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The Chemokine Receptor CCR2 Mediates the Binding and
Internalization of Monocyte Chemoattractant Protein-1 along
Brain Microvessels
Kirk A. Dzenko,
1
Anuska V. Andjelkovic,
1
William A. Kuziel,
2
and Joel S. Pachter
1
1
Blood–Brain Barrier Laboratory, Department of Pharmacology, University of Connecticut Health Center, Farmington,
Connecticut 06030, and
2
Department of Molecular Genetics and Microbiology and Institute for Cellular and Molecular
Biology, University of Texas, Austin, Texas 78712
Previous results from this laboratory revealed the presence of
high-affinity saturable binding sites for monocyte chemoattrac-
tant protein-1 (MCP-1) along human brain microvessels (And-
jelkovic et al., 1999; Andjelkovic and Pachter, 2000), which
suggested that CC chemokine receptor 2 (CCR2), the recog-
nized receptor for this chemokine, was expressed by the brain
microvascular endothelium. To test the role of CCR2 directly in
mediating MCP-1 interactions with the brain microvasculature,
we assessed MCP-1 binding activity in murine brain microves-
sels isolated from wild-type mice and from CCR2 (⫺/⫺) mice
engineered to lack this receptor. Results demonstrate that
MCP-1 binding is greatly attenuated in microvessels prepared
from CCR2 (⫺/⫺) mice compared with wild-type controls.
Moreover, microvessels from wild-type mice exhibited MCP-1-
induced downmodulation in MCP-1 binding and a recovery of
binding activity that was not dependent on de novo protein
synthesis. Furthermore, MCP-1 was shown to be internalized
within wild-type microvessels, but not within microvessels ob-
tained from CCR2 (⫺/⫺) mice, additionally demonstrating that
CCR2 is obligatory for MCP-1 endocytosis. Last, internalization
of MCP-1, but not transferrin, was observed to be inhibited by
disruption of caveolae. Internalized MCP-1 also colocalized at
some sites with caveolin-1, a major protein of caveolae, imply-
ing that this chemokine is endocytosed, in part, via nonclathrin-
coated vesicles. These results prompt consideration that
MCP-1 signals may be relayed across the blood–brain barrier
by highly specialized interactions of this chemokine with its
cognate receptor, CCR2, along brain microvascular endo-
thelial cells.
Key words: MCP-1; receptors; brain; microvessels; blood–
brain barrier; endothelial cells
Chemokines, which are specialized cytokines, belong to a super-
family of proteins that is composed of four recognized classes
known as
␣
,

,
␥
, and
␦
, or CXC, CC, C, and CXXXC, respec-
tively, by which C refers to conserved cysteine residues, and X
refers to the number of amino acids between cysteines (Rossi and
Zlotnick, 2000). As their name implies, chemokines act as che-
motactic factors that direct the emigration of leukocytes to select
tissue locales during inflammatory episodes. The

chemokine
monocyte chemoattractant protein-1 (MCP-1), newly termed
CCL2 (Rossi and Zlotnick, 2000), has been implicated in medi-
ating the infiltration of mononuclear leukocytes into the CNS
during a variety of neuroinflammatory conditions (Ghirnikar et
al., 1998; Miller and Meucci, 1999; Huang et al., 2000). However,
the mechanism or mechanisms by which this chemokine effects
extravasation into the CNS remain unclear. Confounding this
issue is that astrocytes, which project their foot processes onto the
microvessels comprising the blood–brain barrier (BBB; Lass-
mann et al., 1991), are the predominant source of MCP-1 in
several neuroinflammatory conditions (Ransohoff et al., 1993;
Berman et al., 1996; Glabinski et al., 1996; Van Der Voorn et al.,
1999; Sauder et al., 2000; Sharafeldin et al., 2000). That MCP-1
primarily originates from a source located behind the BBB begs
the following question: How does MCP-1 reach leukocytes resid-
ing in the microvessel lumen? Because chemokines might guide
cells other than leukocytes to the neural parenchyma (Rezaie and
Male, 1999; Silverman et al., 2000), the significance of this query
extends beyond matters related solely to inflammation.
Although MCP-1 might reach intravascular leukocytes by dif-
fusing between endothelial cells, this avenue is likely to be
restricted severely by high-resistance tight junctions of the BBB
(Rubin and Staddon, 1999; Kniesel and Wolburg, 2000). In fact,
other

chemokines, e.g., macrophage inflammatory protein-1
␣
(MIP-1
␣
) and MIP-1

, undergo only minimal passage via this
route (Banks and Kastin, 1996). Alternatively, MCP-1 might bind
to the abluminal endothelial surface and then be conveyed by
transcytosis to the luminal side and/or stimulate a signal trans-
duction event that fosters leukocyte extravasation. Consistent
with these possibilities, the
␣
chemokine interleukin-8 (IL-8) has
been reported to undergo abluminal-to-luminal to transcytosis
across dermal microvessels (Middleton et al., 1997), and BBB
endothelial cells harbor highly selective transport systems for the
vectorial movement of cytokines and other ligands into and out of
the brain (Banks and Kastin, 1991; Friden, 1993; Gutierrez et al.,
1993, 1994; Banks et al., 1994; Abbott and Romero, 1996; De-
houck et al., 1997; Makic et al., 1998; Rose and Audus, 1998).
Moreover, cytokines evoke adhesion molecules (Dobbie et al.,
1999; Wong et al., 1999) and heighten permeability (Megyeri et
Received March 19, 2001; revised July 20, 2001; accepted Sept. 10, 2001.
This work was supported in part by National Institute of Mental Health Grant
1RO1-MH54718-01A1 and National Multiple Sclerosis Society Grant RG2633-A-
1/3 to J.S.P.
Correspondence should be addressed to J. S. Pachter, Blood–Brain Barrier Labo-
ratory, Department of Pharmacology, University of Connecticut Health Center, 263
Farmington Avenue, Farmington, CT 06030. E-mail: pachter@nso1.uchc.edu.
Copyright © 2001 Society for Neuroscience 0270-6474/01/219214-10$15.00/0
The Journal of Neuroscience, December 1, 2001, 21(23):9214–9223
al., 1992; Deli et al., 1995) at the BBB, conditions that could
predispose toward leukocyte extravasation.
