Departments of 1 Biochemistry and Molecular Biology and 2 Pathology, Keck School of Medicine, University of Southern California, Los Angeles, California 90033; 3 Center for Aging, University of Rochester, Rochester, New York 14642; and 4 Department of Physiology, Columbia University, New York, New York 10032
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ABSTRACT |
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During normal aging and amyloid
-peptide (A
) disorders such as Alzheimer's disease (AD),
one finds increased deposition of A
and activated
monocytes/microglial cells in the brain. Our previous studies show that
A
interaction with a monolayer of normal human brain microvascular
endothelial cells results in increased adherence and transmigration of
monocytes. Relatively little is known of the role of A
accumulated
in the AD brain in mediating trafficking of peripheral blood monocytes
(PBM) across the blood-brain barrier (BBB) and concomitant accumulation
of monocytes/microglia in the AD brain. In this study, we showed that
interaction of A
1
40 with apical surface of monolayer of brain endothelial cells (BEC), derived either from normal or AD
individuals, resulted in increased transendothelial migration of
monocytic cells (HL-60 and THP-1) and PBM. However, transmigration of
monocytes across the BEC monolayer cultivated in a Transwell chamber
was increased 2.5-fold when A
was added to the basolateral side of
AD compared with normal individual BEC. The A
-induced transmigration
of monocytes was inhibited in both normal and AD-BEC by antibodies to
the putative A
receptor, receptor for advanced glycation end
products (RAGE), and to the endothelial cell junction molecule,
platelet-endothelial cell adhesion molecule-1 (PECAM-1). We conclude
that interaction of A
with the basolateral surface of AD-BEC induces
cellular signaling, promoting transmigration of monocytes from the
apical to basolateral direction. We suggest that A
in the AD brain
parenchyma or cerebrovasculature initiates cellular signaling that
induces PBM to transmigrate across the BBB and accumulate in the brain.
amyloid -peptide; brain endothelial cells; monocytes; platelet-endothelial cell adhesion molecule; receptor for advanced
glycation end products
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INTRODUCTION |
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ALZHEIMER'S DISEASE
(AD) is a progressive neurodegenerative disease and the most frequent
cause of dementia, affecting >5% of the population over the age of 65 yr. The neuropathology of AD is characterized by neuronal loss,
numerous intraneuronal deposits of neurofibrillary tangles (NFT),
senile plaques composed of extracellular proteinaceous deposits around
reactive microglia, neuritic plaques, and cerebrovascular amyloid
deposits (27). The major component of senile plaques and
cerebrovascular deposits is amyloid (A
), consisting of
39-43 amino acid residues, formed by the proteolytic processing of
amyloid precursor protein (APP) (23). The accumulation of
A
is thought to be an early feature of AD (15). The
predominant forms of A
are the 1-40 and 1-42 fragments.
Soluble A
1
40 is the major form of circulating A
,
whereas amyloiodogenic A
1
42, the major constituent of
senile plaques, is present in minor amounts in the circulation
(2, 13, 20, 22).
In A-related cerebral vascular disorders [cerebral amyloid
angiopathy (CAA) and the genetic amyloidogenic disease hereditary cerebral hemorrhage with amyloidosis of the Dutch type (HCHWA-D)] and
AD, one finds not only increased deposition of A
in the brain but
also increased numbers of activated microglial cells in the parenchyma
and monocytes/macrophages in the vessel wall (11, 25, 28,
31). However, relatively less is understood as to how A
in
the circulation and the brain causes accumulation of microglia
(macrophage-like cells) in the AD brain. Microglia are derived from
hematopoietic cells (6, 7). Moreover, recent studies show
that bone marrow-derived cells (monocytes) can migrate across the
blood-brain barrier (BBB) and differentiate into microglia in the brain
parenchyma (3, 16), adding further support to the view
that microglial cells in the brain may be derived from peripheral
hematopoietic cells.
Recent studies (4, 5) show that A induces migration of
monocytes across a monolayer of normal human brain endothelial cells
that serves as a model of the BBB. Additionally, we show that the
interaction of A
with its putative receptor, receptor for advanced
glycation end products (RAGE), causes cellular signaling in normal
human brain-derived microvascular endothelial cells (N-HBMVEC), which
culminates in the expression of cell adhesion molecules and concomitant
adhesion of monocytes. This was followed by the transmigration of
monocytes across the human brain endothelial cell (HBEC) monolayer. We
also observed that transmigration of monocytes was inhibited by
antibody to platelet-endothelial cell adhesion molecule-1
(PECAM-1) (5), a molecule concentrated at endothelial
cell junctions (1, 9). However, it is not clear how
monocytes/microglial cells accumulate to a greater extent in the brain
of AD patients compared with aged-matched controls. We hypothesize that
migration of monocytes across the cerebrovascular endothelium in AD is
augmented as a result of a differential response to A
of the
basolateral surface of the endothelium in AD, compared with normal.
