Effect of endothelial cell polarity on beta -amyloid-induced migration of monocytes across normal and AD endothelium

Ranjit Giri1, Suresh Selvaraj1, Carol A. Miller2, Florence Hofman2, S. D. Yan4, David Stern4, Berislav V. Zlokovic3, and Vijay K. Kalra1

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
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During normal aging and amyloid beta -peptide (Abeta ) disorders such as Alzheimer's disease (AD), one finds increased deposition of Abeta and activated monocytes/microglial cells in the brain. Our previous studies show that Abeta 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 Abeta 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 Abeta 140 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 Abeta was added to the basolateral side of AD compared with normal individual BEC. The Abeta -induced transmigration of monocytes was inhibited in both normal and AD-BEC by antibodies to the putative Abeta 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 Abeta with the basolateral surface of AD-BEC induces cellular signaling, promoting transmigration of monocytes from the apical to basolateral direction. We suggest that Abeta in the AD brain parenchyma or cerebrovasculature initiates cellular signaling that induces PBM to transmigrate across the BBB and accumulate in the brain.

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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta  (Abeta ), consisting of 39-43 amino acid residues, formed by the proteolytic processing of amyloid precursor protein (APP) (23). The accumulation of Abeta is thought to be an early feature of AD (15). The predominant forms of Abeta are the 1-40 and 1-42 fragments. Soluble Abeta 140 is the major form of circulating Abeta , whereas amyloiodogenic Abeta 142, the major constituent of senile plaques, is present in minor amounts in the circulation (2, 13, 20, 22).

In Abeta -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 Abeta 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 Abeta 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 Abeta 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 Abeta 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 Abeta of the basolateral surface of the endothelium in AD, compared with normal. Previous studies show that high-affinity binding sites for soluble (s) Abeta 140 are preferentially localized to the apical surface of N-HBEC, similar to that observed for insulin (12). Thus interaction of Abeta 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 Abeta binding to its putative receptor on the basolateral side.

We report here that interaction of Abeta 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 Abeta 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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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 express gamma -glutamyl transpeptidase indicative of BEC characteristics. Moreover, these HBEC populations contained <1% pericytes or glial cells, as determined by labeling with anti-smooth muscle actin and anti-glial fibrillary acidic protein, respectively (12). No microglial presence was detected as assessed by staining with anti-CD11b. HBEC were used between passages 2 and 5.

Cell 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 × 10-7 M 1alpha -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 Omega  · 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 Abeta 140 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 Abeta 140, 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 Abeta 140 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) beta -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. 1alpha ,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 (Abeta 140) 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 Abeta -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 Abeta -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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Effect of interaction of Abeta 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 Abeta -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 Abeta 140 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 Abeta 140 was 125 nM, which did not affect morphology or cause toxicity to HBEC. As shown in Fig. 1A, addition of Abeta 140 (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 Abeta 140 (125 nM). In AD-HBEC, Abeta 140 (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 Abeta 140 when the same AD patient-derived BEC were used at either passage 4 or 5. These studies demonstrate that Abeta 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, Abeta 140-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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Fig. 1.   Effect of interaction of amyloid beta -peptide (Abeta )140 on the apical and basolateral surface of human brain endothelial cells (HBEC) on the transmigration of THP-1 monocytic cells. HBEC derived from normal (N) and Alzheimer's disease (AD) subjects were grown to confluence on a fibronectin-coated porous membrane of Transwell inserts. To the top compartment of the Transwell chamber, containing monolayer of either N-HBEC or AD-HBEC, was added THP-1 (0.5 × 105 cells/well) followed by the addition of Abeta (125 nM) to either the top compartment (A) or the bottom compartment (B). At the indicated time point, aliquots (50 µl) from the bottom compartment of the Transwell chamber were removed, and transmigrated THP-1 cells were counted in a hemocytometer. Data are means ± SD of individual experiments run in triplicate. Abeta 140-treated AD-HBEC vs. Abeta 140-treated N-HBEC: *** P < 0.001, ** P < 0.01 (n = 3).

Effect of interaction of Abeta with basolateral side of N- and AD-BEC monolayer on the transmigration of monocytic cells. With Abeta peptide in the AD brain increased toward the basolateral side of the cerebrovascular endothelium, we hypothesized that interaction of Abeta with the basolateral surface of the brain vascular endothelial cell monolayer would augment migration of monocytes. As shown in Fig. 1B, addition of Abeta 140 (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 Abeta 140 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 Abeta 140 (125 nM) at 2 h (Abeta 140-treated AD-HBEC vs. Abeta -treated N-HBEC; P < 0.01). Similar results were observed at time points of 3 and 4 h.