Functional binding of MCP-1 along the BBB likely would
require the expression of selective receptors for this chemokine.
Recently, this laboratory identified separate, high-affinity binding
sites for MCP-1 along the abluminal surface of endothelial cells
within human brain microvessels (Andjelkovic et al., 1999; And-
jelkovic and Pachter, 2000). Although these sites resemble CCR2,
the G-protein-coupled receptor (GPCR) for MCP-1 that is
present on leukocytes and other hemopoietic cells (Rossi and
Zlotnick, 2000), direct evidence of CCR2 participation in MCP-1
interactions with brain microvessels has not been confirmed.
Because other putative receptors for MCP-1 have been proposed
(Boddeke et al., 2000), there is a need to clarify the role of CCR2
in MCP-1 binding at the BBB. Consequently, efforts were under-
taken here to test the hypothesis that CCR2 is obligatory for
MCP-1 binding and postbinding events along brain microvessels.
Results indicate that such vessels from mice lacking CCR2 do not
exhibit these activities. In addition, internalization of MCP-1
apparently occurs partly via a caveolae-dependent process. Be-
cause caveolae participate in both signal transduction and trans-
port (Shaul and Anderson, 1998; Feng et al., 1999), engagement
of MCP-1 by CCR2 on the abluminal microvascular surface may be
critical in relaying the signal of this chemokine across the BBB.
MATERIALS AND METHODS
Mice. Mice lacking CCR2, i.e., CCR2 (⫺/⫺), were generated by homol-
ogous recombination as reported previously (Kuziel et al., 1997). Both
CCR2 (⫺/⫺) and wild-type mice were of the genetic background
C57BL/6J⫻129P3/J.
Isolation of murine microvessels. Animals were killed with CO
2
in
accordance with measures stipulated by the Animal Care and Use Guide-
lines of the University of Connecticut Health C enter (Animal Welfare
Assurance A3471-01). Immediately after death, craniotomy was per-
formed, and the entire cerebrum was removed and dissected free of
meninges. Microvessels were isolated from brain tissue by the procedure
detailed in recent publications (Andjelkovic et al., 1999; Andjelkovic and
Pachter, 2000). Briefly, this procedure involved initially separating mi-
crovessels from parenchymal tissue by gentle Dounce homogenization
and then pressing the tissue extract through graded sieves of smaller
porosity to remove macrovascular segments. Microvessels subsequently
were isolated from the dissociated material by centrifugation through
sequential dextran and Percoll gradients. Because enzymatic treatment
was avoided during the isolation protocol, the basement membrane
surrounding the resultant microvascular fragments was left relatively
intact. Isolated microvessels were washed in PBS, pH 7.4, and were used
immediately.
Binding e xperiments. Chemokine binding to brain microvessels was
conducted as described previously (Andjelkovic et al., 1999; Andjelkovic
and Pachter, 2000). In brief, purified microvessels were reacted with
biotinylated recombinant murine (biot.-rm) MCP-1 or biot.-rmMI P-1
␣
(Fluorokine kits, R&D Systems, Minneapolis, MN) in the absence or
presence of increasing concentrations of unlabeled rmMCP-1 (R&D
Systems) or unlabeled rmMIP-1
␣
(Peprotech, Norwood, MA) at speci-
fied temperatures. In some experiments the microvessels were reacted
with biotinylated transferrin or biotinylated cholera toxin subunit B (both
from Sigma, St. Louis, MO). After 2 hr of incubation with biotinylated
ligands the microvessels were reacted with avidin-fluorescein (R&D
Systems) for an additional 1 hr at 4°C. For negative controls the mi-
crovessels were incubated with the irrelevant biotinylated soybean tryp-
sin inhibitor (R&D Systems). Samples were viewed with a Zeiss LSM
410 confocal microscope (Oberkochen, Germany) equipped with an
argon–krypton laser [excitation at 488 nm; emission at 515 nm (long-pass
filter)], and images were obtained and processed with Adobe Photoshop
3.0 software (Adobe Systems, San Jose, CA) as detailed previously
(Andjelkovic et al., 1999; Andjelkovic and Pachter, 2000). To quantify
the extent of labeled chemokine binding, we recorded the values of mean
pixel intensity from a total of 25 randomly chosen areas (192 pixels each)
along at least 10 individual microvessels from each sample. Mean pixel
values were obtained similarly from negative controls, and an average
value of this parameter was subtracted from each of the 25 pixel inten-
sities obtained from all of the different chemokine binding conditions.
This correction served to remove background “noise” caused by indis-
criminate avidin-fluorescein binding. Corrected pixel intensities then
were averaged, with the resulting value representing the relative degree
of specific chemokine binding along the microvessels.
To detect internalized ligands, we fixed the microvessels in 4% para-
formaldehyde immediately after incubation with biotinylated chemokine,
transferrin, or cholera toxin and then permeabilized them by incubation
in PBS containing 0.5% (w/v) Tween 20 (Sigma). Then the ligands were
revealed by subsequent reaction with avidin-fluorescein.
Ligand-induced downmodulation/recovery of MCP-1 binding sites. To
assess the loss of biot.-rmMCP-1 surface binding because of downmodu-
lation, we preexposed the microvessels to unlabeled rmMCP-1 (2.5
g/ml) for varying times at 37°C. Next the samples were washed with
PBS and subjected to the standard binding assay by using biot.-rmMCP-
1/avidin-fluorescein at 4°C.
For evaluation of the recovery of biot.-rmMCP-1 binding after down-
modulation, the microvessels were incubated first with unlabeled
rmMCP-1 (2.5
g/ml) for 2 hr at 37°C. Then the samples were washed
with PBS and allowed to recover for varying periods of time in PBS
containing 10 m
MD-glucose (Sigma) at 37°C, after which the standard
binding assay was performed. To determine whether the reexpression of
MCP-1 binding activity along the brain microvessel surface required de
novo protein synthesis, we exposed the microvessels to 10
g/ml cyclo-
heximide (Sigma) throughout the recovery phase. Specifically, cyclohex-
imide was introduced during the last 30 min of exposure of the microves-
sels to unlabeled rmMCP-1. At the end of this period the microvessels
were washed with PBS containing cycloheximide and then resuspended
in fresh PBS/cycloheximide/
D-glucose and incubated at 37°C for 90 min
(recovery phase). The standard binding assay was conducted after this
recovery phase.
