1 Departments of Biochemistry and Molecular Biology, and 4 Neurosurgery, University of Southern California Keck School of Medicine, and 2 Division of Infectious Disease in the Department of Pediatrics at Childrens Hospital-University of Southern California, Los Angeles, California 90033; and 3 Department of Physiology, College of Physicians and Surgeons of Columbia University, New York, New York 10032
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ABSTRACT |
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In patients with
amyloid -related cerebrovascular disorders, e.g., Alzheimer's
disease, one finds increased deposition of amyloid peptide (A
) and
increased presence of monocyte/microglia cells in the brain. However,
relatively little is known of the role of A
in the trafficking of
monocytes across the blood-brain barrier (BBB). Our studies show that
interaction of A
1-40 with monolayer of human brain
endothelial cells results in augmented adhesion and transendothelial
migration of monocytic cells (THP-1 and HL-60) and peripheral blood
monocytes. The A
-mediated migration of monocytes was inhibited by
antibody to A
receptor (RAGE) and platelet endothelial cell adhesion
molecule (PECAM-1). Additionally, A
-induced transendothelial
migration of monocytes were inhibited by protein kinase C inhibitor and
augmented by phosphatase inhibitor. We conclude that interaction of
A
with RAGE expressed on brain endothelial cells initiates cellular
signaling leading to the transendothelial migration of monocytes. We
suggest that increased diapedesis of monocytes across the BBB in
response to A
present either in the peripheral circulation or in the
brain parenchyma may play a role in the pathophysiology of A
-related
vascular disorder.
amyloid -peptide; brain endothelial cells; platelet endothelial
cell adhesion molecule; receptor for advanced glycation end product
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INTRODUCTION |
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DURING NORMAL AGING,
deposition of amyloid -peptide (A
) occurs in the brain and
cerebral blood vessels and is accelerated in diseases such as
Alzheimer's disease (AD), cerebral amyloid angiopathy (CAA), and the
genetic amyloidogenic disease hereditary cerebral hemorrhage with
amyloidosis of the Dutch type (HCHWA-D) (28). The A
, a
heterogeneous, 39- to 43-amino acid peptide, the major constituent of
senile plaques and cerebrovascular deposits (28), is
thought to play a significant role in the pathophysiology of AD due to
its cytotoxic properties (16). The predominant forms of
A
are the 1-40 and 1-42 fragments
(A
1-40 and A
1-42) formed by the
normal proteolytic processing of amyloid precursor protein (APP)
(19). The A
precursor protein is present in many cells,
including vascular smooth muscle cells, perivascular cells, platelets,
endothelial cells, neuronal cells, and blood cells (11, 12,
19). The soluble A
1-40 is the major form of
circulating
-amyloid and cerebral vascular amyloid, whereas the more
amyloidogenic A
1-42, which is present in only minor
amounts in the circulation, constitutes a major component of senile
plaques (3, 15, 19, 21).
In A-related cerebral vascular disorders (CAA and HCHWA-D) and AD,
one finds not only the increased deposition of A
in both the brain
parenchyma and cerebral vasculature, but also an increased presence of
monocytes/macrophages in the vessel wall and activated microglial cells
in the parenchyma (12, 26, 28). Relatively little is known
of A
's putative relationship to the accumulation of these
inflammatory cells in the brain. Early studies of Hickey and coworkers
(6, 7) showed that microglial cells in the brain were
derived from hematopoietic cells. Recent studies (4) have
shown that peripheral hematopoietic cells (e.g., monocytes) can cross
the blood-brain barrier (BBB) and then undergo differentiation into
microglial cells in the brain parenchyma, thus supporting the
contention that microglial cells can arise from peripheral hematopoietic cells.
