Expression of Membrane-Bound and Soluble Cell Adhesion Molecules by Human Brain Microvessel Endothelial Cells

Microvascular Research 57, 52– 60 (1999) Article ID mvre.1998.2115, available online at http://www.idealibrary.com on

Expression of Membrane-Bound and Soluble Cell Adhesion Molecules by Human Brain Microvessel Endothelial Cells Monika Vastag, Judit Skopa´l, Zolta´n Voko, E´va Csonka, and Zolta´n Nagy National Stroke Center, Huˆvo¨svo¨lgyi u´t 116, Budapest H-1021, Hungary Received March 3, 1998

Expression of membrane-bound (mb) and soluble (s) forms of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) induced by tumor necrosis factor-a (TNF-a) has been measured by enzyme-linked immunosorbent assay in cultured human brain microvessel endothelial cells. Both the mb and the s forms of VCAM-1 and ICAM-1 were upregulated by TNF-a; however, the stimulation of the s forms was delayed in time. When piracetam, a neuroprotective drug, was added to the tissue culture medium simultaneously with TNF-a, the expression of mbVCAM-1 and ICAM-1 was lowered. Differential upregulation of mb and s forms of adhesion molecules and a novel effect of piracetam have been demonstrated in human brain microvessel endothelial cell cultures. © 1999 Academic Press Key Words: human brain microvessel endothelium; adhesion molecules; tumor necrosis factor; piracetam.

INTRODUCTION Ischemic/hypoxic episodes result in a metabolic crisis of brain tissue, which is followed by a second insult in the course of reperfusion. In the postischemic reperfusion, the inflammatory response is localized mostly in the microvasculature (Hallenbeck et al., 1990; del Zoppo et al., 1994). Increased expression of cell adhesion molecules and adherence of leukocytes to the

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microvessel endothelium in the reperfused brain areas have been described (Lefer et al., 1994; Okada et al., 1994; Lindsberg et al., 1996). Leukocytes potentiate reperfusion injury by clogging the microcirculation after endothelial adhesion and by infiltration into the brain parenchyma, where they exacerbate brain damage, releasing lipid-derived mediators (e.g., arachidonic acid, leukotrienes), cytokines, free radicals, and proteases (Pozzilli et al., 1985; Springer, 1995; Feuerstein et al., 1996). The firm adhesion and transmigration of leukocytes are directed by several classes of inducible cell surface glycoproteins (Akopov et al., 1996). These include the members of the immunoglobulin supergene family: vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and the leukocyte integrins (CD11/CD18) (Stad et al., 1994). VCAM-1 and ICAM-1 can be upregulated on endothelial cells by inflammatory cytokines like tumor necrosis factor-a (TNF-a), and interleukin-1 (Wong et al., 1992, 1995; McCarron et al., 1995; Shen et al., 1995). Blocking of leukocyte adhesion to the endothelium using specific antibodies binding to either the CD18 leukocyte adhesion molecule or its endothelial ligand ICAM-1 can protect against leukocyte-mediated reperfusion injury after ischemia in animal models (Clark et al., 1991; Bowes et al., 1993; Chopp et al., 1994; Zhang et al., 1994; Clark et al., 1995a). ICAM-1 and VCAM-1 are transmembrane gly0026-2862/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.

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Adhesion Molecules in Brain Endothelium

coproteins, but their soluble forms have been described in cell supernatants (Leeuwenberg et al., 1992; Pigott et al., 1992; Rieckmann et al., 1995). Circulating/ soluble adhesion molecules are present in normal human sera, and their elevated levels have been demonstrated in different immune and inflammatory disorders (Sheth et al., 1991; Gearing et al., 1992, 1993; Hartung et al., 1995). In stroke patients increased levels of soluble (s) VCAM-1 and sICAM-1 were documented (Fassbender et al., 1995); however, the membrane-bound (mb) adhesion molecules are believed to be involved in the reperfusion injury. Generally, the soluble cell adhesion molecules are thought to be the shedding fragments of the cell surface-bound isoforms. Soluble adhesion molecules may function as competitive inhibitors of the membrane-bound forms and thereby downregulate cell adhesion (Dunlop et al., 1992; Leeuwenberg et al., 1992). On the other hand, soluble endothelial-leukocyte adhesion molecule-1 (sELAM-1) has been reported to promote adhesive activity by stimulation of leukocyte integrins (Lo et al., 1991). The biological/clinical significance of circulating adhesion molecules is still not clear, but their serum levels may be useful markers in disease states. Currently, limited experimental/clinical data are available on the soluble/circulating adhesion molecules, and there is a paucity of information about their relation to the membrane-bound variants. Piracetam (2-oxopyrrolidinacetamide), a low-molecular-weight derivative of g-aminobutyric acid, is widely used in clinical practice as a nootropic agent. The neuroprotective effect of piracetam in the early therapy of acute hemispheric stroke has been reported (De Deyn et al., 1997). A therapeutic window for piracetam therapy appears to be the first few hours following stroke, when the cytokine–adhesine cascade is believed to be stimulated in the reperfused microvessels. The effect of piracetam on enhancement of compromised regional cerebral blood flow has been reported (Platt et al., 1992). Its neuroprotective properties may be mediated through the effects on cell membrane; however, the exact mechanism of action of this compound is not well understood. Cell culture of brain microvessel endothelium is an extensively used method to study endothelial cell activity (Tao-cheng et al., 1987). The endothelial cell cul-