Filipin III treatment. To assess the contribution of caveolae to the
process of MCP-1 internalization, we added filipin III (5
g/ml; Sigma)
to the microvessels for a 30 min pretreatment before the addition of
biotinylated ligand. Then the microvessels were washed with PBS, and
binding reactions were performed in the absence of filipin III. Exposure
to filipin III was minimized intentionally to avoid toxicity, and the
microvessels remained viable throughout this protocol as judged by
trypan blue exclusion.
Heparinase treatment. So that the role of heparan sulfate in MCP-1
binding could be investigated, the microvessels were digested with 176
U/ml Heparinase I (Sigma) for 45 min at 37°C. This procedure served to
remove all immunodetectable heparan sulfate but did not affect adversely
the gross integrity of the microvessels (Andjelkovic et al., 1999). After
digestion the samples were washed with PBS and were submitted to the
standard binding assay.
Histochemistry/immunoc ytochemistry. To gauge the distribution of
bound/internalized biot.-rmMCP-1 relative to the endothelial plasma
membrane, we fixed the microvessels in 4% paraformaldehyde, perme-
abilized them after reaction with chemokine, and then costained them
with rhodamine-conjugated lectin wheat germ agglutinin (rho.-WGA;
Vector Laboratories, Burlingame, CA) at 5
g/ml.
To assess the colocalization of internalized MCP-1 with caveolin-1, we
reacted the microvessels with biot.-rmMCP-1 at 37°C for 10 min, which
was sufficient time to observe chemokine transport across the endothelial
plasma membrane (as judged separately by rho.-WGA staining). This
time point thus was considered appropriate to maximize the detection of
internalized MCP-1 with caveolin-1-containing vesicles at the earliest
stages of their invagination. After incubation with chemokine the mi-
crovessels were fixed with 4% paraformaldehyde and permeabilized.
Then the samples were reacted with Alexa Fluor 488-conjugated avidin
(Molecular Probes, Eugene, OR) at 5
g/ml. The reason for using the
Alexa Fluor 488-derivative here, instead of fluorescein-conjugated avi-
din, is that the former fluorophore has a higher quantum yield. Because
the signal associated with transported biot.-rmMCP-1 at this early time
point was low, this necessitated the use of the brighter fluorophore. After
reaction with Alexa 488 the samples were rinsed in PBS and incubated
with polyclonal anti-caveolin-1 antibody (2
g/ml; Transduction Labs,
Lexington, KY). Next the microvessels were washed again in PBS and
reacted with Texas Red-conjugated goat anti-rabbit IgG (10
g/ml;
Vector Laboratories). Lectin and anti-caveolin-1-stained samples were
viewed with a Zeiss 410 LS confocal microscope, using the after-filter
configurations fluorescein and Alexa Fluor 488 [excitation at 488 nm,
emission at 515–540 nm (bandpass filter)], rhodamine [excitation at 568
Dzenko et al. • CCR2 on Brain Microvessels J. Neurosci., December 1, 2001, 21(23):9214–9223 9215
nm, emission at 590 nm (long-pass filter)], and Texas Red [excitation at
568 nm, emission at 610 nm (long-pass filter)].
Three-dimensional renderings of biot.rmMCP-/rho.-WGA costained
microvessels were generated from a z-series of confocal images through
the entirety of microvessel samples, using VoxelView software (Vital
Images, Fairfield, IA) as described previously (Andjelkovic et al., 1999;
Andjelkovic and Pachter, 2000).
Statistical analysis. To determine the significance of the effects of
varied treatments on biot.rm-MCP-1 binding to brain microvessels, we
performed a one-way ANOVA, followed by a Bonferroni multiple com-
parisons test.
RESULTS
MCP-1 binding along brain microvessels
Figure 1 shows the binding of biot.-rmMCP-1 along the abluminal
surface of isolated murine brain microvessels. In microvessel
samples prepared from wild-type mice, the distribution of labeled
MCP-1 appears to be relatively continuous along the microvas-
cular surface, as has been described recently for the correspond-
ing human chemokine derivative along human brain microvessels
(Andjelkovic et al., 1999). Only negligible signal was detected in
samples exposed to biot.-soybean trypsin inhibitor (negative con-
trol), reflecting the specificity of the binding reaction. Heparan
sulfate moieties along either the endothelial surface or attendant
basement membrane were not major factors in biot.-rmMCP-1
binding, because digestion of the microvessels with heparinase I
[sufficient to eliminate all heparan sulfate immunocytochemical
reactivity (Andjelkovic et al., 1999; data not shown)] failed to
eliminate the chemokine signal and only very slightly depressed it.
Thus, although heparan sulfate can bind MCP-1, albeit with an
affinity 2–3 log units less than CCR2 (Hoogewerf et al., 1997), it
is not the primary binding domain for this chemokine on brain
microvessels under the conditions that were used. Because these
new findings recapitulated those previously observed with human
microvessels, it established the murine system as a suitable para-
digm to investigate the role of CCR2 in mediating MCP-1 inter-
actions at the human BBB.
In marked contrast to the situation observed with wild-type
vessels, significantly diminished binding of biot.-rmMCP-1 was
detected on brain microvessels from CCR2 (⫺/⫺) mice. What-
ever little binding was observed in the CCR2 (⫺/⫺) samples may
derive, at least partially, from heparan sulfate, because hepari-
nase digestion nearly completely eradicated the detectable signal.
Importantly, binding of biot.-rmMIP-1
␣
was nearly invariant
between microvessels from wild-type and CCR2 (⫺/⫺) mice,
exhibiting a similar punctate pattern previously observed with
human tissue (Andjelkovic et al., 1999; Andjelkovic and Pachter,
2000) and indicating that CCR2 ablation generally did not affect
chemokine binding. These findings provide the first direct evi-
dence that the binding of MCP-1 along brain microvessels is
linked inextricably to the expression of CCR2.
