21%;
CXCR4,
41%
and
45%,
and
CCR5,
6%
and
4%.
In
HUVEC,
the
results
were
7
%
with
CCR3,
48%
with
CXCR4,
and
8%
with
CCR5.
The
flow-cytometric
diagrams
of
a
typical
experiment
illustrate
the
above
receptor
densi-
ties,
as
follows:
in
BMVEC
the
order
was
CCR3
=
CXCR4
>
CCR5
(Fig.
4A);
in
CAEC
the
order
was
CXCR4
>
CCR3
>
CCR5
(Fig.
4B);
and
in
HUVEC
the
order
was
CXCR4>
CCR5
and
CCR3

0.
Berger
et
al.:
Chemokine
Receptors
on
Endothelia
(A)
(B)
(C)
Blak
solid:
IpG
control
Black
open:
CCR3
Gray
solid:
CCRS
Gray
open:
CXCR44
_
A-
f__j
v
J
Lb-
w
*
*-w_
6
s
P~~~~~~~~~~~~~~~..
..VW~
Fig.
4.
Chemokine
receptor
expression
in
CAEC,
BMVEC,
and
HUVEC
evaluated
by
flow
cytometry.
Endothelial
cells
BMVEC
(A),
CAEC
(B),
and
HUVEC
(C)
were
lifted
from
the
flask
in
a
cell-dissociation
buffer,
washed,
and
stained
using
the
monoclonal
antibody
to
CCR3,
CCR5,
or
CXCR4,
and
analyzed
by
flow
cytometry
as
described
in
Ma-
terials
and
Methods.
(Fig.
4C).
When
the
cells
were
fixed
first
and
stained
second,
the
percentage
of
staining
in-
creased.
CCR3,
CXCR4,
CCR5,
and
CCR2A
Are
Expressed
in
Coronary
Endothelia
of
the
Heart
Immunohistochemical
staining
of
the
human
heart
sections
was
performed
with
three
heart
specimens.
Typical
results
revealed
a
strong
(4+)
staining
of
small
venules
and
capillaries
in
the
myocardium
with
goat
polyclonal
antiserum
to
CCR2A
(Fig.
SD),
a
moderately
strong
(3+)
staining
with
a
monoclonal
antibody
to
CXCR4
(Fig.
5C),
a
moderate
(2+)
staining
with
mono-
clonal
anti-CCR5
(Fig.
SB),
and
a
weak
(1+)
staining
with
mouse
MAb
to
CCR3
(Fig.
5A).
A
positive
staining
reaction
appeared
to
involve
the
endothelial
cell
cytoplasm
as
well
as
the
cell
membrane
(Fig.
SC,
D).
IgG
isotype
control
was
negative
(Fig.
SF).
Preincubation
of
anti-CCR2A
with
its
blocking
peptide
completely
inhibited
the
staining
reaction
(compare
Fig.
SD
with
Fig.
SE).
The
results
of
immunostaining
of
the
heart
tissues
with
anti-CCRI,
CCR2B,
and
CCR4
were
negative.
RANTES,
MIP-lIa,
and
MIP-1f3
Stimulate
Migration
of
Coronary
Endothelia
CAEC
were
tested
for
the
chemotactic
responses
to
the
CC
chemokines
RANTES,
MIP-la,
and
MIP-
I,B.
Each
of
these
chemokines
produced
en-
dothelial
cell
migration
at
a
concentration
above
12.5
,ug/ml
(Fig.
6).
A
second
experiment
con-
firmed
the
chemotactic
responses
to
these
che-
mokines
but
the
threshold
for
chemotactic
activ-
ity
was
one
dilution
lower
(12.5
,ug/ml)
with
each
chemokine.
Discussion
We
have
shown
that
human
coronary
and
brain
endothelia
express
to
a
different
degree
the
CC
chemokine
receptors
CCR3
and
CCR5,
and
the
CXC
receptor
CXCR4.
