Differential monocyte adhesion and adhesion molecule expression in venous and arterial endothelial cells

Theodore J. Kalogeris1, Christopher G. Kevil2, F. Stephen Laroux2, Laura L. Coe2, Travis J. Phifer1, and J. Steven Alexander2

Departments of 1 Surgery and 2 Molecular and Cellular Physiology, Louisiana State University Medical Center, Shreveport, Louisiana 71130

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

We compared U-937 cell adhesion and adhesion molecule expression in human umbilical venous (HUVECs) and arterial (HUAECs) endothelial cells exposed to tumor necrosis factor (TNF), interleukin-1, and lipopolysaccharide (LPS). TNF and LPS stimulated vascular cell adhesion molecule (VCAM)-1 surface expression and adhesion of U-937 monocyte-like cells to HUVECs but not to HUAECs. Antibody studies demonstrated that in HUVECs at least 75% of the adhesion response is VCAM-1 mediated. Interleukin-1 stimulated U-937 cell adhesion to and VCAM-1 surface expression in both HUVECs and HUAECs. Pyrrolidinedithiocarbamate and the proteasome inhibitor MG-132 blocked TNF- and LPS-stimulated U-937 cell adhesion to HUVECs. These agents also significantly decreased TNF- and LPS-stimulated increases in HUVEC surface VCAM-1. TNF increased VCAM-1 protein and mRNA in HUVECs that was blocked by pyrrolidinedithiocarbamate. However, neither TNF or LPS stimulated VCAM-1 expression in HUAECs. TNF stimulated expression of both intercellular adhesion molecule-1 and E-selectin in HUVECs, but in HUAECs, only intercellular adhesion molecule-1 was increased. Electrophoretic mobility shift assays demonstrated no difference in the pattern of TNF-stimulated nuclear factor-kappa B activation between HUVECs and HUAECs. These studies demonstrate a novel and striking insensitivity of arterial endothelium to the effects of TNF and LPS and indicate a dissociation between the ability of HUAECs to upregulate nuclear factor-kappa B and VCAM-1.

inflammatory mediators; cell culture; leukocyte-endothelial interactions; E-selectin; vascular cell adhesion molecule-1; intercellular adhesion molecule-1; nuclear factor-kappa B

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

LEUKOCYTE-ENDOTHELIAL ADHESION is an early step in many inflammatory disorders including sepsis (38), acute respiratory distress syndrome (8), allograft rejection (24), ischemia-reperfusion injury (14), and atherosclerosis (34). The mechanisms regulating neutrophil adhesion in several of these disorders have been intensively studied, in part because neutrophil-endothelial adhesion is the first step in the acute phase of inflammation. However, with the exception of atherosclerosis (where monocyte recruitment and adhesion is a very early event in lesion development) (31), the role of monocyte adhesion in most other inflammatory pathologies is not as well understood.

Similar to neutrophils, adhesion of monocytes to endothelial cells is mediated by endothelial cell adhesion molecules (ECAMs) such as intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin. The transcription and cell surface mobilization of ECAMs are induced by proinflammatory stimuli including cytokines, endotoxin, oxidized lipoproteins, and reactive oxygen metabolites (9, 19, 23, 29, 30, 37). The ECAMs are among numerous genes induced through the action of the transcription factor nuclear factor-kappa B (NF-kappa B) (19, 23, 29, 37). It is assumed that the adhesion of monocytes also depends on activation of NF-kappa B. However, a test of this hypothesis has not yet been reported.

Most evidence indicates that monocyte adhesion to endothelial cells is mediated by VCAM-1. First, VCAM-1 binds to the beta 1-integrin very late activating antigen-4 (VLA-4) expressed on monocytes but not on neutrophils (20). Second, expression of VCAM-1 in atherosclerotic lesions precedes monocyte adhesion (27). Third, VCAM-1 is expressed under control by oxidant signals, with parallel activation of NF-kappa B (19, 37). Finally, the p65 subunit of NF-kappa B is a potent activator of the VCAM-1 promoter (1). These findings implicate NF-kappa B-mediated upregulation of VCAM-1 as a critical determinant for monocyte adhesion to endothelium. However, to prove this hypothesis, it must be shown that activation of endothelial NF-kappa B is required for monocyte adhesion. This has not been reported but has only been inferred from experiments showing that MG-132 (a peptide aldehyde proteasome inhibitor that blocks NF-kappa B activation) abolishes both tumor necrosis factor (TNF)-stimulated neutrophil and lymphocyte adhesion to endothelial cells (29).