Previous studies show that high-affinity binding sites for soluble (s)
A
1
40 are preferentially localized to the apical
surface of N-HBEC, similar to that observed for insulin
(12). Thus interaction of A
with its receptor on the basolateral surface in the AD brain may favor migration of monocytes from the blood into the brain but may be diminished in normal brain
vascular endothelium because of reduced or absent A
binding to its
putative receptor on the basolateral side.
We report here that interaction of A with either apical or
basolateral surfaces of an HBEC monolayer, when derived from either normal or AD individuals, differentially affects the transmigration of
monocytes. Furthermore, we show here that interaction of A
with its
putative receptor RAGE expressed on the basolateral surface of AD-HBEC
initiates cellular signaling to allow monocytes to transmigrate from an
apical to basolateral direction. Additionally, the transmigration of
monocytes is blocked by antibody to PECAM-1, indicating the role of
PECAM-1 junction molecule in mediating the transmigration of monocytes.
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METHODS |
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Isolation and characterization of HBEC. Human brain capillaries were isolated from small fragments of cerebral cortex obtained from post mortem surgical resections from adults with seizure disorder and AD (66-92 yr) through the Neuropathology Core of the University of Southern California (USC) Alzheimer Disease Research Center (ADRC) as previously described (5). Brain specimens were cut into small pieces and homogenized in DMEM containing 2% fetal bovine serum (DMEM-S) with the use of a Dounce homogenizer with a loose fitting. The homogenate was centrifuged in 15% dextran in DMEM-S for 10 min at 10,000 g. The pellet containing crude microvessels was further digested in a solution containing 1 mg/ml collagenase-dispase in DMEM-S for 1 h at 37°C. Brain endothelial cells (BEC) were fluorescence-activated cell sorter (FACS) sorted using acetylated low-density lipoprotein (LDL) labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (diI-Acetyl LDL) as previously described (12).
Human brain microvessels were plated on gelatin-coated tissue culture dishes or glass coverslips and cultured in RPMI 1640-based medium with 10% fetal bovine serum (FBS), 10% NuSerum, endothelial cell growth supplement (30 µg/ml) (Collaborative Biomedical Products; B&D, Bedford, MA), heparin (100 µg/ml), L-glutamine (2 mM), sodium pyruvate (1 mM), MEM nonessential amino acids, MEM vitamins, penicillin and streptomycin (100 U/ml; Irvine Scientific, Irvine, CA). Cultures were incubated at 37°C in a humid atmosphere of 5% CO2 and characterized as described previously (12). The BEC exhibit a cobblestone morphology and express factor VIII antigen and CD105 (12). These HBEC expressCell culture of monocytes.
The promyelocytic cell line HL-60 and human monocytic cell line THP-1
(American Type Culture Collection, Rockville, MD) were cultured in RPMI
1640 containing 10% heat-inactivated fetal calf serum (FCS). HL-60
cells differentiate to a monocyte-like phenotype after treatment with
1 × 107 M 1
-25-dihydroxyvitamin D3
(Biomol Research Laboratories, Plymouth Meeting, PA) for 3-4 days
under culture conditions as described previously (19). We
used these cells in the differentiated state for transmigration studies.
Isolation of peripheral blood monocytes. Peripheral blood human monocytes (PBM) were isolated from blood collected in EDTA as the anticoagulant as previously described (5). Briefly, 10 vol of blood sample (30 ml) were mixed with 1 vol (3 ml) of a solution composed of 6% dextran-500 in 0.9% NaCl in a 50-ml conical tube. The tube was allowed to stand at room temperature for 45 min, which resulted in the sedimentation of erythrocytes. The leukocyte-rich plasma was harvested, layered over Nyco-prep media, density 1.068 g/ml (Accurate Chemical and Scientific, Westbury, NY) at a ratio of 2:1, and centrifuged at 600 g for 15 min. The monocyte fraction was further purified according to the manufacturer's instructions. Monocytes isolated by this procedure had purity in the range of 80-95% and a yield of ~60%, as assessed by labeling with CD11b-FITC antibody (Coulter Diagnostics, Hialeah, FL). T lymphocytes were isolated from leukocyte-rich plasma by passing through a sterilized column of Nytex as previously described (5).