Whereas
in
CAEC
usually
only
CXCR4
achieved
a
high
density,
in
BMVEC,
CCR3
and
CXCR4
were
both
highly
expressed.
CCR5
was
expressed
at
a
low
density
in
both
endothelia.
The
effect
of
successive
in
vitro
pas-
sages
of
the
endothelia
appeared
to
decrease
the
chemokine
receptor
density.
The
pattern
of
a
strong
expression
of
CXCR4
and
a
weak
expres-
sion
of
CCR3
was
also
found
in
the
coronary
vessel
endothelia
in
the
heart.
Two
other
chemo-
kine
receptors,
CCR2A
and
CCR4,
were
detected
by
immunostaining
in
both
CAEC
and
BMVEC,
and
CXCR1
and
CXCR3
were
found
in
BMVEC
only.
In
addition,
CCRS
and
CXCR4
mRNAs
were
detected
in
BMVEC
by
reverse
transcriptase
polymerase
chain
reaction
(RT-PCR)
with
five
times
greater
expression
of
CXCR4
mRNA
(C.
Gujuluva
et
al.,
unpublished
results).
The
pres-
ence
of
multiple
chemokine
receptors
in
endo-
thelia
is
supported
by
the
RT-PCR
data
of
Gupta
et
al.
(20)
in
HUVEC
and
BMVEC,
which
showed
an
abundant
transcription
of
CXCR4
and
a
I
801
I
i

802
Molecular
Medicine,
Volume
5,
Number
12,
December
1999
Fig.
5.
Coronary
endothe-
lia
display
CCR2A,
CXCR4,
CCR5,
and
CCR3
(in
this
order).
Cryostat
sections
of
the
heart
were
stained
by
the
peroxidase
technique
(DAKO)
using
the
antibody
to
CCR3
(A),
CCR5
(B),
CXCR4
(C),
IgG
isotype
control
(F),
CCR2A
(D),
or
CCR2A
absorbed
with
the
peptide
KSIGRAPEASLQD-
KEGA
(E).
weaker
transcription
of
CXCR1,
CCR1,
and
CCR2.
A
recent
RT-PCR
study
of
HUVEC
noted
the
presence
of
CXC
chemokine
mRNAs
but
not
CC
chemokine
mRNAs
(21),
and
the
authors
speculated
that
endothelial
cells
do
not
express
CC
chemokine
receptors.
However,
their
conclu-
sions
were
based
on
transcriptional
studies
and
involved
only
HUVEC.
Our
flow-cytometric
data
demonstrate
that
whereas
CXCR4
and
CCR3
are
both
highly
expressed
in
BMVEC,
only
CXCR4
is
highly
expressed
in
CAEC
and
HUVEC.
These
results
were
subjected
to
quantitative
variation,
with
preservation
of
these
relationships
between
the
receptors,
due
to
the
effects
of
cell
passage
and
fixation.
The
fixation
before
staining
in-
creased
the
staining
density
because
of
staining
of
intracellular
receptors.
These
results
strongly
suggest
that
brain,
coronary,
and
umbilical
en-
dothelia
differ
from
each
other
with
respect
to
chemokine
receptor
expression.
The
histochem-
ical
study
of
the
heart
provided
further
evidence
of
the
relevance
of
chemokine
receptors
to
in-
flammatory
responses
of
coronary
endothelia,
as
four
chemokine
receptors,
CCR2A,
CCR3,
CCR5,
and
CXCR4,
were
found
on
the
coronary
vessels
but
only
CXCR4
and
CCR2A
were
expressed
at
a
high
level,
in
agreement
with
the
above
results.
Chemokine
HIV-
1
coreceptors,
CCR5
and
CXCR4,
play
a
crucial
role
in
HIV-
1
entry
into
the
blood
cells
(36).
On
leukocytes,
CXCR4
serves
as
a
coreceptor
for
T
cell
line
tropic
"X"
strains,
whereas
the
CC
chemokine
receptor
CCR5
is
a
main
coreceptor
for
M-tropic
"R"
strains.