Almost all the currently available information on NF-kappa B-dependent adhesion molecule expression (and, presumably, monocyte adhesion) has been obtained with venous cells. In view of the apparent differences in leukocyte adhesiveness to arterial and venous endothelia under proinflammatory stress in vivo (14), it is important to examine the mechanisms controlling adhesion in both cell types under identical conditions.

One interesting, albeit poorly understood, observation is that venous endothelium is more adhesive for leukocytes (especially neutrophils) than arterial endothelium (14). Differences in dynamic forces (i.e., shear rate, shear stress) are not sufficient to explain this phenomenon (22, 26). Recently, it was suggested (7) that differential expression of ECAMs causes these differences. However, direct comparisons of both adhesion molecule expression and monocyte adhesion in venous versus arterial endothelial cells have not been reported. Moreover, whether monocytes behave similarly as neutrophils (i.e., showing a preference for adhesion to venous endothelium) is unknown.

Here we compared cytokine- and lipopolysaccharide (LPS)-stimulated U-937 cell adhesion and ECAM expression in both venous and arterial endothelial cells. We also examined whether proteasome inhibition has similar effects on adhesion and ECAM expression in these cell types. Importantly, in this study, we compared cell types from the same tissue and at an identical passage. Our results show that, quite unlike venous cells, exposure of arterial cells to either TNF or LPS does not stimulate U-937 cell adhesion. Arterial insensitivity to these inflammatory mediators may be due to an inability of TNF and LPS to stimulate gene expression and cell surface expression of VCAM-1. Our data support the hypothesis that venous endothelium may be more predisposed to inflammation than arterial endothelium because of a greater ability to upregulate ECAMs in response to proinflammatory stimuli.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Reagents

A monoclonal antibody to VLA-4 (HP1/2) was generously provided by Dr. Roy Lobb (Biogen Research, Cambridge, MA). Antibodies to VCAM-1 (clone 1.G11B1, C-19), ICAM-1 (clone 15.2), and E-selectin (clone 1.2B6) were purchased from Southern Biotechnology Associates (Birmingham, AL) and Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal rabbit anti-human antisera to NF-kappa B subunits p50 and p65 were purchased from Rockland (Gilbertsville, PA). Horseradish peroxidase-coupled goat anti-rabbit IgG was purchased from PharMingen (San Diego, CA). VCAM-1 cDNA probe was provided by Dr. Laurelee Osborn (Biogen Research). Pyrrolidinedithiocarbamate (PDTC) was purchased from Sigma (St. Louis, MO). The proteasome inhibitor carbobenzyl-leucinyl-leucinyl-leucinyl-H (MG-132) was obtained from Proscript (Cambridge, MA).

Cell Culture

Venous and arterial cells were isolated from human umbilical cords with a modification of the procedures described by Jaffe et al. (16). These primary cultures were seeded in T-25 flasks in endothelial cell growth medium plus bovine pituitary brain extract (Clonetics, San Diego, CA). For experiments, they were trypsinized and passaged to either 24-well plates (U-937 cell adhesion and VCAM-1 surface expression measurements) or 100-mm dishes (Western and Northern analyses of VCAM). Due to a substantially lower yield of human umbilical arterial endothelial cells (HUAECs) on primary isolation, it was necessary to expand their population through four passages to obtain enough cells for experiments. Because preliminary experiments showed that endothelial responses to TNF and LPS were similar from the first through the sixth passages, the experiments reported herein used human umbilical venous endothelial cells (HUVECs) and HUAECs at the fifth passage. Preliminary experiments indicated that U-937 cell adhesion was similar from 1 to 8 days after confluence was attained; data reported herein are from cells at 4 days postconfluence. Human promonocytic U-937 cells (35) were obtained at unknown passage from American Type Culture Collection (Manassas, VA) and maintained in suspension culture in RPMI 1640 medium (Sigma) plus 10% fetal bovine serum (Atlanta Biologicals, Atlanta, GA) plus 2 mM L-glutamine (GIBCO BRL, Gaithersburg, MD). They were passaged every 3 days after a density of 4 × 105 cells/ml was achieved.