Transendothelial migration assay of monocytes.
HBEC were grown to confluence on fibronectin-coated porous membranes in
a Transwell chamber (3.0 micron; Collaborative Biomedical Products).
After 5-6 days in incubation, HBEC exhibited a transendothelial electrical resistance of 120-180 · cm
2
and were used at that time. To the top chamber was added 1.0 ml
of either monocytic cells (0.5 × 105 cells/well) or
PBM (0.5 × 105 cells/well) in RPMI 1640 medium
containing 2% FCS. To the bottom compartment of the Transwell chamber
was added 1 ml of RPMI 1640 plus 2% FCS, as previously described
(5). To either the upper chamber (top or apical side) or
to the lower chamber (bottom or basolateral side) was then added
A
1
40 synthetic peptide (125 nM), and the contents
were incubated at 37°C for time periods ranging from 30 min to 4 h. At the indicated time points, 50-µl aliquots were removed from the
bottom compartment of the well. Cells were stained with 0.2% trypan
blue, and trypan blue-excluded transmigrated monocytic cells were
counted microscopically with the use of a hemacytometer grid.
To keep the volume constant in the bottom compartment, an equal amount
(50 µl) of medium was added after each removal of monocytes. For
inhibition experiments, the pharmacological inhibitors were added 45 min before addition of A
1
40, to either the top
compartment or the bottom compartment of the Transwell chamber.
32P labeling of cells and immunoprecipitation.
HBEC grown to confluence in 100 × 15-mm tissue culture dishes
were washed with prewarmed phosphate-free RPMI 1640 (GIBCO/BRL) and
radiolabeled with 0.20 mCi 32P (carrier free; ICN
Biomedical, Irvine, CA) in 3 ml of the same medium for 4 h at
37°C as previously described (10). Briefly, the
32P-labeled monolayer of HBEC was washed with
phosphate-free medium and incubated in 3 ml of the same medium in the
presence or absence of A1
40 for the indicated time
periods. At the end of incubation, cells were washed, and cell lysate
was prepared using 1 ml of lyses buffer containing 50 mM
Tris · HCl, pH 8.0, 150 mM NaCl, 0.1% SDS, 1% Nonidet P-40
(NP-40), 0.1 mM dithiothreitol (DTT), 0.5% sodium deoxycholate, 2 mM
sodium orthovanadate, 50 mM sodium fluoride, 10 mM EDTA, 100 µg/ml
phenylmethylsulfonyl fluoride (PMSF), 1 µg/ml pepstatin A, and 1 µg/ml leupeptin. The lysate was centrifuged at 3,000 g for
10 min. PECAM-1 was immunoprecipitated from the supernatant by a
polyclonal antibody to human PECAM-1. The immunocomplex was collected
by centrifugation, washed three times with 300 µl of lysis buffer,
and then solubilized in electrophoresis buffer [50 mM
Tris · HCl, pH 6.8, 2% SDS, 5% (vol/vol)
-mercaptoethanol, 10% (vol/vol) glycerol, and 0.1% bromphenol blue]. The solubilized sample was subjected to electrophoresis on a 10% SDS-polyacrylamide gel followed by autoradiography. Radioactivity in the band
corresponding to PECAM-1 (130 kDa) was quantitated by scanning with an
Ambis Radioanalytic Imaging Systems scanner (model 101; San Diego, CA).
Immunohistochemical analysis of the human central nervous system. AD patients and neurologically normal age-matched controls from the ADRC of USC were evaluated clinically and followed to autopsy. Included were males and females, ranging in age from 66 to 92 yr. Tissue blocks (1 cm3) were obtained postmortem (range 4-7 h; mean 5 h), fixed in 10% neutral buffered Formalin (pH 7.3; Sigma Chemical), and embedded in paraffin or snap-frozen in liquid nitrogen-chilled isopentane. Tissues were sampled from the superior and middle frontal gyrus (Brodmann's area 10) and cerebellum. Sections were stained with hematoxylin and eosin, thioflavin S, or the Gallyas modification of the Bielschowsky silver impregnation method. Thioflavin S-stained sections were viewed through a Zeiss fluorescence microscope with a narrow-band blue/violet filter at 400-455 nm. Diagnosis of AD was according to a modified protocol from the Consortium to Establish a Registry for Alzheimer's Disease (18).