Due to the accumulation of A in the cerebral vasculature, one
frequently observes disruption of basement membrane, degeneration of
the endothelium, and diminished size of the lumen of the blood vessel
(8). The interaction of A
with its receptor advanced glycation end products (RAGE) expressed on endothelial cells, neurons,
and microglia has been shown to initiate cellular signaling leading to
the generation of oxidant stress as determined by the formation of
lipid peroxides and activation of the transcription factor nuclear
factor-
B (30). Other studies showed that mononuclear phagocytes, including microglia, which express class A scavenger receptor, interact with A
and generate reactive oxygen species (5). However, relatively little is understood about how
the accumulation of A
in the cerebral vasculature causes dysfunction of the endothelium leading to migration of peripheral blood cells across BBB.
We hypothesize that the interaction of soluble A with cerebral
vascular endothelial cells initiates cellular signaling leading to the
increased migration of monocytes across the endothelial cell monolayer,
an index of dysfunction of endothelial cell-cell junction permeability.
In this study, we examine the effect of A
1-40 on
the migration of vitamin D3-differentiated HL-60 cells
(referred to hereafter as monocytes) across human brain endothelial
cell monolayer cultivated in Transwell chambers. We also examine
whether monocyte migration in response to A
1-40 is
mediated via the platelet endothelial cell adhesion
molecule-1 (PECAM-1). Previous studies have shown that PECAM-1
concentrated at endothelial cell intercellular junctions plays an
important role in cell-to-cell adhesion and is implicated in mediating
the transendothelial migration of monocytes and neutrophils (17, 27).
We show here that synthetic A1-40 interaction with
putative receptor RAGE expressed on cultured human brain microvascular endothelial cells (HBMVEC) initiates cellular signaling resulting in
adhesion and severalfold increase in the migration of monocyte-like cells (HL-60 and THP-1) and peripheral blood monocytes across the
confluent monolayer of HBMVEC. The migration of monocytes is blocked by
an antibody to PECAM-1, indicating the role of PECAM-1 junction
molecule in mediating the migration of monocytes.
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METHODS |
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Cell cultures. Primary HBMVEC were isolated from solid neural tissue samples of adult patients as previously described (24). Briefly, microdissection of the microvascular fragments was carried out followed by unit gravity sedimentation for 2-5 min to allow separation of nonvascular elements. The resulting pellet was subjected to this procedure two to three times. The microvascular fragments were seeded on gelatin-coated tissue culture dishes in a 1:1 DMEM/Ham's F-12 nutrient mixture (Sigma Chemical, St. Louis, MO) containing 10% heat-inactivated fetal bovine serum and 25 mM HEPES, pH 7.2, endothelial cell growth supplement (50 µg/ml) (Collaborative Biochemical Products), and heparin (100 µg/ml). The outgrowing cultures were cultured in a 5% CO2 atmosphere in a tissue culture incubator maintained at 37°C. Cells were expanded using trypsin-EDTA. The brain endothelial cells exhibit a cobblestone morphology, express factor VIII antigen, take up acetylated low-density lipoprotein, and are positive for Ulex Europaeous Agglutinin I (24). These HBMVEC express gamma glutamyl transpeptidase, indicating brain endothelial cell characteristics. Moreover, these HBMVEC are minimally contaminated with pericytes and glial cells, as determined by labeling with anti-smooth muscle actin and anti-glial fibrillary acidic protein, respectively (24). HBMVEC were used between passages 3 and 6.
The promyelocytic cell line HL-60 and THP-1 (human monocytic cell line) obtained from American Type Culture Collection (Rockville, MD) were cultured in RPMI 1640 containing 20% heat-inactivated fetal calf serum (FCS). HL-60 cells differentiate to a monocyte-like phenotype after treatment with 1 × 10Cell viability and assessment of cell injury.