Additional evidence supporting the assertion that CCR2 medi-
ates MCP-1 binding to brain microvessels was provided by a
competitive ligand-binding assay (Fig. 2). As described recently
for human brain microvessels (Andjelkovic et al., 1999; Andjel-
kovic and Pachter, 2000), the binding of biot.-rmMCP-1 could be
antagonized by its unlabeled homolog, but not by unlabeled
rmMIP-1
␣
, the latter a ligand for chemokine receptors CCR1
and CCR5, but not for CCR2. The residual binding exhibited in
the presence of competitor ligand, as for the case with brain
microvessels from CCR2-deficient mice, might reflect the small
contribution from nonreceptor-mediated association of biotinyl-
ated chemokine with heparan sulfate and/or other glycosamino-
glycans (GAGs; Hoogewerf et al., 1997; Kuschert et al., 1999).
Figure 1. Biotinylated chemokine
binding along murine brain microves-
sels. Top, Representative examples of
fluorescent detection of chemokines at-
tached to the brain microvascular sur-
face. Detection of biot.-rmMCP-1 and
biot.-rmMI P-1
␣
binding was performed
at 4°C as described in Materials and
Methods. Diminished binding of biot.-
rmMCP-1 to microvessels from CCR2
(⫺/⫺) mice, compared with those from
wild-type mice (WT), is readily appar-
ent. No such difference between these
respective microvessel populations is
observed for biot.-rmMIP-1
␣
binding,
and heparinase treatment failed to ab-
rogate biot.-rmMCP-1 binding. Scale
bar, 50
m. Bottom, Quantitative analy-
sis of biotinylated chemokine binding.
Mean pixel intensities ⫾ SEM, reflect-
ing relative chemokine binding, were
determined as described in Materials
and Methods and were corrected for
background noise by subtraction of in-
tensity values associated with negative
controls. Values represent those deter-
mined from at least three different ex-
periments. *p ⬍ 0.001 when contrasted
with corresponding wild-type value.
9216 J. Neurosci., December 1, 2001, 21(23):9214–9223 Dzenko et al. • CCR2 on Brain Microvessels
Downmodulation and recovery of MCP-1 binding
Because CCR2 on leukocytes and transfected cells has been
reported to undergo downmodulation in response MCP-1 expo-
sure (Sarau et al., 1997; Aragay et al., 1998; Fantuzzi et al., 1999),
it was reasoned that if the interaction of this chemokine with
brain microvessels were to be mediated predominantly or exclu-
sively by CCR2, then preexposure to unlabeled MCP-1 should
lessen the degree of biot.-rmMCP-1 binding along the microvas-
cular endothelial surface. Figure 3 shows that, when microvessels
were preexposed to unlabeled MCP-1 for varying lengths of time,
washed free of unbound chemokine, and then reexposed to biot.-
rmMCP-1, the degree of labeled MCP-1 binding to the microves-
sel surface gradually decreased. A nearly 50% attenuation in
“maximal binding” (i.e., the signal detected when there was no
preexposure to unlabeled ligand) was achieved after 15 min of
preexposure to unlabeled MCP-1. This loss of MCP-1 binding
activity along the abluminal brain microvascular surface could be
recovered within minutes, similar to the time scale reported for
the ligand-induced loss of expression of several chemokine recep-
tors on other cell types (Madani et al., 1998; Feniger-Barish et al.,
1999). In this case the recovery of 50% of maximal biot.-
rmMCP-1 binding was attained within 30 min, and complete
recovery was attained within 60 min after removal of the unla-
beled chemokine. It was determined further that the restoration
of MCP-1 binding was not dependent on de novo protein synthe-
sis, because recovery proceeded unabated in the presence of
cycloheximide (Fig. 4). These features are consistent with the
interpretation that, like other chemokine receptors stimulated by
their respective ligands, MCP-1 binding sites are internalized and
then recycled to the endothelial cell surface as a consequence of
MCP-1 exposure (Mack and Schlondorff, 2000).
Internalization of MCP-1
To confirm whether MCP-1 is, in fact, internalized after binding
to the brain microvascular surface, we performed the binding of
biot.-rmMCP-1 at 37°C, followed by interaction with avidin-
fluorescein at 4°C. Figure 5, top, indicates that under these con-
ditions the detection of biot.-rmMCP-1 on the microvascular
surface was reduced significantly compared with that seen when
chemokine binding was performed at 4°C. However, when mi-
crovessels incubated with biot.-rmMCP-1 at 37°C were fixed and
then permeabilized before interaction with avidin-fluorescein,
the fluorescent signal was detected within the endothelial cell
cytoplasm. The staining pattern revealed by permeabilization
also appeared to be more “patchy” than that observed with
samples exposed to chemokine at 4°C (also compare with Fig. 1).
These results are taken to indicate that biotinylated chemokine
Figure 2. Competition of biotinylated MCP-1 binding along murine
brain microvessels. Competition studies were performed at 4°C with a
constant concentration of biot.-rmMCP-1 and increasing concentrations
of unlabeled chemokines (indicated by symbols). Binding was quantitated
as described in Materials and Methods and is reported as the percent-
age ⫾ SEM of maximal binding achieved in the absence of inhibitor
ligand. Values represent those determined from at least three different
experiments.
Figure 3. Loss and recovery of MCP-1 binding sites on murine brain
microvessels after ligand exposure. Microvessels were reacted with unla-
beled MCP-1 at 37°C for 2 hr (recovery) or for varying periods of time
(loss). After exposure to unlabeled chemokine the microvessels were
washed in PBS and either were exposed immediately to biot.-rmMC P-1
for2hrat4°C (loss) or were incubated at 37°C for varying periods of time
and then exposed to biot.-rmMCP-1 (recovery). After reaction with
biot.-rmMCP-1 the samples were subjected to the standard binding assay
conditions, and binding intensity along the microvessels was analyzed as
described in Materials and Methods. The extent of both loss and recovery
of biot.-rmMCP-1 binding is indicated as the percentage ⫾ SEM of
maximal binding obtained in the absence of any previous exposure of the
microvessels to unlabeled MCP-1. Values represent those determined
from at least three different experiments.