Materials.
1,25-Dihydroxyvitamin D3 and calyculin A were obtained
from Biomol Research Laboratories; GF-109203X [protein kinase C
(PKC) inhibitor] was obtained from Calbiochem-Novabiochem (San Diego, CA). diI-acetyl LDL was purchased from Biomedical Technologies (Stoughton, MA). An antibody to bovine PECAM-1 (XVD2 as
ascites fluid) was developed in our laboratory (8),
monoclonal antibody to human PECAM-1 (5.6 E) was obtained from
Immunotech (Westbrook, ME), and monoclonal antibody to human leukocyte
antigen (HLA)-ABC antigen (class I, W6/32) was obtained from
Dako (Carpinteria, CA). Polyclonal antibody to human PECAM-1 (SEW 16)
was kindly provided by Dr. Peter Newman (Blood Research Institute,
Milwaukee, WI). Polyclonal antibody to RAGE in rabbits was developed in
our laboratory (21). All other chemicals, unless otherwise
mentioned, were obtained from Sigma (St. Louis, MO). X-ray film (Kodak
X-Omat AR) was obtained from Eastman-Kodak (Rochester, NY). Amyloid
peptide (A
1
40) was custom synthesized at the W. M. Keck facility at Yale University, purified, and characterized by
analytical reverse phase HPLC, amino acid analysis, and laser
desorption spectrophotometry as described earlier (12). It
was dissolved in PBS before use.
Statistical analysis.
Statistical analysis of the responses obtained from control and
A-treated HBEC was carried out by one-way analysis of variance (ANOVA), using Instat 2 (Graphpad, San Diego, CA) software program. The
effects of inhibitors on A
-induced responses were analyzed by
comparing the response of endothelial cells in the presence and absence
of inhibitor. Student's t-test was used for multiple comparisons. P values < 0.05 were considered significant.
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RESULTS |
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Effect of interaction of A with the apical surface of N- and
AD-BEC monolayer on the transmigration of monocytic cells.
With increased monocytes/microglial cells in cerebrovasculature and
brain parenchyma of A
-related cerebral vascular disorders, including
AD, we compared migration characteristics of monocytes across the
monolayer of endothelial cells derived from either age-matched controls
or AD patients. Our previous studies (5) show that
A
1
40 augmented the adhesion and migration of either
monocytic cells (THP-1 or differentiated HL-60 cells) or PBM across the
monolayer of N-HBEC. The optimal concentration of
A
1
40 was 125 nM, which did not affect morphology or
cause toxicity to HBEC. As shown in Fig.
1A, addition of
A
1
40 (125 nM) to the top compartment of the Transwell
chamber, containing N-HBEC monolayer (apical side), resulted in a
time-dependent increase in the migration of THP-1 monocytic cells.
There was an ~2.5-fold increase above the basal level in the
migration of THP-1 monocytic cells by the 2-h time point in response to
treatment with A
1
40 (125 nM). In AD-HBEC,
A
1
40 (125 nM) also caused a time-dependent increase
in the migration of THP-1 cells and showed an approximately twofold
increase in transmigration above the basal level by the 2-h time point
(Fig. 1A). There was <10% difference in the extent of
transmigration of monocytes in response to A
1
40 when the same AD patient-derived BEC were used at either passage
4 or 5. These studies demonstrate that A
interaction
with the apical surface of N-HBEC and AD-HBEC can mediate the migration
of monocytes from the apical to the basolateral side. Moreover,
A
1
40-induced transmigration of monocytes across the
monolayer of AD-HBEC was relatively higher compared with N-HBEC at time
points from 2 to 4 h (P value < 0.001).
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Effect of interaction of A with basolateral side of N- and
AD-BEC monolayer on the transmigration of monocytic cells.
With A
peptide in the AD brain increased toward the basolateral side
of the cerebrovascular endothelium, we hypothesized that interaction of
A
with the basolateral surface of the brain vascular endothelial
cell monolayer would augment migration of monocytes. As shown in Fig.
1B, addition of A
1
40 (125 nM) to the
bottom compartment of the N-HBEC monolayer, cultivated in a Transwell
chamber, caused a 30% increase in the transmigration of THP-1
monocytes above the basal level. However, when A
1
40 was added to the bottom compartment of the AD-HBEC monolayer, there was
a time-dependent increase in the migration of THP-1 monocytes by 2 h. In AD-HBEC, there was an ~400% increase in the transmigration of monocytes in response to A
1
40 (125 nM) at 2 h (A
1
40-treated AD-HBEC vs.