ture prepared from human brain microvessels offers a sensitive model system for the analysis of endothelial adhesine responses (Dorovini-Zis et al., 1991; McCarron et al., 1995). In this study, we characterize the expression of cell surface-bound and soluble forms of ICAM-1 and VCAM-1 under basal conditions and in response to cytokine stimulus in human brain microvessel endothelial cell (HBEC) culture. Furthermore, we test the effect of piracetam on the cytokine-stimulated adhesion expression.

MATERIALS AND METHODS Cell Culture The present method (Vastag and Nagy, 1997) is a modification of the previously described procedure for the isolation of bovine brain capillary endothelium (Bowman et al., 1983). Briefly, capillaries were isolated from the brain of human subjects autopsied because of sudden death due to cardiac arrest. The gray matter was collected and homogenized. The homogenate was purified by centrifugation (10g, 10 min at 20°C) and the pellet was digested with 0.5% collagenase in Dulbecco’s phosphate-buffered saline/Dulbecco’s modified essential medium (DPBS/DMEM), for 30 min at 37°C. Collagenase was removed by repeated centrifugation and the pellet was resuspended in 15% dextran in DPBS and centrifuged (4000g, 30 min at 4°C). The resulting pellet was layered on 35% Percoll in DPBS and centrifuged (20,000g, 30 min at 10°C). Finally, capillaries and single endothelial cells were collected in the culture medium [20% fetal bovine serum (FBS), endothelial mitogen, 5 mg/ml, DMEM/Ham’s F-12 1/1 and penicillin/streptomycin, 100 units/ml and 100 mg/ml, respectively]. This procedure resulted in a highly purified endothelial cell culture. The endothelial cell content was enriched to 95–98% at the beginning of cell proliferation by selection and removal of cell clusters containing atypical cell types. The cells were characterized by the uptake of acetylated low-density lipoprotein (DiI-Ac-LDL) (Voyta et al., 1984) and

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immunoperoxidase staining for the visualization of von Willenbrand factor (Dorovini-Zis et al., 1991). For the experiments the cells were seeded (104 cells/ ml) in 96-well microplates or on glass coverslips and grown till confluence. The cells were used for studies at passages 2 and 3.

Immunocytochemistry Surface expression of ICAM-1 and VCAM-1 was assessed by an immunocytochemical method. HBEC grown on glass coverslips were washed in DPBS and fixed at 4°C with 4% paraformaldehyde in DPBS for 20 min. The nonspecific protein binding sites were blocked by a 30-min incubation at room temperature with 10% FBS in DPBS. This step was followed by the incubation of the cells with antibodies (mouse anti-human ICAM-1 or mouse anti-human VCAM-1) in 10% FBS in DPBS for 60 min at room temperature. The cells were washed three times with DPBS and incubated for 1 h at room temperature with rabbit anti-mouse biotinylated immunoglobulins in 10% FBS in DPBS. The endogenous peroxidase activity was blocked by a 30-min incubation with 0.2% H2O2 in methanol at room temperature. The cells were washed in DPBS and incubated for 1 h at room temperature with avidin/HRP. Finally, the peroxidase reaction was performed for 15–20 min at room temperature with 0.05% DAB (3,39-diaminobenzidine tetrachloride) 0.02% H2O2 in DPBS. Replacing the mixture with water stopped the reaction. Evaluation of immunocytochemical staining was accomplished by microscopic examination of the coverslips.