Figure 4. Effect of protein synthesis inhibition on the recovery of MCP-1
binding sites along murine brain microvessels. Microvessels were exposed
to unlabeled MCP-1 as described in Figure 3, except that in the last 30
min the protein synthesis inhibitor cycloheximide (10
g/ml) either was
added to the incubation mixture or was not. After chemokine exposure
the microvessels treated with cycloheximide were allowed to recover for
90 min in the continued presence of protein synthesis inhibitor while the
control samples continued their recovery in the absence of cycloheximide.
Binding was quantitated as described in Materials and Methods and is
reported as the percentage ⫾ SEM of maximal binding obtained in the
absence of any previous exposure of the microvessels to unlabeled MCP-1.
Values represent those determined from three different experiments.
Dzenko et al. • CCR2 on Brain Microvessels J. Neurosci., December 1, 2001, 21(23):9214–9223 9217
had been internalized at the elevated temperature and was not
available to interact with the avidin-fluorophore in the nonper-
meabilized condition. This interpretation is supported in Figure
5, bottom, which depicts three-dimensional renderings of mi-
crovessels and the relative distribution of labeled MCP-1 to the
endothelial surface. In this case the combined visualization of
biot.-rmMCP-1 and rho.-WGA, the latter marking endothelial
plasma membrane (Vorbrodt et al., 1994), reveals the chemokine
to lie external to the membrane at 4°C and internal to it at 37°C.
To establish that internalization of biot.-rmMCP-1 most likely
is mediated by CCR2, we also performed an identical binding
paradigm at 37°C with brain microvessels from CCR2 (⫺/⫺)
mice. In this case there was little signal detected in the cytoplasm
of either permeabilized or nonpermeabilized vessels. In fact,
biot.-rmMCP-1 staining of these two populations of vessels did
not differ much from each other or from microvessels of
CCR2-deficient mice exposed to MCP-1 at 4°C, also demon-
strating that non-CCR2 binding sites for MCP-1 are not sub-
ject to internalization.
Last, the mechanism by which MCP-1 is internalized along
brain microvessels was investigated. Two pathways for GPCR-
mediated internalization of agonist ligands currently are recog-
nized: one using clathrin-coated pits (Bohm et al., 1997; Lefko-
witz, 1998) and the other using nonclathrin-coated plasmalemmal
vesicles called caveolae (Anderson, 1998; Okamoto et al., 1998).
However, several facts regarding caveolae, along with the need to
reconcile how MCP-1 deposited in the perivascular space can
exert its effect on leukocytes on the other side of the BBB, lead us
to focus on the caveolar pathway at this time: (1) caveolae are
particularly prominent in endothelial cells, including those of
brain microvessels (Ikezu et al., 1998); (2) caveolae mediate the
vectorial movement of low-density lipoprotein across cultured
endothelial cells derived from brain microvessels (Dehouck et al.,
1997); (3) caveolae have been suggested to be involved possibly in
the abluminal-to-luminal transcytosis of the
␣
chemokine IL-8
across dermal venules (Middleton et al., 1997); and (4) caveolae
have been implicated in signal transduction events (Lisanti et al.,
1994; Shaul and Anderson, 1998). Thus several precedents al-
ready have been set highlighting the possibility that caveolae
could relay MCP-1 signals across the BBB. To investigate use of
caveolae in the internalization process, we pretreated brain mi-
crovessels with the agent filipin III before their exposure to
labeled MCP-1 at 37°C. Filipin III is a sterol-binding agent that
removes cholesterol from membranes and, thus, selectively dis-
rupts caveolar microdomains (Schnitzer et al., 1994). Figure 6
reveals that previous treatment of microvessels with filipin III
enabled the detection of biot.-rmMCP-1 when samples were
exposed to chemokine at 37°C and then fixed, without the need of
permeabilization before the application of avidin-fluorescein. In
marked contrast to the near-complete loss of detectable staining
of nonpermeabilized control microvessels, the staining of perme-
abilized and nonpermeabilized filipin III-treated samples ap-
Figure 5. Internalization of MCP-1
binding sites along murine brain mi-
crovessels. Top, Microvessels were re-
acted with biot.-rmMCP-1 at 37°C, fixed
with 4% paraformaldehyde, and then ei-
ther reacted directly with avidin-
fluorescein (nonpermeabilized) or per-
meabilized with Tween 20 before
reaction with avidin-fluorescein, as de-
scribed in Materials and Methods. Com-
pared with Figure 1, wild-type microves-
sels (WT) that were reacted with labeled
chemokine at 37°C and that were not
permeabilized reveal a greatly attenuated
signal. Permeabilization, however, re-
stores the detection of biot.-rmMCP-1,
suggesting that the labeled chemokine
had been internalized within wild-type
microvessels at the elevated temperature.
Microvessels from CCR2 (⫺/⫺) mice, in
contrast, demonstrated only a weak signal
regardless of whether or not they had
been permeabilized, also implying that
they do not internalize MCP-1. Scale bar,
50
m. Bottom, Three-dimensional ren-
derings of microvessels from wild-type
mice depicting the distribution of biot.-
rmMCP-1 relative to the endothelial
plasma membrane. In accordance with
the procedures that were described in
Materials and Methods, the microvessels
were reacted with biot.-rmMCP-1 at ei-
ther4or37°C and then were fixed and
permeabilized. Next the samples were
stained consecutively with avidin-
fluorescein to reveal chemokine localiza-
tion ( green) and then with rho.-WGA to
indicate endothelial plasma membrane
(red). Samples were subject to confocal
microscopy and three-dimensional rendering as described in Materials and Methods, and the images are oriented so that viewer is looking “on face”
toward the microvascular lumen. Chemokine staining clearly lies external to the plasma membrane at 4°C and internal to it at 37°C (some chemokine
staining actually might reflect complete transit into the lumen at the elevated temperature).
9218 J. Neurosci., December 1, 2001, 21(23):9214–9223 Dzenko et al. • CCR2 on Brain Microvessels
peared to be similar to each other in pattern and intensity.