HBMVEC seeded in six-well culture plates (2 × 105
cells per well) were grown to confluence in RPMI 1640 containing 20%
FCS (24). Cells were incubated with
A1-40 (50-250 nM) for time periods ranging
from 1 to 6 h. Experiments were done in duplicate. Cell viability
was measured by trypan blue exclusion. Briefly, cell suspensions were
prepared by trypsinization followed by washing and resuspension in
Hanks' balanced salt solution. An aliquot of the cell suspension was
mixed with 0.5% trypan blue, and cells were counted by a
hemocytometer. Cell viability was calculated as the percentage of
viable (unstained) cells. The injury to endothelial cells in response
to exposure to A
1-40 (50-250 nM) was determined by the release of 51Cr from radiolabeled HBMVEC.
Briefly, confluent monolayers of HBMVEC grown in 24-well plates were
labeled with 51Cr (2 µCi/ml) and washed three times with
PBS. 51Cr-labeled HBMVEC in RPMI 1640 culture medium (1 ml)
were incubated in triplicate wells without and with
A
1-40 (50-250 nM) for time periods of 30, 60, 120, and 240 min. At these time points, the supernatant was removed for
counting the radioactivity. The cell-bound 51Cr was
determined after solubilization of the monolayer with 0.5 ml of 1 N
NaOH. The percentage 51Cr release was determined as a
percentage of total radioactivities present in the radiolabeled
monolayer. The morphology of the monolayer after treatment with
A
1-40 was determined by phase-contrast microscopy
(Olympus IMT-2 microscope).
Adherence assay.
HBMVEC were cultured in 24-well dishes to confluence. The wells were
rinsed twice with serum-free medium, and 51Cr-labeled THP-1
monocytes (2 X 105 cells) in 5% FBS medium containing RPMI
1640 were added to each well in the absence and presence of
A1-40 (125 nM). After 30 min, nonadherent
monocytes were removed by aspiration and the wells were washed twice
with PBS (1 ml) containing 0.5% BSA. The radioactivity in the cell
monolayer containing adherent monocytes was determined, and the
percentage of adherent monocytes calculated (22). To
delineate the functional role of RAGE, VCAM-1 and VLA-4 (CD 49 d) in monocytes binding corresponding monoclonal antibodies were incubated with endothelial cell monolayers for 30 min before incubation with monocytes.
Transendothelial migration assay of monocytes.
HBMVEC were grown to confluence on fibronectin-coated porous membranes
in a Transwell chamber (3.0 µm; Collaborative Biomedical Products,
Bedford, MA). Transendothelial resistance was measured daily on the
filters with sterilized electrodes using an EVOM voltammeter (World
Precision Instruments, Sarasota, FL). The monolayer after 5-6 days
in culture exhibited transendothelial electrical resistance (TEER) of
120-180 /cm2 and was used at that time for
migration studies. To the confluent monolayer was added an aliquot
(0.1 × 106 cells/well) of monocytes in 0.5 ml of RPMI
1640 plus 20% FCS, as previously described (22), whereas
the lower compartment of the Transwell chamber contained 1 ml of RPMI
1640 plus 20% FCS. A
1-40 synthetic peptide was
then added to the upper chamber, and the contents were incubated at
37°C for time periods ranging from 30 min to 4 h. At the
indicated time points, aliquots of 50 µl were removed from the bottom
compartment of the well, and cells were stained with 0.2% trypan blue
for microscopic counting of trypan blue-excluded transmigrated
monocytes, using a hemocytometer grid. To keep the volume constant in
the lower compartment, an equal amount (50 µl) of medium was added at
each removal of monocytes. In inhibition experiments, the
pharmacological inhibitors were added at 45 min, unless otherwise
indicated, before the addition of A
1-40 to the
upper compartment of the Transwell chamber.
Expression of cell adhesion molecules by ELISA.