In Situ Cellular ELISA The in situ cellular ELISA allows a quantitative analysis of reaction products which reflects the amount of membrane-bound adhesion molecules (Tanaka et al., 1990). The cells in 96-well microplates were washed three times with DPBS. After the wash, the cells were fixed at 4°C with 4% paraformaldehyde in DPBS for 20 min. The endogenous peroxidase activity was blocked by a 30-min incubation with 0.2% H2O2 in methanol at room temperature. The nonspecific protein binding

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Vastag et al.

sites were blocked by a 60-min incubation at room temperature with 10% FBS in DPBS. This step was followed by the incubation of the cells with antibodies (mouse anti-human ICAM-1 or mouse anti-human VCAM-1) in 10% FBS in DPBS for 60 min at room temperature. The cells were washed three times with 0.1% Tween in DPBS and incubated overnight at 4°C with peroxidase-labeled rabbit anti-mouse immunoglobulin in 10% FBS in DPBS. Finally, the peroxidase reaction was performed for 20 min at room temperature with 1% 3,39,5,59-tetramethylbenzidine/0.02% H2O2 in 0.1 mol/L citrate buffer, pH 5.0. Adding 6 mol/L H2SO4 stopped the reaction, and the absorbances were read at 450 nm (ref. 620 nm) using an LP 400 Microplate reader (Sanofi Diagnostics Pasteur, France).

Determination of Soluble Cell Adhesion Molecules by Standard ELISA Soluble adhesion molecules, sICAM-1 and sVCAM-1, were quantified from the endothelial cell culture medium using soluble ELISA kits (Bender Medsystems (Austria) and BioSource International Cytoscreen (U.S.A.).

Reagents Fetal bovine serum, DMEM, Ham’s F-12 nutrient mixture, penicillin/streptomycin solution, Fungizone, and collagenase (type I) were purchased from Life Technologies (GIBCO BRL, Austria). Endothelial mitogen (bovine hypothalamus) and DiI-AcLDL were obtained from Biomedical Technologies, Inc. (U.S.A.). Physiological salt solution (Salsol A) and DPBS without Ca21 and Mg21 were prepared at the laboratory of the National Stroke Institute. Monoclonal antibodies against human ICAM-1 and VCAM-1 were purchased from Serotec (United Kingdom) and rabbit anti-mouse immunoglobulin/ biotinylated avidin/HRP and peroxidase-labeled rabbit anti-mouse immunoglobulin from Dako A/S (Germany). Piracetam was obtained from UCB (Belgium); TNF-a, anti-human von Willenbrand factor, and DAB were from Sigma–Aldrich (U.S.A.); and paraformaldehyde was from Merck (Germany). Sol-

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FIG. 1. Immunoperoxidase staining of HBEC for membranous VCAM-1 and mbICAM-1. Slight expression of mbVCAM-1 (A) and mbICAM-1 (B) in nonstimulated cells. Strong upregulation of mbVCAM-1 with intense cytoplasmic staining (C) and a weaker coloration for mbICAM-1 (D) in TNF-a (5 ng/ml for 24 h)-treated cells (3300).

uble ICAM-1 immunoassay kits were purchased from Bender Medsystems (Austria) and sVCAM-1 immunoassay kits were obtained from the BioSource International Cytoscreen (U.S.A.).

Statistics

Stimulation of HBEC

RESULTS

Confluent monolayers of endothelial cells were exposed to TNF-a (5 ng/ml) alone or TNF-a (5 ng/ml) plus piracetam (1 mmol/L–10 mmol/L) in DMEM containing 10% FBS for 4 –24 h at 37°C. Soluble and surface-bound adhesion molecules were determined in parallel from the same wells. Supernatants were collected and kept at 220°C until detection of soluble cell adhesion molecules, while immunoperoxidase staining or in situ cellular ELISA for detection of cell surface bound forms was performed immediately. The absorbances (450 nm) were proportional to the amount of the expressed cell adhesion molecules. Each experiment was repeated three times and run in five parallel wells.

Human brain microvessel endothelial cells express membranous vascular cell adhesion molecule-1 in cultures as determined by both immunohistochemical examination and in situ cellular ELISA. Immunohistochemistry revealed intense cytoplasmic staining of TNF-a (5 ng/ml for 24 h)-treated cells (Fig. 1C). Nontreated cells expressed less mbVCAM (Fig. 1A). ELISA results showed that exposure to TNF-a (5 ng/ml) upregulated significantly the mbVCAM-1 expression as early as 4 h after stimulus, compared to the nonstimulated controls (MWW test P , 0.01; Fig. 2A). The membranous VCAM-1 upregulation preceded any elevation in the level of soluble VCAM-1, which could

Mann–Whitney–Wilcoxon (MWW) and Cuzick (C) tests were used for statistical analysis (Cuzick, 1985).