To determine whether this effect was unique to BEC derived from the two AD patients or was common to other AD patients or specific to THP-1 monocytic cells, we studied the transmigration of vitamin D3-differentiated HL-60 monocytic cells in six AD-HBEC and five N-HBEC cultures. As shown in Fig. 2, there was an approximately threefold increase in transmigration of HL-60 monocytic cells in response to interaction of Abeta 140 with N-HBEC (372 ± 139%; n = 11 for HBEC derived from 5 normal individuals) and AD-HBEC (367 ± 142%; n = 10 for HBEC derived from 6 AD individuals) at 2 h. These results indicate that no significant differences in the migration of HL-60 cells between N- and AD-derived HBEC monolayers occur when Abeta 140 is added to the top or apical side of the endothelial cell monolayer (P > 0.05). However, there was a substantial increase in the migration of monocytes across AD-HBEC (458 ± 152%; n = 10 derived from 6 AD individuals) compared with the N-HBEC (220 ± 76, n = 6 for HBEC derived from 5 normal individuals) monolayer in response to the interaction of Abeta with the basolateral surface. The data show a significant difference (<0.01) in the migration of HL-60 cells when Abeta 140 is added to the basolateral side (bottom) between the AD- and N-HBEC monolayers (Fig. 2). These data indicate that Abeta induces a differential response in monocyte migration from the apical to basolateral direction, when Abeta interacts with the basolateral surface of endothelial cell monolayer derived from normal and AD individuals.


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Fig. 2.   Effect of interaction of Abeta 140 with apical (top; T) and basolateral (bottom; B) surface of N-HBEC and AD-HBEC monolayer on the transmigration of HL-60 monocytic cells. The experimental protocol was similar to that in Fig. 1, except that HL-60 cells were used. The data for N-HBEC are for n = 11, which were obtained from the brain endothelial cells (BEC) of 5 different non-AD (N) subjects. The data for AD-HBEC are for n = 10, which were obtained from the endothelial cells of 6 different AD brains. All of these studies were done on BEC cultivated at passages 3-5. Data are expressed as means ± SD.

Involvement of PECAM-1 and RAGE in the transendothelial migration of HL-60 cells in response to interaction of Abeta 140 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 Abeta 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 Abeta 140 (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 Abeta , inhibited Abeta -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 Abeta 140 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 Abeta with the apical side involves both RAGE and PECAM-1.


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Fig. 3.   Effect of antibodies to platelet-endothelial cell adhesion molecule-1 (PECAM-1) and receptor for advanced glycation end products (RAGE) on transendothelial migration of HL-60 human monocytic cells across AD-HBEC monolayer. AD-HBEC grown to confluence on Transwell membranes were preincubated with monoclonal antibody (Ab) to PECAM-1 (Ab-PECAM-1; 5 µg/ml), monoclonal Ab-RAGE (5-10 µg/ml), monoclonal Ab-HLA-ABC (10 µg/ml), and monoclonal Ab to intracellular adhesion molecule-1 (Ab-ICAM-1; 5 µg/ml) for 30 min before the addition of HL-60 monocytic cells (0.5 × 105 cells/well). Abeta 140 (125 nM) was added to either top (A) or bottom compartment (B) of the Transwell chamber. Transmigrated monocytes were counted at 2 h. Data are means ± SD of 3 independent experiments. Abeta 140 vs. in the presence of Ab: P < 0.001.

We further examined whether PECAM-1 and RAGE were involved in the transmigration of HL-60 cells in response to interaction of Abeta with the basolateral side of AD-HBEC. As shown in Fig. 3B, Ab-RAGE and Ab-PECAM-1 inhibited ~80 and 85%, respectively, the transmigration of HL-60 monocytes. Addition of both Ab-RAGE and Ab-PECAM-1 did not augment further inhibition, indicating that the effects of these antibodies were not additive (Fig. 3B). Studies (30) have demonstrated that migration of T cells across activated HBEC is blocked by antibody to intracellular adhesion molecule-1 (Ab-ICAM-1); thus we examined the effect of monoclonal Ab-ICAM-1 on transmigration of HL-60 cells. As shown in Fig. 3B, there was no significant effect by Ab-ICAM-1. Similar findings were obtained using an irrelevant Ab-HLA-ABC. Our results indicate that Abeta -mediated migration of monocytes across a HBEC monolayer from the apical to basolateral direction can occur as a result of the interaction of Abeta with RAGE, which is expressed on both the apical and the basolateral surfaces of AD-HBEC.

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 Abeta -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 Abeta 140 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 Abeta -mediated migration of monocytes. Because Abeta -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)-kappa B, we determined whether pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-kappa 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 Abeta 140 with the basolateral side of an AD-HBEC monolayer. These results indicate that cellular signaling emanating from the interaction of Abeta 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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Fig. 4.   Effect of pharmacological inhibitors of signaling on Abeta -induced transmigration of HL-60 cells in AD-HBEC. AD-HBEC grown to confluence on Transwell membranes were preincubated with inhibitors (25 µg/ml Genistein, 20 nM GF-109203X, and 2 nM calyculin A) and pyrrolidine dithiocarbamate (PDTC; 5 µM) for 30 min before the addition of Abeta 140 (125 nM) to the bottom compartment of the Transwell chamber. This was followed by the addition of HL-60 cells to the top compartment. Transmigrated monocyte cells were counted at 2 h. Data are means ± SD of 3 independent experiments. Abeta 140 vs. in the presence of inhibitors: P < 0.001.

