beta -Amyloid-induced migration of monocytes across human brain endothelial cells involves RAGE and PECAM-1

Ranjit Giri1, Yamin Shen1, Monique Stins2, Shi Du Yan3, Ann Marie Schmidt3, David Stern3, Kwang-Sik Kim2, Berislav Zlokovic4, and Vijay K. Kalra1

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
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In patients with amyloid beta -related cerebrovascular disorders, e.g., Alzheimer's disease, one finds increased deposition of amyloid peptide (Abeta ) and increased presence of monocyte/microglia cells in the brain. However, relatively little is known of the role of Abeta in the trafficking of monocytes across the blood-brain barrier (BBB). Our studies show that interaction of Abeta 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 Abeta -mediated migration of monocytes was inhibited by antibody to Abeta receptor (RAGE) and platelet endothelial cell adhesion molecule (PECAM-1). Additionally, Abeta -induced transendothelial migration of monocytes were inhibited by protein kinase C inhibitor and augmented by phosphatase inhibitor. We conclude that interaction of Abeta 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 Abeta present either in the peripheral circulation or in the brain parenchyma may play a role in the pathophysiology of Abeta -related vascular disorder.

amyloid beta -peptide; brain endothelial cells; platelet endothelial cell adhesion molecule; receptor for advanced glycation end product


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

DURING NORMAL AGING, deposition of amyloid beta -peptide (Abeta ) 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 Abeta , 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 Abeta are the 1-40 and 1-42 fragments (Abeta 1-40 and Abeta 1-42) formed by the normal proteolytic processing of amyloid precursor protein (APP) (19). The Abeta 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 Abeta 1-40 is the major form of circulating beta -amyloid and cerebral vascular amyloid, whereas the more amyloidogenic Abeta 1-42, which is present in only minor amounts in the circulation, constitutes a major component of senile plaques (3, 15, 19, 21).

In Abeta -related cerebral vascular disorders (CAA and HCHWA-D) and AD, one finds not only the increased deposition of Abeta 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 Abeta '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 Abeta 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 Abeta 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-kappa B (30). Other studies showed that mononuclear phagocytes, including microglia, which express class A scavenger receptor, interact with Abeta and generate reactive oxygen species (5). However, relatively little is understood about how the accumulation of Abeta 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 Abeta 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 Abeta 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 Abeta 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 Abeta 1-40 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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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 × 10-7 M 1alpha ,25-dihydroxyvitamin D3 (Biomol Research Laboratories, Plymouth Meeting, PA) for 3-4 days under culture conditions as described previously (10). We used these cells in the differentiated state for transmigration studies. Peripheral blood human monocytes were isolated from blood collected in EDTA as the anticoagulant. Briefly, 10 volumes of blood sample (30 ml) were mixed with 1 volume (3 ml) of a solution composed of 6% dextran 500 in 0.9% NaCl. 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 manufacturer's instructions. Monocytes isolated by this procedure had purity in the range of 75-95% as assessed by labeling with CD11b-FITC antibody (Coulter Diagnostics, Hialeah, FL) and yield of 55-75%. T cells were isolated from leukocyte-rich plasma by passing through a sterilized column of nylon.

Cell 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 Abeta 1-40 (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 Abeta 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 Abeta 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 Abeta 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 Abeta 1-40 (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 Omega /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. Abeta 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 Abeta 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 Abeta 1-40 (125 nM) at 37°C. This concentration of Abeta 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 Omega /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 Abeta 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 Abeta 1-40, antibody to RAGE, antibody to PECAM-1, and peripheral blood monocytes (PBM) to the upper compartment of the Transwell chamber.

Materials. 1alpha ,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 Abeta -treated endothelial cells was carried out by one-way ANOVA using Instat 2 (Graphpad, San Diego, CA) software program. The effects of inhibitors on Abeta -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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REFERENCES

Effect of Abeta on the viability and injury to cultured human brain endothelial cells. Because Abeta 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 Abeta i.e., Abeta 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 Abeta 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 (Abeta 1-40 treated), there was no discernible change in the morphology of HBMVEC monolayer on treatment with 125 nM Abeta 1-40 at 4 h. However, a higher concentration (250 nM) of Abeta 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 Abeta for 8 h did not affect either cell viability or cause injury, we used this concentration (125 nM) of Abeta 1-40 and time period (4-8 h) for most of the experiments described here.


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Fig. 1.   Morphology of human brain microvascular endothelial cells (HBMVEC) and HL-60 monocyte-like cells. Phase contrast micrograph of HBMVEC cultured for 3 days (×100) (A), HBMVEC treated with amyloid beta  peptide (Abeta ) for 4 h (×100) (B), monocyte-like HL-60 cells (×400) (C), monocyte-like HL-60 cells transmigrated at 2 h in response to Abeta (×400) (D).