Particularly noteworthy is that the staining of both filipin III-
treated samples was delineated along the microvascular surface,
distinct from the patchy cytoplasmic pattern manifested by con-
trol samples (Figs. 5, 6). The interpretation here is that, by
disrupting caveolae-based internalization, filipin III treatment
allowed surface-bound biot.-rmMCP-1 to remain on the cell
surface and, thus, accessible to reaction with avidin-fluorescein in
the absence of membrane permeabilization.
To preclude the possibility that treatment with filipin III merely
resulted in cellular toxicity or adversely affected all endocytic
processes, we also evaluated the effect of this agent on transferrin
internalization, which is mediated by clathrin-coated vesicles
(Benlimame et al., 1998) (Fig. 7). As was the case with labeled
MCP-1, the detection of biot.-transferrin at 37
C was achieved
only when microvessels were fixed and then permeabilized before
exposure to avidin-fluorescein. Failure to permeabilize the mi-
crovessels resulted in a lack of biot.-transferrin detection, imply-
ing sequestration of this ligand behind the plasma membrane and
validating that transferrin internalization had occurred. Contrary
to the effects on biot.-rmMCP-1 detection, however, previous
treatment of microvessels with filipin III did not prevent the
diminished detection of the biot.-transferrin signal at 37°C. Both
filipin III-treated and control samples required detergent perme-
abilization to reveal biot.-transferrin bound at 37°C, implying that
exposure to filipin III did not prevent transferrin internalization.
To confirm additionally that the action of filipin III was not
peculiar to MCP-1, we investigated the effect of this agent on the
internalization of cholera toxin, a ligand reported to be endocy-
tosed by endothelial cells via the caveolar route (Orlandi and
Fishman, 1998). In this case (Fig. 7) the filipin III treatment
yielded results identical to those observed with MCP-1: it enabled
the detection of cholera toxin when the samples were exposed to
ligand at 37°C and then fixed, without the need of membrane
permeabilization. The interpretation here too is that, by blocking
caveolae-mediated internalization, filipin III caused cholera toxin
to remain on the microvessel surface.
Last, colocalization of internalized MCP-1 with caveolin-1, a
major structural protein of caveolae (Rothberg et al., 1992; Schle-
gel and Lisanti, 2001), was examined by double-label confocal
microscopy to corroborate further an association of internalized
chemokine with the caveolar pathway. Figure 8 shows the pat-
terns of distribution of biot.-rmMCP-1 and caveolin-1 along brain
microvessels after chemokine exposure at 4 and 37°C. At 4°C both
biot.-rmMCP-1 and caveolin-1 staining appeared to be concen-
trated at or near the abluminal microvascular surface, although
segregated from each other. The lack of a significant cytoplasmic
chemokine signal is in accord with there being little or no inter-
nalization at this temperature, and the distribution of caveolin-1
is consistent with the presence of caveolae at the plasmalemma
(Kurzchalia and Parton, 1999). After exposure at 37°C for 10 min
the distribution of biot.-rmMCP-1 colocalized with that of
caveolin-1 at some sites, as indicated by the yellow fluorescent
signal. In addition, cytoplasmic staining of biot.-rmMCP-1 ap-
peared to be more intense at 37°C than at 4°C, compatible with a
Figure 6. Effect of filipin III on internalization of MCP-1 along murine
brain microvessels. To gauge whether MCP-1 internalization may be
mediated by caveolae, we exposed the microvessels to the caveolae-
disrupting agent filipin III before incubation with biot.-rmMCP-1 at 37°C.
Contrary to nontreated controls, the microvessels pretreated with filipin
III did not require permeabilization to enable the detection of labeled
chemokine, suggesting that biot.-rmMCP-1 remained on the cell surface
as a consequence of caveolar disruption. Scale bar, 50
m.
Figure 7. Effect of filipin III on internalization of transferrin and cholera
toxin along murine brain microvessels. Microvessels received (⫾) filipin
III treatment, were exposed to biot.-transferrin or biot.-cholera toxin, and
then were processed as described in Figure 6. Both control and filipin
III-treated microvessels required permeabilization to detect biot.-
transferrin, indicating that the process of transferrin internalization pro-
ceeded despite disruption of the caveolae. In contrast, biot.-cholera toxin
could be observed along the surface of filipin III-treated microvessels both
with and without permeabilization, reflecting filipin III-mediated inter-
ference with the internalization of this ligand. Scale bar, 50
m.
Dzenko et al. • CCR2 on Brain Microvessels J. Neurosci., December 1, 2001, 21(23):9214–9223 9219
significantly higher degree of endocytosis occurring at the ele-
vated temperature. Pretreatment of microvessels with filipin III
caused disruption in the pattern of caveolin-1 staining, resulting
in a lessening in intensity along the microvessel contour and a
redistribution to the cytoplasmic compartment. This change is
consistent with that reported by others (John et al., 2001; Rose-
berry and Hosey, 2001) and possibly reflects the cytoplasmic
dispersion of caveolin-1 liberated from plasma membrane-
associated caveolae. Collectively, these results suggest that the
caveolar pathway is used in some capacity during the internaliza-
tion of MCP-1 by endothelial cells of brain microvessels.
DISCUSSION
Previous studies from this laboratory have demonstrated the
presence of high-affinity saturable binding sites for MCP-1 along
human brain microvessels and have shown that these sites display
pharmacological and biochemical properties similar to those re-
ported for CCR2 (Andjelkovic et al., 1999; Andjelkovic and
Pachter, 2000). Here we substantiated these findings with murine
brain microvessels and further established that the expression of
CCR2 by brain microvascular endothelial cells is obligatory for
the manifestation of MCP-1 binding properties. Specifically,
biot.-rmMCP-1 bound to the abluminal surface of isolated brain
microvessels from wild-type mice by a process that was inhibited
by unlabeled MCP-1, but not unlabeled MIP-1
␣
, and also was
primarily independent of the presence of heparan sulfate. Such
biot.-rmMCP-1 binding was reduced greatly along microvessels
isolated from CCR2 (⫺/⫺) mice, whereas the binding of biot.-
rmMIP-1
␣
occurred to similar extents under wild-type and
CCR2 knock-out conditions. As also reported for human mi-
crovessels, biot.-rmMCP-1 was internalized within endothelial
cells of isolated murine vessels after binding at 37
C, but endo
-
cytosis of this chemokine was not observed in microvascular
tissue from CCR2 (⫺/⫺) mice. Last, internalization of biot.-
rmMCP-1 and biot.-cholera toxin, but not biot.-transferrin, ap-
parently was prevented by the use of the caveolae-disrupting
agent filipin III. These results collectively argue that CCR2 is
predominantly, if not solely, responsible for MCP-1 binding along
the abluminal brain microvascular surface and that such receptor-
mediated binding leads, in some proportion, to the internaliza-
tion of ligands via a pathway known to be associated with trans-
cytotic and signal transduction events.