A
-treated N-HBEC; P < 0.01). Similar results were
observed at time points of 3 and 4 h.
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Involvement of PECAM-1 and RAGE in the transendothelial migration
of HL-60 cells in response to interaction of A1
40
with the apical or basolateral side of endothelial cells.
Our recent studies (5) showed that migration of monocytes
across the N-HBEC monolayer, mediated by the interaction of A
with
the apical side, was abrogated by an antibody (Ab) to PECAM-1 (Ab-PECAM-1), indicating the role of PECAM-1 in regulating the flux
of monocytes across N-HBEC. We examined whether PECAM-1 also affected the transmigration of monocytes across the AD-HBEC monolayer. As shown in Fig. 3A, addition
of monoclonal Ab-PECAM-1 caused 80 ± 10% inhibition in the
transmigration of HL-60 cells when A
1
40 (125 nM) was
added to the apical side, data similar to those observed with N-HBEC
(5). Similar results were obtained with polyclonal
Ab-PECAM-1 (SEW 16; data not shown). Additionally, we previously
observed that Ab-RAGE, a putative receptor for A
, inhibited
A
-induced migration of monocytes across the N-HBEC monolayer. Thus
we examined whether Ab-RAGE had any effect in AD-HBEC. Addition of
Ab-RAGE (5 µg/ml) reduced by 75 ± 20% the transmigration of
HL-60 cells in response to the addition of A
1
40 to
the apical side (Fig. 3A). Doubling the amount of Ab-RAGE
(10 µg/ml) did not further reduce the transmigration of monocytes (data not shown). Addition of an irrelevant Ab-HLA-ABC, used as a
negative control, had no effect on the transmigration. These results
indicate that in AD-HBEC, the transmigration of HL-60 monocytes
mediated by the interaction of A
with the apical side involves both
RAGE and PECAM-1.
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Effect of tyrosine kinase and protein kinase inhibitors on the
transmigration of HL-60 monocytes across the AD-HBEC monolayer.
Our recent studies (5) reveal that inhibitors of PKC and
tyrosine kinase blocked A-mediated migration of monocytes across the
N-HBEC monolayer. As shown in Fig. 4,
Genistein, a tyrosine kinase inhibitor, blocked (~65%) the migration
of HL-60 cells in response to the interaction of A
1
40
with the basolateral side of the AD-HBEC monolayer. Additionally, we
observed that the PKC inhibitor GF-109203X blocked (~50%) the
transmigration of HL-60 monocytes. However, the protein phosphatase
inhibitor calyculin A did not significantly affect the A
-mediated
migration of monocytes. Because A
-mediated signaling in N-HBEC
(5) resulted in increased expression of cell adhesion
molecules [CAMs; ICAM-1, vascular CAM-1 (VCAM-1), and E-selectin]
through the activation of the transcription factor nuclear factor
(NF)-
B, we determined whether pyrrolidine dithiocarbamate (PDTC), an
inhibitor of NF-
B activation, would affect the transmigration of
monocytes. As shown in Fig. 4, PDTC reduced by ~70% the
transmigration of monocytes in response to the interaction of
A
1
40 with the basolateral side of an AD-HBEC
monolayer. These results indicate that cellular signaling emanating
from the interaction of A
with RAGE expressed on the basolateral
surface in AD-HBEC involves downstream activation of tyrosine kinase
and PKC, culminating in amplified transmigration of monocytes.
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A1
40-induced phosphorylation of PECAM-1 in
HBEC.
Treatment of 32P-labeled HBMVEC with A
1
40
(125 nM) resulted in a time-dependent (0.5-4 h) increase in
32P incorporation into PECAM-1 (130-kDa band; data not
shown). The phosphorylation of PECAM-1 increased to a maximal level at
the 2-h time point after exposure to A
1
40 (125 nM)
and then decreased by the 4-h point. The incorporation of
32P into PECAM-1 at 2 h was approximately fivefold
higher relative to untreated HBMVEC at the zero time point (data not
shown). As shown in Fig. 5, the addition
of GF-109203X (20 nM), a selective PKC inhibitor, reduced by ~90%
the A
1
40-induced incorporation of 32P
into PECAM-1 (P < 0.001). As previously
observed (10) in endothelial cells exposed to hypoxia, the
addition of phosphatase inhibitor calyculin A (2 nM) with
A
1
40 resulted in an approximately twofold increase in
the incorporation of 32P into PECAM-1 relative to
A
1
40 alone-treated HBMVEC (Fig. 5). Moreover, we
observed that A
1
40-induced phosphorylation of PECAM-1
was blocked by Ab-RAGE but not with irrelevant Ab-HLA-ABC (Fig. 5),
indicating that phosphorylation of PECAM-1 emanates as a result of
cellular signaling on interaction of A
1
40 with
receptor RAGE expressed on HBMVEC (12). The
effect is specific for A
1
40, as amyloid peptide with
reverse orientation A
40
1 had a minimal effect on the
phosphorylation.