Abeta 1-40-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 Abeta 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 Abeta (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 Abeta 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 Abeta 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 Abeta 1-40-induced expression of VCAM-1.


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Fig. 2.   Effect of Abeta on the adherence of THP-1 monocytic cells to HBMVEC. HBMVEC grown to confluence in 24-well dish were preincubated with monoclonal antibody to RAGE (receptor for advanced glycation end products; 10 µg/ml), VCAM-1 (5 µg/ml), VLA-4 (5 µg/ml), and HLA-antigen (5 µg/ml) for 30 min before the addition of Abeta . The data shown are relative adherence of monocytes to endothelial cells using 100% adherence of monocytes to HBMVEC in the absence of Abeta . Data are expressed as means ± SD for n = 3. Abeta 1-40 vs. in the presence of antibody: ***P < 0.001; ns, not significant.



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Fig. 3.   Time course of cell adhesion molecule (CAMs) expression in response to incubation with Abeta in HBMVEC. HBMVEC grown to confluence in 24-well plates were incubated with Abeta 1-40 (125 nM) for the indicated time periods (2, 4, 8, 12, and 24 h). ICAM-1 and VCAM-1 expression was determined by ELISA. Results are expressed as means ± SD of optical density (OD) at 405 nm of triplicate determinations (n = 3).



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Fig. 4.   Effect of inhibitors on VCAM-1 expression induced by Abeta in HBMVEC. HBMVEC grown to confluence in 24-well plates were incubated with Abeta (125 nM) in the presence and absence of inhibitors (PD-98059, 10 µM; vitamin E, 25 µM; MAb-RAGE, 5 µg/ml; Ab-HLA-antigen, 10 µg/ml) for 4 h. VCAM-1 expression was determined by ELISA. Results are expressed as means ± SD of optical density at 405 nm of triplicate determinations for n = 3. Abeta 1-40 vs. in the presence of either inhibitor or antibody: ***P < 0.001.

Effect of beta -amyloid on migration of monocytes across human brain endothelial cell monolayer. We examined whether incubation of HBMVEC with Abeta 1-40 would result in an increased migration of monocytes across the monolayer. As shown in Fig. 5A, addition of Abeta 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, Abeta 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 Abeta 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 Abeta 1-40 (see Fig. 8A). In previous studies (2) it was observed that treatment of porcine pulmonary aortic endothelial cells with Abeta 25-35 resulted in an increased permeability to albumin. However, such effect was observed only with micromolar concentration of Abeta . Additionally, we observed that Abeta 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 Abeta 1-40 (125 nM) (data not shown).


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Fig. 5.   Effect of amyloid synthetic peptide on the transmigration of monocyte-like HL-60 cells across HBMVEC monolayer. HBMVEC were grown to confluence on fibronectin-coated porous membrane (3.0-µm pore size, Transwell, catalog 40492, Biocoat cell culture insert, Collaborative Biomedical Products, Bedford, MA). To the upper chamber of the Transwell chamber containing monolayer of HBMVEC was added vitamin D3 differentiated monocyte-like HL-60 cells (0.1 × 106 cells/well) and Abeta at indicated concentration. At the indicated time point, aliquots (50 µl) from the lower compartment of the Transwell chamber were removed, and transmigrated HL-60 cells were counted in a hemocytometer. A: time course of migration of HL-60 monocytes. Data are means ± SD of 3 individual experiments. None vs. Abeta 1-40 (125 nM) at same time point; ***P < 0.001; *P < 0.05. B: dose response effect of Abeta 1-40 on the transmigration of monocyte-like HL-60 cells across HBMVEC monolayer. Data are means ± SD of 3 individual experiments. None vs. Abeta 1-40 treated; ***P < 0.001; **P < 0.05. C: effect of various Abeta and lipopolysaccharide (LPS) on the transendothelial migration of monocyte-like HL-60 cells. HBMVEC monolayers in Transwell chambers were incubated with Abeta (Abeta 1-40, Abeta 1-42, or Abeta 40-1) at a concentration of 125 nM or LPS (100 ng/ml) plus 5% human serum in the absence or presence of Polymyxin B (5 µg/ml). Data are means ± SD of 3 independent experiments. ***P < 0.001, none vs. Abeta treated. **P < 0.05 between indicated samples. D: effect of antibody to PECAM-1 and RAGE on transendothelial migration of monocyte-like HL-60 cells. HBMVEC grown to confluence on Transwell membranes were preincubated with monoclonal antibody to PECAM-1 (XVD2; Ref. 9; 10 µg/ml), monoclonal antibody to ICAM-1 (10 µg/ml), monoclonal antibody to HLA-ABC (10 µg/ml), and monoclonal antibody to RAGE (5 or10 µg/ml) for 30 min before the addition of HL-60 cells and 125 nM Abeta 1-40. Abeta 1-40 treated vs. in the presence of antibody. ***P < 0.001; **P < 0.05 (n = 3).