That specific biot.-rmMCP-1 binding to murine brain mi-
crovessels was observed is an important validation of similar
results recently reported with human ligands and tissue (Andjel-
kovic et al., 1999; Andjelkovic and Pachter, 2000) and allays the
prospect that this interaction is restricted to human tissue and/or
is attributable to some artifact uniquely associated with the pro-
curement thereof. On the contrary, it points to the expression of
endothelial MCP-1 binding sites as possibly being a common
mammalian feature. Supporting this interpretation are findings
from other laboratories of the expression of CCR2 as well as
other chemokine receptors by a variety of endothelial cell types
from human, macaque, bovine, and rodent tissues (Edinger et al.,
1997; Rottman et al., 1997; Feil and Augustin, 1998; Gupta et
al., 1998; Sanders et al., 1998; Volin et al., 1998; Andjelkovic
et al., 1999; Berger et al., 1999; Murdoch et al., 1999; Shaw and
Grieg, 1999; Weber et al., 1999; Andjelkovic and Pachter, 2000;
Molino et al., 2000; Salcedo et al., 2000).
Because brain microvessels from CCR2 (⫺/⫺) mice failed to
exhibit the binding of MCP-1 but retained that of MIP-1
␣
, this
report is the first to link the expression of a specific chemokine
receptor with selective chemokine binding to endothelial cells.
Thus, whereas competitor chemokine binding assays revealed a
pharmacological profile consistent with CCR2 activity along hu-
Figure 8. Colocalization of internalized
MCP-1 with caveolin-1. Microvessels were
pretreated (⫾) with filipin III. Then the
samples were exposed to biot.-rmMCP-1
at4or37°C, fixed/permeabilized, and
stained to reveal labeled chemokine
( green) and caveolin-1 (red) localization,
as described in Materials and Methods.
Then confocal images were obtained at a
level approximately midway through the
interior of the microvascular samples, re-
vealing the relative distribution patterns
of labeled chemokine and caveolin-1. In
the control sample exposed to biot.-
rmMCP-1 at 37°C, caveolin-1 staining can
be seen concentrated around the periph-
ery of the microvascular segment (arrows),
with chemokine apparently present dif-
fusely in the cytoplasm. Areas of yellow
fluorescence (asterisks) indicate presumed
sites of biot.-rmMCP-1/caveolin-1 colocal-
ization. In the filipin III-treated sample
exposed to chemokine at 37°C, no sites of
colocalization are detected readily, and
biot.-rmMCP-1 appears to be confined to
the membrane surface (arrowheads), with
caveolin-1 expression heightened in some
cytoplasmic locales (arrows). Microvessels
exposed to chemokine at 4°C(⫾) filipin
III pretreatment also fail to show any areas of biot.-rmMCP-1/caveolin-1 colocalization. These samples also do not demonstrate as strong a cytoplasmic
distribution of labeled chemokine as that observed in the control sample at 37°C but seemingly manifest a more peripheral chemokine staining
(arrowheads), possibly restricted to the membrane surface. As with the samples exposed to chemokine at 37°C, caveolin-1 distribution appears to be
concentrated along the periphery of the microvessel in the control (arrowheads) but is dispersed more cytoplasmically in the filipin III-treated sample.
Arrows, Caveolin-1; arrowheads, biot.-rmMCP-1; asterisks, biot.-rmMCP-1/caveolin-1 colocalization. Scale bar, 10
m.
9220 J. Neurosci., December 1, 2001, 21(23):9214–9223 Dzenko et al. • CCR2 on Brain Microvessels
man brain microvessels (Andjelkovic et al., 1999; Andjelkovic
and Pachter, 2000), results from this report verify the microvas-
cular expression of this receptor and its role in MCP-1 binding.
The reduction in MCP-1 binding associated with CCR2 absence
also lessens the possibility that binding is attributable to another
receptor with CCR2-like properties (Boddeke et al., 2000).
Hence, bearing any unforeseen ablation in the knock-out mice of
a gene encoding such a similarly functioning receptor for MCP-1,
these findings offer persuasive evidence that CCR2 is singularly
responsible for the binding of MCP-1 along the abluminal surface
of brain microvessels.
In addition to mediating MCP-1 binding, CCR2 is also oblig-
atory for the endocytosis of MCP-1 within endothelial cells, as
was indicated by the inability of brain microvessels from CCR2
(⫺/⫺) mice to internalize this chemokine. Despite some low
degree of MCP-1 binding to heparan sulfate moieties along the
abluminal microvascular surface [in both wild-type and CCR2
(⫺/⫺) mice], ligand engagement at this level does not result in
internalization and probably reflects low-affinity interaction of
the chemokine with GAGs. Weaker binding of this latter type
may serve the purpose of concentrating chemokines in the
perivascular space and/or enabling proper chemokine presenta-
tion to CCR2-bearing cells in or entering the brain parenchyma
(Hoogewerf et al., 1997; Kuschert et al., 1999).
The kinetics of MCP-1-induced loss and recovery of murine
microvascular chemokine binding reported here is similar to that
previously described with human brain microvessels (Andjelkovic
et al., 1999; Andjelkovic and Pachter, 2000) and mirrors the time
course for ligand-stimulated downmodulation and reexpression of
chemokine receptors in other systems (Madani et al., 1998;
Feniger-Barish et al., 1999). Given the relatively rapid times that
are required to achieve half-maximal loss or recovery of MCP-1
binding, the lack of effect of protein synthesis inhibition on the
recovery process, and the dependency of internalization on CCR2
expression, ligand-induced alteration in MCP-1 binding along
brain microvessels is likely to occur by receptor-mediated inter-
nalization and recycling.