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Effect of interaction of A with the apical and basolateral
surfaces of N- and AD-HBEC monolayers on the transmigration of PBM.
Studies were undertaken to determine whether transmigration of PBM
behaved in the same way as monocytic cell lines (THP-1 and
HL-60) in response to A
. As shown in Fig.
6A, addition of A
to the apical side of N-HBEC resulted in an approximately threefold increase in the transmigration of PBM at 2 h. However, addition of
A
1
40 to the bottom compartment of the Transwell
chamber, i.e., the basolateral side of the endothelial cell monolayer
(N-HBEC), resulted in an ~1.6-fold increase in the transmigration of
PBM (Fig. 6A). However, addition of A
to either the
apical or the basolateral side of the AD-BEC monolayer from two
different AD patients yielded a significant time-dependent increase in
the transmigration of PBM (Fig. 6B). There was an
approximately five- and sixfold increase in the transmigration of PBM
across the AD-HBEC monolayer at 2 h in response to A
added to
the apical (top) and basolateral (bottom) side of the endothelial cell
monolayer, respectively (Fig. 6B). To examine whether this
effect was specific for monocytes, we utilized T cells from a normal
human donor. As shown in Fig. 6C, A
added from either the
apical or the basolateral side did not significantly increase the
migration of resting T cells across the AD-HBEC monolayer.
|
Role of RAGE and PECAM-1 in the transmigration of PBM.
Because we observed that transmigration of PBM across AD-HBEC was
significantly augmented in response to the addition of A to the
basolateral side of the monolayer, we determined whether RAGE and
PECAM-1 were involved in this process. As shown in Fig. 6D , the addition of Ab-RAGE and Ab-PECAM-1 reduced transmigration of PBM by
~60 and 80%, respectively. However, a control Ab-HLA-ABC had no
significant effect on the transmigration of PBM. These studies indicate
that A
interaction with RAGE expressed on the basolateral side of
the AD-HBEC may cause cellular signaling culminating in the
transmigration of PBM. An Ab-PECAM-1, an adhesion molecule concentrated
at endothelial cell junctions, blocks this effect.
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DISCUSSION |
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In AD and A-related cerebral vascular disorders (CAA and
HCHWA-D), increased deposition of amyloid peptide (A
) in both the brain parenchyma and the cerebral vasculature occurs. These changes are
accompanied by an accumulation of monocytes/macrophages in the vessel
wall and activated microglial cells in the adjacent parenchyma
(11, 25). Relatively little is known of the putative relationship of A
to the accumulation of these inflammatory cells in
the brain. There are two major views regarding the developmental origin
of microglial cells, one being that they are derived from neuroepithelial cells (17) and the other that they are
derived from hematopoietic cells (7). Early studies of
Hickey and Kimura (7) showed that peripheral hematopoietic
cells (e.g., monocytes) cross the BBB and then differentiate to
microglial cells in the brain parenchyma. These observations support
the idea that microglial cells can arise from peripheral hematopoietic
cells. Recent studies of Eglitis and Mezey (3) give
further credence that microglial cells in the brain are most likely
derived from hematopoietic cells. They showed that implantation of male
bone marrow-derived cells into female mice resulted in the appearance
of these male donor cells in the brain 3 days after the
implant. After a few weeks, these cells differentiated into microglial
cells in the brain as revealed by immunoreactivity toward glial
antigenic marker. These studies thus provide compelling evidence that
hematopoietic cells cross the BBB and act as microglial progenitor
cells. In vivo studies confirm that A
induces the activation and
migration of monocytes across a rat mesenteric vascular bed
(24), indicating that a similar phenomenon occurs in the
brain vasculature.
We previously demonstrated (5) that interaction of A at
nanomolar concentrations with a monolayer of N-HBEC initiates cellular
signaling, leading to adhesion and transmigration of monocytes.
Significantly, the intracellular mechanisms of BEC responses to A
culminate in the upregulation of CAMs (ICAM-1, VCAM-1, and E-selectin).