Because the concentration of Abeta 1-40 used may not be optimal, we examined the dose-response (10-250 nM) effect of Abeta 1-40 on the transendothelial migration of monocyte-like HL-60 cells. As shown in Fig. 5B, Abeta 1-40 at a concentration of 62.5 nM increased by 55% the transendothelial migration of monocyte-like HL-60 cells above the basal level. Lower concentration of Abeta 1-40 (25 nM) was not effective in augmenting the monocytes transendothelial migration. However, Abeta 1-40 (125 nM) exhibited twofold increase in inducing the migration of monocyte-like HL-60 cells above the basal level of untreated HBMVEC (P < 0.001), whereas a higher concentration of Abeta 1-40, i.e., 250 nM, was not significantly different than Abeta 1-40 at 125 nM. Because Abeta 1-40 at 125 nM was most effective in mediating the transmigration of monocytes across the endothelial cell monolayer without causing injury to endothelial cells, we used this concentration of Abeta 1-40 in the studies described here. As shown in Fig. 5C, the isoform of Abeta , i.e., Abeta 1-42, at a concentration of 125 nM was equally as effective as Abeta 1-40 in mediating the transendothelial migration of monocytes. However Abeta peptide with reverse orientation, i.e., Abeta 40-1, did not significantly affect the migration of monocyte-like HL-60 cells across the HBMVEC monolayer (P > 0.05, not significant). The increase in transendothelial migration of monocyte-like HL-60 cells was not due to contamination of synthetic Abeta 1-40 preparation with endotoxin, because Polymyxin B (5 µg/ml), an inhibitor of endotoxin, did not significantly inhibit the Abeta -induced transendothelial migration of monocyte-like cells (Fig. 5C). Similarly, Polymyxin B did not affect Abeta -induced transendothelial migration of peripheral blood monocytes (Fig. 7B). As a positive control, Polymyxin B (5 µg/ml) completely abrogated LPS (100 ng/ml)-induced increase in the transendothelial migration of monocyte-like HL-60 cells (Fig. 5C).

Involvement of PECAM-1 and RAGE in Abeta 1-40-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 Abeta 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 Abeta 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).

Recent studies (30) have shown that Abeta binds to receptor RAGE expressed on neuronal cells, smooth muscle cells, and endothelial cells (30, 31); thus we examined the role of RAGE in Abeta 1-40-induced transendothelial migration of monocytes. As shown in Fig. 5D, antibody to human RAGE (5 µg/ml) reduced by ~60% the transendothelial migration of monocytes induced by Abeta 1-40. A higher amount of RAGE antibody (10 µg/ml) did not cause extra inhibition. The effect of antibody to RAGE was specific for Abeta 1-40-induced transmigration of monocytes, as this antibody had minimal effect on LPS-induced transmigration of monocytes (data not shown).

Additionally, we wanted to determine whether activation of either monocytes or endothelial cells in response to Abeta 1-40 was sufficient to induce transendothelial migration of monocytes. For this purpose, monocytes were pretreated with Abeta 1-40 (125 nM) for 2 h followed by a wash with PBS. Then Abeta -treated monocytes were added to the untreated HBMVEC monolayer. We observed ~50% increase in the transmigration of monocytes at the 2-h time period, above the basal level (Fig. 6). However, when HBMVEC were treated with Abeta for 1 or 2 h followed by a wash with PBS and addition of monocytes to the upper chamber of Transwell chamber, there was an approximately twofold increase above the untreated control (Fig. 6). The morphology of transmigrated monocytes in response to Abeta (Fig. 1D) did not differ much from that of untreated control monocytes (Fig. 1C). Our results indicate that both cell types are involved in mediating the transmigration of monocytes, although activation of brain endothelial cells with Abeta 1-40 is sufficient to induce the transmigration of monocytes. It is pertinent to mention that, in vivo, both cell types are probably bathed in milieu of amyloid peptide and both express RAGE, implying that costimulation may occur to mediate transmigration across cerebrovascular endothelium.


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Fig. 6.   Effect of pretreatment of HBMVEC and monocytes with Abeta 1-40 on the transmigration. HBMVEC cultivated in Transwell chamber were pretreated with Abeta (125 nM) for either 1 or 2 h, followed by wash with buffer and then incubation with HL-60 cells for transmigration studies. Alternatively, monocytes were pretreated with Abeta for 2 h, washed with buffer, and then added to HBMVEC monolayer. Data are expressed as percent change (means ± SD of n = 3) relative to migration of monocytes in untreated HBMVEC.