The ability of filipin III to abrogate internalization of MCP-1
and cholera toxin, but not transferrin, further prompts intriguing
suggestions as to the fate of this chemokine after its binding to
the abluminal surface of brain microvessels. Specifically, the col-
lective evidence points to some portion of internalized MCP-1
entering the caveolae. Caveolae are membrane specializations
enriched in G-protein-coupled receptors, heterotrimeric GTP
binding proteins, IP
3
receptor-like protein, Ca
2⫹
ATPase, and
several protein kinase C isoforms (Isshiki and Anderson, 1999)
and are recognized to transport molecules across endothelial cells
(Anderson, 1998). As such, the association of MCP-1 with this
membrane system may intimate that this chemokine stimulates a
signal transduction pathway or pathways and/or is transferred to
the luminal surface in the process of effecting its actions at the
BBB. Both types of responses have, in fact, been demonstrated in
endothelial cells after exposure to IL-8 (Middleton et al., 1997;
Schraufstatter et al., 2001). Interestingly, previous reports regard-
ing ligand-induced internalization of other chemokine receptors
have described only the use of the clathrin-dependent pathway in
nonvascular cell types (Amara et al., 1997; Mack et al., 1998;
Yang et al., 1999). Our findings thus might suggest a propensity
for endothelial cells, and possibly those of brain microvessels in
particular, to use caveolae conditionally. Indeed, such a charac-
teristic would be consistent with the expression of highly selective
transporter systems at the BBB (Banks and Kastin, 1991; Friden,
1993; Gutierrez et al., 1993, 1994; Banks et al., 1994; Abbott and
Romero, 1996; Dehouck et al., 1997; Makic et al., 1998; Rose and
Audus, 1998). This interpretation is not meant to exclude a role
for the clathrin-dependent pathway in the internalization of che-
mokines and their receptors at the brain microvascular endothe-
lium but only to impart recognition of the caveolar pathway. Both
endocytic venues, in fact, might operate routinely in the action of
MCP-1 along the brain microvascular endothelium, with the
relative involvement of each depending on factors including cel-
lular cholesterol oxidation state (Okamoto et al., 2000) and/or
degree of receptor phosphorylation (Roettger et al., 1995). Dual
pathways of internalization have, in fact, been suggested for
several peptide ligands including growth hormone (Lobie et al.,
1999), insulin (King and Johnson, 1985; Schnitzer et al., 1994),
and cholecystokinin (Roettger et al., 1995).
Although the physiological relevance of MCP-1 binding/inter-
nalization along brain microvessels remains to be ascertained, it
raises the prospect that leukocyte extravasation into the CNS
might involve chemokine engagement along the abluminal micro-
vascular surface. In what may be viewed as an analogous situation
to the perivascular deposition of MCP-1 by astrocytes in vivo, the
application of MCP-1 to the basolateral surface (akin to the
abluminal surface in vivo), but not to the apical surface (repre-
senting the luminal surface in vivo), of cultured brain microvas-
cular endothelial cells (BMEC) has been observed to stimulate
monocyte transendothelial migration (Andjelkovic et al., 2001).
Assuming there is minimal paracellular leakage of MCP-1 be-
tween the BMEC, which retain their highly restrictive BBB
phenotype under the conditions that have been assayed (Biegel et
al., 1995), a seemingly a posteriori conclusion is that the primary
site of MCP-1 action is along the basolateral BMEC surface,
possibly at CCR2. It thus is intriguing to speculate that reduced
sensitivity to experimental autoimmune encephalomyelitis
(EAE) in CCR2 (⫺/⫺) mice, as well as diminished capacity of
these receptor-deficient animals to support stimulated mononu-
clear cell infiltration into the CNS (Fife et al., 2000; Izikson et al.,
2000), might stem at least partially from the absence of CCR2
expression by the brain microvascular endothelium. Such a hy-
pothesis is supported by the finding that adoptive transfer of
myelin oligodendrocyte glycoprotein-sensitized T-cells from
wild-type mice exhibiting EAE to naive CCR2 (⫺/⫺) recipients
failed to produce disease symptomatology and yielded a dramat-
ically reduced mononuclear CNS infiltrate (Fife et al., 2000).
Perhaps reflecting a similar need for chemokine receptor expres-
sion by a tissue barrier, neutrophil transuroepithelial migration in
a model of urinary tract infection has been reported to be depen-
dent on epithelial expression of CXCR1, a receptor for the
␣
chemokine IL-8 (Godaly et al., 2000). In vitro paradigms of
leukocyte transendothelial migration, wherein BMEC and leuko-
cytes from wild-type and receptor-deficient mice are mixed and
matched, would be extremely valuable in resolving the specific
contribution of endothelial chemokine receptors to the extrava-
sation process at the BBB.
With the spectrum of chemokine targets in the CNS, including
all resident and many transient cell types, chemokine activity is
likely to affect homeostatic, developmental, and pathological pro-
cesses (Asensio and Campbell, 1999; Hesselgesser and Horuk,
1999; Bacon and Harrison, 2000). Although this discussion fo-
cused on the role of endothelial CCR2 in mediating leukocyte
extravasation, it well may be that brain microvascular expression
of this receptor also features prominently in the migration of
other cell types across the BBB during ontogeny and adulthood
Dzenko et al. • CCR2 on Brain Microvessels J. Neurosci., December 1, 2001, 21(23):9214–9223 9221
(Rezaie and Male, 1999; Silverman et al., 2000). Endothelial
CCR2 additionally may play a role in angiogenesis in the brain, as
suggested by studies showing MCP-1-induced endothelial chemo-
taxis in vitro (Weber et al., 1999; Salcedo et al., 2000) and
formation of blood vessels in vivo (Salcedo et al., 2000). Accord-
ingly, the endothelium may prove to be an effective therapeutic
target for modifying CNS effects associated with aberrant MCP-1
activity.
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