HBMVEC were grown to confluence in 24-well tissue culture plates,
rinsed with RPMI 1640 medium, and incubated with serum-free RPMI 1640 in the presence or absence of A1-40 (125 nM) at
37°C. This concentration of A
1-40 was found to be
nontoxic to cells and exhibited optimal response in transmigration
assay of monocytes. At the end of indicated incubation periods, ranging from 30 min to 24 h, the medium was aspirated and the wells were washed twice with 1 ml of PBS. To each well was added 500 µl of 2.5%
paraformaldehyde to fix the cells. Cell surface expression of ICAM-1
and VCAM-1 in HBMVEC was assayed by separate incubations with
monoclonal antibodies to ICAM-1 and VCAM-1. Incubations were carried
out at room temperature for 2 h at a saturating concentration of
antibody (1:500 dilution in 0.5 ml of PBS). Cells were washed and then
incubated with alkaline phosphatase-conjugated goat anti-mouse IgG
(diluted 1:1,000 in PBS) for 60 min. Cells were washed twice with 1 ml
of PBS. Binding of the secondary antibody was determined by the
addition of 200 µl of p-nitrophenyl phosphate substrate (1 mg/ml in 0.2 M Tris, pH 9.8, containing 5 mM MgCl2). Plates were incubated for 30 min in the dark. The reaction was terminated by
the addition of 50 µl of 3 N NaOH. The absorbency was read at 405 nm
in an ELISA plate reader (Cambridge Technology model 7520, Watertown,
MA) interfaced with an IBM personal computer. The surface expression of
cell adhesion molecules is shown as the mean ± SD of the
optical density (OD) after subtracting the blank value, that is the OD
in the absence of primary antibody as previously described
(22).
Permeability and electrical resistance of HBMVEC monolayer.
HBMVEC were grown to confluence on fibronectin-coated porous membranes
in a Transwell chamber (3.0 µm; Collaborative Biomedical Products).
The monolayer after 5-6 days in culture exhibited TEER of
120-180 /cm2. The HBMVEC monolayer was preincubated
for 30 min in RPMI culture medium containing 10% fetal bovine serum
(1.0 ml) at 37°C. [14C]inulin and
[3H]dextran was added to the upper chamber
followed by the addition of A
1-40 (125 nM). At 60 min, an aliquot (100 µl) was removed from the bottom compartment of
the Transwell chamber for counting of the radioactivity, using Beckman
LS-8000 liquid scintillation counter. The amount of radiolabeled inulin
or dextran transported across HBMVEC was expressed as the percent of
their respective radioactivity added to the upper chamber as previously
described (13). The electrical resistance of HBMVEC
monolayer was measured at 60 min after the addition of
A
1-40, antibody to RAGE, antibody to PECAM-1, and
peripheral blood monocytes (PBM) to the upper compartment of the
Transwell chamber.
Materials.
1,25-dihydroxyvitamin D3 and Calyculin A were obtained
from Biomol Research Laboratories, Plymouth Meeting, PA; GF-109203X (PKC inhibitor) was obtained from Calbiochem-Novabiochem, San Diego,
CA. PD-98059 (MEK I inhibitor) was obtained from New England Biolabs,
Beverly, MA. An antibody to bovine PECAM-1 (XVD2
as ascites fluid) was developed in author's laboratory
(9); monoclonal antibody to human PECAM-1 (5.6 E), ICAM-1,
and VCAM-1 were obtained from Immunotech, Westbrook, ME; monoclonal
antibody to 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. Polyclonal antibody to RAGE in
rabbits was developed in author's laboratory (20).
[14C]inulin (specific activity = 2.2 mCi/g) and
[3H]dextran (specific activity 322 mCi/g) were obtained
from NEN DuPont (Boston, MA) and Amersham (Arlington Heights, IL),
respectively. 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.
Statistical analysis.
Statistical analysis of the responses obtained from control and
A-treated endothelial cells was carried out by one-way 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.
Dunnett's test was used for multiple comparisons. P values
<0.05 were considered significant.
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RESULTS |
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Effect of A on the viability and injury to cultured human brain
endothelial cells.