In
blood-derived
dendritic
cells,
which
express
CXCR4
and
CCR5
mRNAs
(37),
the
en-
try
of
M-tropic
strains
is
inhibited
by
the
chemo-
kine
RANTES
and
the
entry
of
the
IIIB
strain
by
the
chemokine
SDF-1.
CD34+
progenitors
are

0.
Berger
et
al.:
Chemokine
Receptors
on
Endothelia
803
(A)
0
_
I
I
E
z
(B)
p
300
X
250
r
_
200
*a3150
3
100
E
z
50
(C)
5
I
E
z
0
12.5
25
50
100
RANTES
Concenftron
(nghnl)
0
12.5
25
50
MIP-l
a
Concentaton
(ngiml)
100
MIP-1p
Concenration
(nglml)
Fig.
6.
Endothelial
cell
migration
across
a
Neu-
roProbe
chamber
filter.
One
thousand
CAEC
were
seeded
on
top
of
each
well
in
a
NeuroProbe
chamber
(8
,um
pore
diameter)
with
each
tested
chemokine,
RANTES
(A),
MIP-la
(B),
or
MIP-113
(C),
at
the
indicated
concentration
in
the
lower
chamber.
The
endothelia
were
allowed
to
migrate
for
48
hr,
then
the
filter
was
removed
and
the
cells
on
the
top
of
each
well
were
scraped.
After
staining
by
crystal
violet,
the
transmigrated
cells
on
the
lower
surface
of
each
well
were
determined
by
a
mi-
croscopic
count
as
described
in
Materials
and
Meth-
ods.
susceptible
to
infection
by
various
HIV-1
strains
and
cognate
ligands
for
CXCR4
and
CCR5
mod-
ulate
HIV-1
infection
(38).
CC
and
CXC
chemokine
receptors
belong
to
a
large
family
of
cell
surface,
seven-transmem-
brane
domain,
G
protein-coupled
receptors
that
bind
and
signal
upon
binding
of
one
or
more
CC
or
CXC
chemokines
(3).
CCR2
is
a
receptor
pre-
viously
detected
in
monocytes,
dendritic
cells,
natural
killer
(NK)
cells,
and
T
lymphocytes,
but
not
in
neutrophils,
and
is
thus
considered
to
be
important
in
chronic
inflammation,
including
atherosclerosis.
CCR2
has
recently
been
shown
to
function
as
a
receptor
for
MCP-
1
on
endothe-
lia,
inducing
their
chemotactic
migration
and
possibly
endothelial
wound
repair
(39).
CCR3
is
found
in
brain
microglia,
in
which
it
mediates
HIV-1
entry
(40).
CCR5
is
a
major
HIV-1
core-
ceptor,
which
plays
a
crucial
role
in
HIV
infection
of
peripheral
blood-derived
dendritic
cells,
CD34+
hematopoeitic
progenitor
cells,
and
acti-
vated/memory
Thl
lymphocytes
(41,42).
CCR5
expression
on
freshly
isolated
T
cells
is
increased
by
activation
with
IL-2
and
other
stimuli
(41).
In
contrast
to
the
pivotal
significance
of
CCR5
on
blood
cells
for
HIV-1
infection
(as
well
as
other
infections),
the
role
of
CCR5
in
neurons,
astro-
cytes,
and
other
cells
has
not
been
established.
CXCR4
is
the
major
chemokine
coreceptor
for
T-tropic
HIV-1
strains
that
is
widely
distributed
on
most
hematopoietic
cell
types,
endothelial
cells
(20),
neurons
in
both
the
peripheral
and
central
nervous
systems
(43),
microglia,
and
as-
trocytes
(40).
CXCR4
appears
to
play
an
essential
role
in
development,
as
shown
in
mice
(22).