These CAMs participate in the adhesion of monocytes. The expression of
CAMs mediated by A
is blocked by Ab-RAGE (5). These
results suggest that interaction of A
with its putative receptor
RAGE, expressed on HBEC, results in cellular signaling that promotes
monocytes to adhere to CAMs. These results are in accordance with
previous findings (12) in which binding of
125I-labeled A
1
40 to a monolayer of
cultured endothelial cells derived from normal brain was abrogated by
Ab-RAGE. Moreover, these studies (12) also showed that
binding of 125I-A
1
40 occurred on the
apical side of the monolayer of HBEC. These observations imply that
interaction of circulating sA
with the vessel wall could induce
signaling and allow PBM to adhere to the cerebrovasculature and
subsequently induce their diapedesis into the brain.
The A-induced migration of monocytes across the N-HBEC monolayer was
blocked by Ab-PECAM-1 (platelet-endothelial cell junction molecule-1).
These results indicate the involvement of PECAM-1 in mediating the
transmigration. Our previous studies (19) show that
oxidant stress-induced transmigration of monocytes across monolayers of
human umbilical vein endothelial cells (HUVEC) was inhibited by
Ab-PECAM-1. Thus PECAM-1 plays a role in the transmigration of
monocytes. Vaporciyan et al. (26) report that PECAM-1 is concentrated at endothelial cell junctions and plays a role in the
transmigration of both neutrophils and monocytes in vivo. The fact that
the PECAM-1 molecule is also expressed in both normal and AD brain
vasculature (data not shown) and in HBEC (29) suggests that circulating A
can induce the migration of PBM across the vasculature of the BBB via endothelial cell junction molecule PECAM-1.
The present work documents that interaction of A1
40
with the apical surface of AD-derived BEC also mediates migration of
both PBM and human monocytic cells (THP-1 and vitamin
D3-differentiated HL-60 cells), as has been previously
observed with a monolayer of N-HBEC (5). In both N- and
AD-derived HBEC, the extent of increase in transmigration of monocytes,
in response to interaction of A
1
40 with the apical
surface, was not significantly different. There was an approximately
threefold increase (368 ± 141%) in the transmigration of
monocytes from the apical to basolateral direction in response to
interaction of A
1
40 with the apical surface of the
AD-derived HBEC monolayer. Similarly, in the N-HBEC monolayer there was
an approximately threefold increase (372 ± 139%) above the basal
level in response to the interaction of A
1
40 with the
apical surface of N-HBEC. Moreover, transmigration of monocytes across
the AD-HBEC monolayer, in response to the interaction of
A
1
40 with apical surface, was inhibited by both
Ab-RAGE and Ab-PECAM-1, as we have previously (5) observed
with N-HBEC. Our data suggest that in both normal and AD brain
vasculature, circulating sA
1
40 interaction with RAGE
expressed on brain endothelium can induce cellular signaling that
stimulates monocytes to transmigrate. The cellular signaling involves
activation of redox-sensitive pathways, as
A
1
40-mediated transmigration of monocytes in HBEC has
been previously shown to be attenuated by the antioxidants probucol and
vitamin E (5). The exact sequence of cellular signaling
events downstream of A
-RAGE interaction remains to be elucidated.
However, increased numbers of monocytes/macrophages and the clustering
of microglia in perivascular space of the cerebrovasculature of
patients with AD and A-related disorders (CAA and HCHWA-D) remain
intriguing as does the mechanism by which these inflammatory cells
accumulate in these diseases (11, 25, 31).
Because in AD and A
-related disorders (CAA and HCHWA-D) one finds
(11) increased amounts of A
associated with the
basolateral side of the vascular endothelium, we hypothesized that this
may have a significant influence on the accumulation of inflammatory
cells from the peripheral blood circulation in the vasculature and
eventually into the brain parenchyma.