Because A
at micromolar concentration has been shown to cause
necrosis or cell death in bovine aortic endothelial cells
(25) and apoptosis in porcine pulmonary aortic endothelial
cells (2), we first examined the effect of various
concentrations (50-250 nM) of synthetic A
i.e.,
A
1-40, on the release of 51Cr from
radiolabeled HBMVEC, an index of cellular injury. After a 2- to 4-h
exposure of HBMVEC with A
1-40 (125 nM), the extent of 51Cr release was not significantly different from
untreated control at time points of 0.5-8 h (data not shown). As
shown in Fig. 1, A (untreated)
and B (A
1-40 treated), there was no
discernible change in the morphology of HBMVEC monolayer on
treatment with 125 nM A
1-40 at 4 h. However, a
higher concentration (250 nM) of A
1-40 caused
10-15% increase in the release of 51Cr from
radiolabeled HBMVEC at the 4-h time period. Because exposure of HBMVEC
to 125 nM A
for 8 h did not affect either cell viability or
cause injury, we used this concentration (125 nM) of
A
1-40 and time period (4-8 h) for most of the
experiments described here.
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A1-40-mediated adherence of monocytes to
HBMVEC.
Because adhesion of monocytes is an initial event before diapedesis
across the endothelium, studies were carried out to determine whether
A
1-40 interaction with endothelium augmented
adherence of monocytes. The adherence of THP-1 monocytes to HBMVEC
monolayer was augmented approximately twofold on treatment of HBMVEC
with A
(Fig. 2). Antibody to RAGE
blocked ~60% of the adhesion of THP-1 monocytes. To discern whether
VCAM-1 molecule expressed on endothelial cells and counter receptor
VLA-4 expressed on monocytes were responsible for the adhesion, we used
corresponding blocking antibodies in the adherence assay. As shown in
Fig. 2, antibody to VCAM-1 and VLA-4 reduced by ~60% the adherence
of THP-1 monocytic cells, whereas a control antibody HLA-ABC had no
effect. Because VCAM-1 appears to be involved in the adherence of THP-1
monocytes, we examined the surface expression of VCAM-1 on HBMVEC in
response to A
1-40. As shown in Fig.
3, there was a time-dependent increase in
the expression of ICAM-1 and VCAM-1. The optimal expression of VCAM-1
occurred between the 4- and 6-h time period. Moreover, the
A
1-40-induced expression of VCAM-1 was blocked by
MAP kinase inhibitor (PD-98059), antioxidant (vitamin E), and an
antibody to RAGE (Fig. 4). An irrelevant
antibody to HLA-ABC antigen, used as a control, did not affect the
A
1-40-induced expression of VCAM-1.
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Effect of -amyloid on migration of monocytes across human brain
endothelial cell monolayer.
We examined whether incubation of HBMVEC with A
1-40
would result in an increased migration of monocytes across the monolayer. As shown in Fig.
5A, addition of
A
1-40 (125 nM) to confluent monolayer of HBMVEC
cultured in Transwell chambers resulted in an approximately two- and
threefold increase in the migration of monocyte-like HL-60 cells at the
2- and 4-h time point, respectively (P < 0.001).
Similarly, A
1-40 caused two- to threefold increase
in the transmigration of another monocytic cell line, THP-1, and
peripheral blood monocytes (see Fig. 7, A and B).
The increase in the transmigration of monocytes in response to
treatment of HBMVEC monolayer with nanomolar concentration of
A
1-40 was not due to nonspecific disruption of the
barrier property of the brain endothelial cell monolayer, because we
observed that the permeability of either 3H-labeled dextran
(molecular mass 70 kDa) or [14C]inulin
(molecular mass 6.1 kDa) remained unchanged in the absence and presence
of A
1-40 (see Fig. 8A). In previous
studies (2) it was observed that treatment of porcine
pulmonary aortic endothelial cells with A
25-35
resulted in an increased permeability to albumin. However, such effect
was observed only with micromolar concentration of A
. Additionally,
we observed that A
1-40-mediated effect on the
migration of monocytes across HBMVEC monolayer was specific, because
red blood cells, a negative control, did not transmigrate (data not
shown). Moreover, the migration of resting T cells across endothelial
cell monolayer was not augmented by A
1-40 (125 nM)
(data not shown).