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Vastag et al.

and TNF-a (5 ng/ml) modulated the expression of membranous VCAM-1, and the expression of the adhesion molecule was less than with TNF-a treatment alone (MWW test P , 0.01; Fig. 2C). Piracetam (1 mmol/L–10 mmol/L) had a dose-related effect and the trend was analyzed by a nonparametric test (C test, P , 0.05; Fig. 4). Cell-surface-bound ICAM-1 expression was upregulated significantly by TNF-a (5 ng/ml) compared to the nonstimulated controls (MWW test, P , 0.01; Fig. 3A). The membranous upregulation was detected as early as 4 h after cytokine treatment and preceded the increase of soluble ICAM-1 (Figs. 3A–3B). A slight but significant upregulation of sICAM was observed only after 24 h of TNF-a exposure (MWW test, P , 0.05; Fig. 3B). Immunohistochemical examination revealed weak staining for mbICAM-1 in nonstimulated cells (Fig. 1B), while intense staining was shown in the stimulated cultures (Fig. 1D). Piracetam cotreatment downregulated the mbICAM-1 expression stimulated by TNF-a (5 ng/ml) (MWW test, P , 0.05; Fig. 3C). Piracetam showed a dose-related effect (C test, P , 0.05; Fig. 4). Piracetam had no influence on the level of soluble adhesion molecules, and the previously stimulated expression of VCAM-1 or ICAM-1 by TNF-a could not be downregulated by the later addition of piracetam (data not shown). FIG. 2. (A and B) Composite bar graphs demonstrate the tumor necrosis factor-a-induced upregulation of membrane-bound vascular cell adhesion molecule-1 and soluble VCAM-1 by human brain microvessel endothelial cells. A and B illustrate the time course of the effect of TNF (5 ng/ml). This shows upregulated level of mbVCAM-1 from 4 h, while sVCAM increased only by 24 h. Values represent means 1 SEM of absorbances. *Values significantly different (P , 0.05) from background absorbances; **values significantly different (P , 0.01) from nonstimulated control. A450 means absorbance values detected at 450 nm. (C) Simultaneous treatment with piracetam (10 mmol/L) and TNF (5 ng/ml) downregulates membrane-bound VCAM-1 expression at 24 h compared to TNF (5 ng/ml) alone (**P , 0.01).

be detected only after 24 h of cytokine treatment in the cell tissue culture medium (MWW test, P , 0.01; Fig. 2B). A continuous basal sVCAM-1 level was detected during the entire observation period. Simultaneous treatment of the cells with piracetam

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DISCUSSION Endothelial cell cultures prepared from capillaries of human brain cortex have been shown to be an appropriate in vitro model for studying basic characteristics of the capillary wall ( Joo, 1992; Nagy et al., 1995). Previously, we described the upregulation of membranous ICAM-1 by thrombin and various cytokines and downregulation by plasmin and miniplasmin in HBEC (Nagy et al., 1996). In this study, we measured the expression of membrane-bound and soluble adhesion molecules VCAM-1 and ICAM-1 in TNF-a-stimulated microvessel endothelium isolated and cultured from human brain, and we described the effect of piracetam on the

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adhesin expression. Although several in vitro studies have shown the expression of membranous VCAM-1 and mbICAM-1 under basal conditions and their increased levels in cytokine-stimulated human microvascular endothelial cell cultures (Shen et al., 1995; Wong et al., 1995; McCarron et al., 1995), the soluble

FIG. 4. Dose-related effect of piracetam on adhesion molecule expression in TNF-a-treated HBEC. Different concentrations of piracetam were added in the presence of TNF-a (5 ng/ml) for 24 h, and membranous VCAM-1 (F) and mbICAM-1 () were determined by in situ cellular ELISA. Absorbance values detected at 450 nm reflect the amount of membrane-bound VCAM-1 or mbICAM-1. Symbols and vertical bars represent means 6 SEM of three experiments (Cuzick test for trend, P , 0.05).