Abeta 1-40-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 Abeta 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, Abeta 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 Abeta 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 Abeta 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 Abeta 1-40. As shown in Fig. 7B, Abeta 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 Abeta (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 Abeta -treated HBMVEC reduces by ~80% the migration of monocytes above the basal level. It is pertinent to mention that addition of either Abeta 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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Fig. 7.   Effect of antibody to PECAM-1 and RAGE on transendothelial migration of THP-1 human monocytic cells and peripheral blood monocytes. HBMVEC grown to confluence on Transwell membranes were preincubated with monoclonal antibody to PECAM-1 (XVD2; Ref. 9; 10 µg/ml), monoclonal antibody to HLA-ABC (10 µg/ml), monoclonal antibody to RAGE (10 µg/ml), and calyculin A (2 nM) for 30 min before the addition of either THP-1 monocytic cells (0.1 × 106 cells/well) (A) or peripheral blood monocytes (0.5 X 105 cells/well) and 125 nM Abeta 1-40 (B). Transmigrated monocytes were counted at 2 h. Data are expressed as means ± SD of 3 independent experiments. Abeta 1-40 vs. in the presence of antibody. ***P < 0.001.



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Fig. 8.   Effect of Abeta on the permeability to dextran and inulin and transendothelial electrical resistance (TEER) in confluent HBMVEC monolayer. A: transport of [14C]inulin and [3H]dextran across HMMVEC monolayer in the absence and presence of Abeta (125 nM) at 60 min. Data are for 2 different monolayers in triplicate. B: TEER of HBMVEC monolayer in the absence and presence of Abeta (125 nM). Where indicated, peripheral blood monocytes (PBM) (0.5 × 105 cells), anti-RAGE (5 µg/ml), and anti-PECAM-1 (5 µg/ml) were added to the upper compartment and TEER measured after 60 min. Data are representative of 2 different monolayers in triplicate.

Effect of inhibitors on Abeta 1-40-induced transendothelial migration of monocytes. We examined whether Abeta 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 Abeta 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 Abeta 1-40 alone (Fig. 9). Because interaction of Abeta 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 Abeta 1-40. We observed that the antioxidants probucol (50 µM) and vitamin E (25 µM) were effective in blocking Abeta 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 Abeta 1-40-induced migration of monocyte-like HL-60 cells (Fig. 9).


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Fig. 9.   Effect of inhibitors, antioxidants, and modulators of glutathione redox cycle on Abeta -induced transmigration of monocyte-like HL-60 cells. HBMVEC grown to confluence on Transwell membranes were preincubated with inhibitors (GF-109203X, 20 nM and Calyculin A, 2 nM), antioxidants (probucol, 50 µM and vitamin E, 25 µM), and GSH modulators (GSH-EE, 0.5 mM) for 30 min before the addition of Abeta 1-40 (125 nM). This was followed by the addition of monocyte-like HL-60 cells. Transmigrated monocyte cells were counted at 2 h. Data are expressed as means ± SD of 3 independent experiments. Abeta 1-40 vs. in the presence of inhibitors: ***P < 0.001.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Pathological examination of brains from patients with AD and cerebral vascular disorders (CAA and HCHWA-D) has shown increased presence/deposition of Abeta 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 Abeta 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 Abeta could induce these events. The potential mechanism(s) may involve interaction of Abeta 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 Abeta 1-40 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 Abeta 1-40. The increase in the migration of monocytes in response to Abeta 1-40 was dose dependent, showing optimal migration with soluble Abeta 1-40 at a concentration of 125 nM. Similar results were obtained with Abeta 1-42, another amyloidogenic peptide, which is generated in vivo during the processing of APP. In contrast, amyloid peptide with a reverse orientation Abeta 40-1 had no significant effect on the migration of monocytes. The effect of Abeta 1-40 in augmenting the migration of monocytes was not due to the presence of endotoxin in the Abeta 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 Abeta 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 Abeta 1-40 (125 nM). Previous studies (2) have shown that treatment of porcine pulmonary aortic endothelial cells with micromolar concentration (5-20 µM) of Abeta 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 Abeta 1-40 (125 nM) does not cause nonspecific disruption of the barrier properties of HBMVEC monolayer to allow monocytes to transmigrate. Because Abeta 1-42 had similar effect as soluble Abeta 1-40 on causing increased migration of monocytes across brain endothelial cell monolayer, we suggest that the presence of Abeta 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 Abeta 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 Abeta 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 Abeta 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 Abeta with RAGE (30) mediated the adhesion and transmigration. We also observed that an antibody to PECAM-1 blocked ~70% and ~85% Abeta -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 Abeta -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 Abeta 1-40-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 Abeta -RAGE interaction remains to be elucidated.

In conclusion, the results of the present study show that interaction of Abeta , 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 Abeta with vascular endothelium of BBB may contribute to the increased presence of inflammatory cells (monocytes/macrophages) and activated microglial cells seen in Abeta -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 Abeta -related disorder and by performing experiments in vivo.


    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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