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Involvement of PECAM-1 and RAGE in
A1-40-mediated transendothelial migration of
monocyte-like HL-60 cells.
Previous studies have shown that PECAM-1 localized at endothelial
cell-cell junctions plays a role in the transendothelial migration of
polymorphonuclear leukocytes and monocytes (10, 17, 27);
thus we examined the effect of an antibody to PECAM-1 on the
transendothelial migration of monocyte-like HL-60 cells. As shown in
Fig. 5D, the addition of monoclonal antibody (5 µg/ml) to
PECAM-1 reduced by 60-75% the transendothelial migration of monocyte-like HL-60 cells induced by A
1-40 (125 nM). Addition of double the amount of antibody did not supplement the
inhibition (data not shown). The monoclonal antibody to PECAM-1 was
~80% effective in blocking LPS induced monocyte migration
across HBMVEC monolayer (data not shown). Similarly, polyclonal
antibody to PECAM-1 (SEW-16) also displayed 60-80% inhibition in
the transendothelial migration of monocytes induced by
A
1-40. However, the addition of an antibody to
ICAM-1, an adhesion molecule expressed on endothelial cells, had no
significant effect on the transendothelial migration of monocyte-like
HL-60 cells. Similarly, the addition of an irrelevant antibody to
HLA-ABC, used as a negative control, had no effect on the
transendothelial migration of monocyte-like HL-60 cells (Fig.
5D).
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A1-40-induced migration of THP-1 monocytic
cells and peripheral blood monocytes across HBMVEC monolayer involves
RAGE and PECAM-1.
Studies were undertaken to determine whether the
A
1-40-mediated migration of monocytes occurred with
monocytic cells other than the differentiated HL-60 cells and with
monocytes derived from peripheral blood. As shown in Fig.
7A, A
1-40
(125 nM) exhibited an approximately threefold increase in the
transendothelial migration of THP-1, another monocytic cell line, at
the 2-h time period. Additionally antibody to RAGE was effective in
inhibiting (~65%) the transendothelial migration of THP-1 monocytes.
Furthermore, antibody to PECAM-1 also blocked (~65%) the
transendothelial migration of THP-1 monocytes. However, a control
antibody, HLA-ABC, did not inhibit A
1-40-induced
migration of THP-1 monocytes. These results show that monocyte
migration, whether THP-1 or HL-60 cells, across the brain endothelial
cell monolayer involves interaction of A
with RAGE, which in turn
causes cellular signaling to allow monocytes to adhere and migrate
through cell-cell junctions involving PECAM-1. Studies were undertaken
to discern whether PBM transmigration also occurred in response to
A
1-40. As shown in Fig. 7B, A
1-40 induced fourfold increase in the
transmigration of PBM, which was reduced by ~85% and ~95% by
antibody to RAGE to PECAM-1, respectively. Furthermore, pretreatment of
HBMVEC with phosphatase inhibitor (Calyculin A) followed by the
addition of A
(125 nM), resulted in an ~40% increase in the
transmigration of PBM. Additionally, the data show (Fig. 7B)
that antibody to PECAM-1 added to Calyculin A plus A
-treated HBMVEC
reduces by ~80% the migration of monocytes above the basal level. It
is pertinent to mention that addition of either A
alone (125 nM) or
peripheral blood monocytes did not alter the TEER across HBMVEC
monolayer (Fig. 8B) indicating
that permeability of the HBMVEC monolayer was not affected
significantly by both amyloid peptide and peripheral blood monocytes.
Additionally, antibody to either RAGE or PECAM-1 did not alter the TEER
of HBMVEC monolayer (Fig. 8B), indicating that antibodies to
these molecules do not disrupt barrier properties of HBMVEC monolayer.