Our
studies
of
the
heart
tissues
showed
that
CCR2A
and
CXCR4,
and,
to
a
lesser
degree,
CCR3
and
CCR5
were
displayed
on
coronary
endothelia.
We
have
not
excluded
a
possibility
that
this
staining
pattern
is
related
to
the
health
status
of
each
donor,
as
the
hearts
were
removed
before
transplantation
of
patients
with
conges-
tive
cardiomyopathy.
The
receptors
were
distrib-
uted
diffusely
in
the
endothelial
cytoplasm
and
on
the
plasma
membrane,
in
agreement
with
the
confocal
microscopic
results
in
coronary
endo-
thelia.
The
receptors
were
not
limited
to
-the
abluminal
side
of
endothelia,
as
found
by
And-
jelkovic
et
al.
(18),
by
staining
isolated
microves-
sels
with
MAb
to
CCR1,
CCR3,
and
CCR5.
The
functional
role
of
chemokine
receptors
on
brain
endothelia,
in
particular
CCR3,
CXCR4,
and,
to
a
lesser
degree,
CCR5,
could
be
signifi-
cant
for
HIV-
1
neuroinvasion.
Macrophage-
tropic
strains
circulating
at
a
very
high
titer
(107
RNA
copies/ml
plasma)
during
the
primary
in-
fection
could
find
early
entry
into
the
brain
be-
cause
of
a
high
expression
on
BMVEC
of
CCR3
and
a
variable
CCR5
expression.
Our
recent
work
strongly
suggests
that
HIV
invades
across
brain
endothelia
via
both
the
paracellular
(30,31)
and
the
transcellular
route
(C.
Gujuluva
et
al.,
unpublished
results)
without
undergoing
replication
in
BMVEC.
However,
we
have
not
I
T
m

804
Molecular
Medicine,
Volume
5,
Number
12,
December
1999
yet
obtained
conclusive
evidence
for
the
role
of
chemokine
receptors
in
viral
transcytosis.
After
the
neuroinvasion,
the
macrophage-tropic
HIV-
1
strains
utilizing
CCR3
and
CCR5
could
effectively
enter
the
microglia
via
CCR3
(40)
and
CCR5
(44).
SIV
infects
simian
brain
endothelial
cells
by
a
CCR5-dependent
mechanism,
which
is
inhib-
ited
by
the
CCR5
ligand
RANTES
(45),
but
this
route
may
be
limited
in
human
brain
endothelia
because
of
the
lower
expression
of
CCR5.
The
presence
of
CXCR4
on
brain
endothelia
may
al-
low
the
T-tropic
strains
emerging
late
in
the
dis-
ease
to
infect
the
brain.
Coronary
endothelia,
unlike
brain
endothelia,
appear
to
have
a
re-
duced
expression
of
CCR3,
which
may
limit
the
ability
of
macrophage-tropic
strains
circulating
early
after
infection
to
infect
the
heart.
The
cor-
onary
endothelia,
however,
express
CXCR4
and
may
be
susceptible
to
T-tropic
strains
that
emerge
late
in
the
course
of
AIDS,
when
HIV-1
cardiomyopathy
is
apt
to
develop.
Acknowledgments
This
work
was
supported
in
part
by
NIH
grant
DA10442
and
a
subcontract
NS
26126
to
M.F.
and
M.C.G.,
HL
48493
to
M.H.W.,
and
RO-1
NS
26310
and
RO-1
HL
61951
to
K.-S.K.A.R.B.
is
supported
in
part
by
grants
from
NIH
HL-42550,
The
Methodist
Hospital
Foundation,
a
Chao
Fel-
lowship,
and
ALA
RG
068.
The
authors
acknowl-
edge
the
UCSD
Center
for
AIDS
Research
and
NIH
DAIDS
2
P30
AI36214-07.
We
thank
Philip
Murphy
for
incisive
comments,
Matthew
Schibler
for
assistance
with
confocal
microscopy,
and
Carol
Appleton
for
technical
assistance
with
illustrations.
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