Abeta 140-induced phosphorylation of PECAM-1 in HBEC. Treatment of 32P-labeled HBMVEC with Abeta 140 (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 Abeta 140 (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 Abeta 140-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 Abeta 140 resulted in an approximately twofold increase in the incorporation of 32P into PECAM-1 relative to Abeta 140 alone-treated HBMVEC (Fig. 5). Moreover, we observed that Abeta 140-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 Abeta 140 with receptor RAGE expressed on HBMVEC (12). The effect is specific for Abeta 140, as amyloid peptide with reverse orientation Abeta 401 had a minimal effect on the phosphorylation.


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Fig. 5.   Effect of inhibitors and antibodies on the 32P incorporation in PECAM-1 in HBEC on incubation with Abeta . 32P-labeled HBEC were incubated with 125 nM of Abeta 140 and either inhibitors (20 nM GF-109203X, 2 nM calyculin A) or antibody (10 µg/ml monoclonal Ab-RAGE, 10 µg/ml Ab-HLA-antigen) for 2 h. HBEC were incubated with 125 nM of Abeta 140 peptide with reverse orientation. Incorporation of 32P in PECAM-1 was determined as shown in Fig. 4. Data are expressed as means ± SD; n = 3. Abeta 140 vs. in the presence of either antibody or inhibitors.

Effect of interaction of Abeta 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 Abeta . As shown in Fig. 6A, addition of Abeta to the apical side of N-HBEC resulted in an approximately threefold increase in the transmigration of PBM at 2 h. However, addition of Abeta 140 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 Abeta 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 Abeta 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, Abeta added from either the apical or the basolateral side did not significantly increase the migration of resting T cells across the AD-HBEC monolayer.


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Fig. 6.   Effect of Abeta interaction with the apical and basolateral surface of the monolayer of N-HBEC and AD-HBEC on the transmigration of peripheral blood monocytes (PBM). HBEC derived from N and AD subjects were grown to confluence on a fibronectin-coated porous membrane of Transwell inserts. To the top compartment of the Transwell chamber, containing monolayer of either N-HBEC or AD-HBEC, was added human PBM (0.5 × 105 cells/well) followed by the addition of Abeta (125 nM). A: effect of addition of Abeta (125 nM) to top compartment (apical surface). Data are means ± SD of 3 individual experiments. B: effect of addition of Abeta (125 nM) to bottom compartment (basolateral surface). Data are means ± SD of 3 individual experiments. C: effect of Abeta (125 nM) on transmigration of resting T cells across AD-HBEC monolayer. To the AD-HBEC monolayer cultivated in inserts on the Transwell chamber were added T cells (0.5 × 105 cells) to the top compartment followed by the addition of Abeta (125 nM) to either the bottom or the top compartment. D: effect of Ab-RAGE and Ab-PECAM-1 on transmigration of PBM in response to interaction of Abeta with basolateral surface of AD-HBEC monolayer. AD-HBEC grown to confluence on Transwell membranes were preincubated with monoclonal Ab-PECAM-1 (10 µg/ml), monoclonal Ab-RAGE (10 µg/ml), and monoclonal Ab-HLA-ABC (10 µg/ml) for 30 min before the addition of PBM (0.5 × 105 cells/well). Abeta 140 (125 nM) was added to bottom compartment of the Transwell chamber. At the indicated time point, aliquots (50 µl) from the bottom compartment of the Transwell chamber were removed, and transmigrated cells were counted in a hemocytometer. Data are means ± SD of 3 individual experiments.

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 Abeta 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 Abeta 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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In AD and Abeta -related cerebral vascular disorders (CAA and HCHWA-D), increased deposition of amyloid peptide (Abeta ) 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 Abeta 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 Abeta 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 Abeta 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 Abeta 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 Abeta is blocked by Ab-RAGE (5). These results suggest that interaction of Abeta 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 Abeta 140 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-Abeta 140 occurred on the apical side of the monolayer of HBEC. These observations imply that interaction of circulating sAbeta with the vessel wall could induce signaling and allow PBM to adhere to the cerebrovasculature and subsequently induce their diapedesis into the brain.

The Abeta -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 Abeta 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 Abeta 140 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 Abeta 140 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 Abeta 140 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 Abeta 140 with the apical surface of N-HBEC. Moreover, transmigration of monocytes across the AD-HBEC monolayer, in response to the interaction of Abeta 140 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 sAbeta 140 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 Abeta 140-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 Abeta -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 Abeta -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 Abeta -related disorders (CAA and HCHWA-D) one finds (11) increased amounts of Abeta 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 Abeta 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 Abeta 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 Abeta 140 and expression of Abeta 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 Abeta 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 Abeta 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 Abeta 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, Abeta -mediated transmigration of monocytes was inhibited by PDTC, an inhibitor of NF-kappa B activation. These studies indicate that Abeta -mediated activation of PKC in BEC may lead to activation of NF-kappa B, as has been shown for tumor necrosis factor-alpha (TNF-alpha )-mediated activation of PKC in Jurkat cells, leading to activation of NF-kappa 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 Abeta 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 Abeta 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 Abeta 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 Abeta 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 Abeta 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 Abeta 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 Abeta -related vascular disorders such as AD.


    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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