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Effect of inhibitors on A1-40-induced
transendothelial migration of monocytes.
We examined whether A
1-40- induced
signaling lead to transendothelial migration of monocyte-like HL-60
cells. As shown in Fig. 9, preincubation
of HBMVEC with GF-109203X (20 nM), a PKC inhibitor, for 30 min followed
by the addition of A
1-40 resulted in an ~75%
decrease in the transendothelial migration of monocyte-like HL-60
cells. Tyrosine kinase inhibitor (genistein) also blocked ~55%
migration of monocytes. Additionally, Calyculin A, a protein
phosphatase inhibitor, augmented transmigration ~200% above the
levels obtained with A
1-40 alone (Fig. 9). Because interaction of A
with neuronal cells has been shown to induce oxidative stress (31), we determined whether antioxidants
and redox-sensitive pathways were involved in mediating transmigration in response to A
1-40. We observed that the
antioxidants probucol (50 µM) and vitamin E (25 µM) were effective
in blocking A
1-40-induced migration of
monocyte-like HL-60 cells. Similarly, treatment of HBMVEC with
glutathione ethylester (GSH-EE), which increases
intracellular levels of GSH, reduced by ~60% the
A
1-40-induced migration of monocyte-like HL-60
cells (Fig. 9).
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DISCUSSION |
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Pathological examination of brains from patients with AD and
cerebral vascular disorders (CAA and HCHWA-D) has shown increased presence/deposition of A in both the brain parenchyma and cerebral vasculature. Additionally, one finds increased presence of inflammatory cells (monocyte/macrophages) in the vessel wall and activated microglial cells around neuritic plaques (12). However, it
is not clear whether A
in the circulation or in the brain is
responsible for increased accumulation of inflammatory cells and
microglial cells in the brain. Involvement of hematopoietic cells in
the formation of microglial cells in the brain is supported by a recent study (4) in which female mice were implanted with donor
bone marrow cells, either male donor cells or cells genetically marked with a retroviral tag. Marrow-derived cells appeared in the brain 3 days after the implant. After a few weeks, the number of marrow-derived cells in the brain increased substantially and showed immunoreactivity toward the antigenic macrophage and glial marker F4/80. These studies
thus indicate that hematopoietic cells can cross the BBB. However,
relatively little is known of the mechanism by which A
could induce
these events. The potential mechanism(s) may involve interaction of
A
with scavenger receptor (RAGE) expressed on the endothelium of the
BBB (14) to initiate cellular signaling to allow monocytes
to transmigrate through cell-cell junctions.
We show here that the interaction of soluble A1-40
with a confluent monolayer of HBMVEC causes increased adherence and
transmigration of monocytic cells (THP-1). Similarly, another monocytic
cell line (HL-60) and peripheral blood monocytes show two- and fourfold
increased transmigration, respectively, in response to
A
1-40. The increase in the migration of monocytes
in response to A
1-40 was dose dependent, showing
optimal migration with soluble A
1-40 at a
concentration of 125 nM. Similar results were obtained with
A
1-42, another amyloidogenic peptide, which is
generated in vivo during the processing of APP. In contrast, amyloid
peptide with a reverse orientation A
40-1 had no
significant effect on the migration of monocytes. The effect of
A
1-40 in augmenting the migration of monocytes was
not due to the presence of endotoxin in the A
1-40
soluble preparation, because the addition of Polymyxin B (5 µg/ml),
an inhibitor of endotoxin, did not reduce the migration of monocytes.