Endothelial cells in vivo are polarized with the apical side
interacting with circulating blood cells and the basolateral side
interacting with matrix components. In the brain microvasculature, the
basolateral surface of endothelial cells can interact with both amyloid
peptides associated with the basolateral surface of vascular
endothelium and soluble/fibrillar forms of amyloid peptide accumulated
in the brain. Here, we show that interaction of A with the
basolateral side of the monolayer of AD-HBEC results in an ~4.5-fold
increase (458 ± 151%) in the transmigration of monocytes. In
contrast, the interaction of A
with the basolateral side of the
monolayer of N-HBEC resulted in an approximately twofold increase
(220 ± 76%) in the transmigration of monocytes. These results
indicate that there are inherent differences in the transmigration of
monocytes across an endothelial cell monolayer derived from normal and
AD individuals, in response to interaction with amyloid peptide. It
remains to be determined whether there are differences in the binding
of A
1
40 and expression of A
putative receptors
(RAGE, Scavenger Receptor, SR-A; Ref. 12) or an alternate signaling mechanism to cause increased migration of monocytes from the
apical to basolateral side in response to the interaction of A
with
the basolateral side of the AD-HBEC monolayer.
The involvement of both RAGE and PECAM-1 in the transmigration of PBM
and monocytic cells in response to the interaction of A with the
basolateral side of the AD-HBEC monolayer is supported by data showing
that Ab-RAGE and Ab-PECAM-1 reduce transmigration by ~60 and 80%,
respectively. Furthermore, our studies show that interaction of A
with the basolateral side of the monolayer of AD-HBEC causes cellular
signaling by activation of both tyrosine kinases and PKC, as the
pharmacological inhibitor of tyrosine kinase (Genistein) and PKC
(GF-109203X) reduce ~65 and 50% of transmigration of monocytic
cells, respectively. Moreover, A
-mediated transmigration of
monocytes was inhibited by PDTC, an inhibitor of NF-
B activation.
These studies indicate that A
-mediated activation of PKC in BEC may
lead to activation of NF-
B, as has been shown for tumor necrosis
factor-
(TNF-
)-mediated activation of PKC in Jurkat cells,
leading to activation of NF-
B (14). Our previous studies (19) have shown that oxidant stress
(t-butyl hydroperoxide) causes an increase in the
transmigration of monocytes across the monolayer of HUVEC and a
concomitant increase in the phosphorylation of PECAM-1. Both
transmigration of monocytes and phosphorylation of PECAM-1 were
inhibited by PKC inhibitor, suggesting that there may be a causal or
direct effect of PECAM-1 phosphorylation in the transmigration of
monocytes. In the present study, we also observed that A
caused an
increase in the phosphorylation of PECAM-1 in HBEC, suggesting that
phosphorylation of PECAM-1 may have a direct or causal relationship in
facilitating migration of monocytes. Overall, these results indicate
that amyloid peptide present on the cerebrovasculature or in the brain
parenchyma of AD may elicit cellular signaling to allow monocytes to
migrate from the lumen side of the BBB, leading to accumulation in the cerebrovasculature and eventually the brain. The migration of monocytes
involves PECAM-1 cell junctional molecule expressed on AD-BEC.
Our results show that interaction of A with either the apical or
basolateral surface of a monolayer of HBEC initiates cellular signaling
leading to transmigration of monocytes from the apical to basolateral
direction. We show that the increase in the transmigration of monocytes
across BEC monolayers derived from normal and AD individuals is not
significantly different in response to the interaction of A
with the
apical surface. These results indicate that trafficking of PBM across
the BBB can occur in response to amyloid peptide present in the
peripheral circulation or because of accumulation of amyloid peptide in
the brain. However, interaction of A
with the basolateral surface of
the monolayer of N-HBEC shows a modest increase (2-fold) compared with
AD-HBEC, which shows a substantial increase (4.5-fold) in the
transmigration of monocytes. The transmigration of monocytes across
AD-HBEC, in response to interaction of amyloid peptide with basolateral surface, occurs as a result of interaction of A
with the receptor RAGE. Furthermore, we show that PECAM-1 localized at endothelial cell-to-cell junctions regulates the trafficking of monocytes in
response to the interaction of amyloid peptide with the apical and
basolateral surface of BEC. We suggest that increased diapedesis of
monocytes brought about by the interaction of A
with the basolateral surface of the vascular endothelium of BBB in AD individuals may contribute to the increased presence of inflammatory cells
(monocytes/macrophages) and activated microglial cells seen in
A
-related vascular disorders such as AD.
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ACKNOWLEDGEMENTS |
---|
This work was supported by National Institute on Aging Grant PO1-AG-16233 (to B. V. Zlokovic).
![]() |
FOOTNOTES |
---|
Address for reprint requests and other correspondence: V. Kalra, HMR 611, Dept. of Biochemistry and Molecular Biology, USC Keck School of Medicine, Los Angeles, CA 90033 (E-mail: vkalra{at}usc.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpcell.00293.2001
Received 29 June 2001; accepted in final form 6 May 2002.
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