FIG. 3. (A and B) Composite bar graphs demonstrate the tumor necrosis factor-a (5 ng/ml)-induced upregulation of membranebound intercellular cell adhesion molecule-1 and soluble ICAM-1 expression by human brain microvessel endothelial cells. The mbICAM-1 was upregulated at 4 h, whereas increases in the level of soluble ICAM appeared only at 24 h. *Values significantly different (P , 0.05) from background absorbances (A) and from control (B); **values significantly different (P , 0.01) from nonstimulated control. (C) Simultaneous treatment with piracetam (10 mmol/L) and TNF (5 ng/ml) downregulates the membrane-bound ICAM-1. C shows values measured after 24-h treatment compared to TNF (5 ng/ml)-stimulated cells (*P , 0.05).

adhesion molecules were demonstrated independently in different cultures (Leeuwenberg et al., 1992; Pigott et al., 1992). In contrast, soluble VCAM-1 and sICAM-1 were measured in parallel with the surfacebound forms in our experiments. Human brain microvessel endothelial cells exhibited easily detectable expression of mbVCAM-1 and mbICAM-1 in TNF-astimulated cultures, while the levels of sVCAM-1 and sICAM-1 were moderate. Histological studies showed that the expression of the adhesion molecules on endothelial cells is minimally present in normal brain; however, it is strongly exposed in areas of focal brain ischemia early after vessel occlusion (Clark et al., 1995b). Changes in the serum level of circulating selectin- and immunoglobulin-type adhesion molecules in patients with acute ischemic stroke have been documented: sELAM-1 and sVCAM-1 are significantly increased as early as 4 h after ischemic insult (Fassbender et al., 1995). It could be hypothesized that elevated levels of the soluble form of adhesins reflect acute upregulation and shedding of adhesion molecules at sites of the ischemic lesion. In our HBEC model both the membranous and the soluble variants of VCAM-1 and ICAM-1 were upregulated by TNF-a;

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however, the expression of membrane-bound adhesion molecules was high from the beginning of stimulation with TNF-a and preceded any significant elevation in the levels of s forms, which increased only by 24 h. These results demonstrate that the soluble forms do not reflect accurately the upregulated adhesin expression on the cell surface of cultured brain endothelial cells and also may not reflect adhesin expression in vivo under pathological conditions. Shedding of the membranous adhesion molecules from the cell surface could be a mechanism for the appearance of soluble adhesion molecules and a function for downregulating cytokine-induced increases in adhesin exposition. However, the presence of soluble adhesion molecules in HBEC was not related to significant reduction of the expression of the membranous forms. The different timing in the increase of the membrane-bound and soluble adhesion molecules in our model may be explained by independent upregulation via various syntheses and membrane translocation of the spliced two variants. Demonstration of distinct mRNA encoding soluble ICAM-1 in human tissue supports the existence of this mechanism (Wakatsuki et al., 1995). Furthermore, a recent study defined an independent expression of mbICAM-1 on endothelial cells of different vascular beds and of sICAM-1 in mouse plasma (Komatsu et al., 1997). Piracetam significantly downregulated the stimulatory effect of TNF-a on membranous ICAM-1 and mbVCAM-1 during simultaneous treatments in HBEC. It did not affect the basal levels of the s and mb forms or the cytokine-stimulated levels of the s forms. The exact mechanism of piracetam as a neuroprotective drug is under intensive research. Its action on the cell membrane is well documented. Piracetam molecules are inserted into the membrane at the level of polar heads of the membrane phospholipids modifying the cell membrane fluidity (Peuvot et al., 1995; Muller et al., 1997). It has rheological and antiaggregant features (Moriau et al., 1993). Most probably the rheological effects of piracetam are at least partly related to the remodeling of the endothelial cell membrane structure, which could affect the interaction of cytokines and their binding sites. Binding of TNF-a upregulates the expression of adhesion molecules, which involves the activation of NFkB-related tran-

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Vastag et al.

scriptional factors that recognize the kB regulatory sequence found in several genes, including VCAM-1, ICAM-1, and E-selectin (Collins et al., 1995; Weber et al., 1996). The initiation of cytokine-related signal transduction could be affected by the “membrane remodeling effect” of piracetam. In our experiments addition of piracetam after cytokine stimuli did not decrease the adhesin expression. Most probably the signal transduction has already been initiated and the adhesion molecule expression taken place, therefore the expression could not be blocked. The results of this in vitro cell culture study agree with the data of the piracetam acute stroke study (PASS I), namely that piracetam seems to be effective in the early treatment of stroke patients (first 7 h following the ischemic insult), but is not effective later (De Deyn et al., 1997). In conclusion, we demonstrated a differential upregulation of membranous and soluble adhesion molecules ICAM-1 and VCAM-1 in cytokine-stimulated human brain microvessel endothelial cultures and downregulation of the membranous forms in the presence of a neuroprotective agent, piracetam.

ACKNOWLEDGMENTS The excellent technical assistance of Maria Lantos and Erzse´bet Badar is greatly appreciated. This study was supported in part by the Hungarian Research Fund (OTKA T-6026) and the UCB Fund.

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