Moreover, the A
1-40-mediated effect on the
transmigration of monocytes was not due to nonspecific disruption of
the barrier properties of HBMVEC monolayer, because the permeability of
the monolayer toward dextran (molecular mass ~70 kDa) and inulin
(molecular mass ~6.1 kDa) remained unaltered in the absence and
presence of A
1-40 (125 nM). Previous studies
(2) have shown that treatment of porcine pulmonary aortic
endothelial cells with micromolar concentration (5-20 µM) of
A
25-35 resulted in apoptosis-induced cell death and
severalfold increase in the permeability of albumin across the EC
monolayer. In the present study, we find that nanomolar concentration
of A
1-40 (125 nM) does not cause nonspecific disruption of the barrier properties of HBMVEC monolayer to allow monocytes to transmigrate. Because A
1-42 had
similar effect as soluble A
1-40 on causing
increased migration of monocytes across brain endothelial cell
monolayer, we suggest that the presence of A
1-42 in
the senile plaques and interstitial fluid may act on the neighboring
cerebrovascular endothelial cells to promote the diapedesis and
accumulation of monocytes in the brain parenchyma, thereby providing a
recruiting stimulus from the endogenous source of amyloid peptide. We
also show that treatment of either monocytes or endothelial cell with
A
promotes migration of monocytes, which leads us to suggest that
such additive actions could occur in an in vivo situation and thus
amplify the transendothelial migration and accumulation of monocytes in vivo.
Recent studies (14, 30) have shown that A binds to an
~50-kDa polypeptide in both endothelial and neuronal cells. This polypeptide is identical to RAGE previously characterized as a cellular
receptor for advanced glycation end products (AGEs). Thus we examined
whether A
binding to RAGE elicited cellular signaling resulting in
the adhesion and migration of monocytes. On the addition of an antibody
to RAGE, there was approximately >60% inhibition in the adhesion and
transmigration of monocytes, indicating that signaling via the
interaction of A
with RAGE (30) mediated the adhesion
and transmigration. We also observed that an antibody to PECAM-1
blocked ~70% and ~85% A
-mediated migration of HL-60 cells and
peripheral blood monocytes, respectively, across the monolayer of
HBMVEC, as has been previously observed for the migration of monocytes
across monolayer of HUVEC in response to AGE and LPS (18,
23). Previous studies (27) have also shown that
PECAM-1 concentrated at endothelial cell intercellular junctions
participates in mediating the transendothelial migration of neutrophils
and monocytes. Because A
-mediated migration of monocyte-like HL-60
cells was not completely abrogated by an antibody to PECAM-1, we
suspect that other adhesion molecules localized at endothelial
cell-to-cell junction (1) may also play a role in the
transendothelial migration of monocytes.
We show that A1-40-mediated transendothelial
migration of monocytes is inhibited by the PKC inhibitor and augmented by protein phosphatase inhibitor Calyculin A, indicating that phosphorylation/dephosphorylation cellular events may play a role in
the flux of monocytes across the human brain endothelial cell monolayer, as has been noted for AGE-induced transendothelial migration
of monocytes (18). The exact sequence of cellular signaling events downstream of A
-RAGE interaction remains to be elucidated.
In conclusion, the results of the present study show that interaction
of A, with its receptor RAGE expressed on human brain endothelial
cells, initiates cellular signaling leading to the transendothelial
migration of monocytes. We further show that PECAM-1 adhesion molecule
concentrated at endothelial cell-cell junctions plays an important role
in regulating the trafficking of monocytes across the monolayer of
human brain endothelial cells. The increased adhesion and diapedesis of
monocytes brought about by the interaction of A
with vascular
endothelium of BBB may contribute to the increased presence of
inflammatory cells (monocytes/macrophages) and activated microglial
cells seen in A
-related vascular disorders, such as CAA, HCHWA-D,
and AD. Further studies are required to delineate this effect in brain
endothelial cells derived from patients with A
-related disorder and
by performing experiments in vivo.
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ACKNOWLEDGEMENTS |
---|
We thank Peter Newman, Blood Research Institute, Milwaukee, WI, for generously providing antibody to human PECAM-1.
![]() |
FOOTNOTES |
---|
This work was supported by National Institutes of Health Grants PO1-AG16233, NS-26310, and HL-61951.
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}hsc.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.
Received 9 February 2000; accepted in final form 25 July 2000.
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