U-937 Cell Adhesion Assay

Adhesion studies were performed with the promonocytic cell line U-937, which has been established as a useful model for monocytes in adhesion studies (9). U-937 cells were washed two times with labeling medium (RPMI 1640 plus 1% fetal bovine serum), then incubated for 1 h (37°C, 5% CO2) with sodium chromate-51 (3-5 µCi/5 × 107 cells, 2-ml incubation volume; Dupont NEN, Boston, MA) with gentle intermittent mixing. Labeled U-937 cells were washed four times with labeling medium, then resuspended in fresh labeling medium at 2 × 107 cells/ml. After control or experimental treatments, endothelial cell monolayers in 24-well plates were washed two times with labeling medium, and then 450 µl of labeling medium were added to each well. For static adhesion assays, 50 µl of labeled U-937 cell suspension (1 × 106 cells) were added to each well of endothelial cells (U-937-to-endothelial cell ratio = 2:1), and the plates were gently agitated and placed in a cell culture incubator for 10 min (preliminary studies indicated that results of adhesion assays conducted for 10 min gave similar results to assays with a 30-min incubation time). At the end of the incubation period, the medium from each well was aspirated and saved for radioactive counting. The monolayer was gently washed three times with cold PBS; collected washes were combined with medium and counted, yielding a measure of nonadherent cells. Preliminary experiments indicated that three washes of the monolayer decreased control or basal adhesion by 50-100% (compared with a single wash) while effecting a minimal decrease (<2%) in TNF-stimulated adhesion. After the final wash, the monolayers were lysed for 1 h with 1 M NaOH; counting of the lysate yielded a measure of adherent U-937 cells. Adhesion is expressed as the percentage of total collected radioactivity present in the lysate {percent adhesion = [cpmlysate/ (cpmlysate + cpmsupernatant+washes)] × 100}, where cpm is counts/min.

Effect of TNF, LPS, and Interleukin-1 on U-937 Cell Adhesion to Endothelial Cell Monolayers

Endothelial cells were seeded at ~1 × 105 cells/well in 24-well plates. At 4 days postconfluence, the medium was aspirated and replaced with fresh medium containing Escherichia coli LPS (1 µg/ml; Sigma), graded doses of TNF (0.001-10 ng/ml, human recombinant; R&D Systems, Minneapolis, MN), interleukin-1 (IL-1; 5.0 ng/ml; Calbiochem, La Jolla, CA), or vehicle control [Hanks' balanced salt solution (HBSS) plus 0.5% BSA]. The monolayers were exposed to these treatments for 4 h, after which adhesion assays were carried out as described in U-937 Cell Adhesion Assay. In some experiments, before the above treatments, HUVECs were pretreated with either the antioxidant PDTC (100 µM in HBSS immediately before TNF or LPS treatment) or the peptide aldehyde proteasome inhibitor MG-132 (in 40 µM dimethylformamide, preincubated for 1 h before TNF or LPS treatment). Working stock solutions of PDTC and MG-132 were prepared immediately before use; they were added to wells such that the volume of vehicle or vehicle plus PDTC or MG-132 was 0.1% of the total incubation volume. Before adhesion assays were conducted, endothelial cell monolayers were gently but thoroughly washed free of all treatment substances with labeling medium. In some studies, radiolabeled, washed U-937 cells were preincubated for 30 min with a monoclonal antibody to VLA-4 (HP1/2, 10 µg/ml) or an isotype-matched control monoclonal antibody before the U-937 cells were added to TNF-treated HUVECs. In other studies, TNF-treated HUVECs were incubated for 30 min with an affinity-purified polyclonal antibody to fibronectin (13) before adhesion studies were conducted.

Effect of TNF and LPS on Endothelial Surface Expression of ECAMs

Surface expression of VCAM-1, E-selectin, and ICAM-1 was assayed with the method of Khan et al. (19). Endothelial cells were grown in 24-well culture plates. After exposure to TNF or LPS (in HUVECs with and without PDTC or MG-132), the wells were gently washed once with PBS, then incubated with antibodies to VCAM-1, E-selectin, or ICAM-1 (C-19, clone 1.2B6, or clone 15.2, respectively; Southern Biotechnology Associates) diluted 1:400 in PBS plus 5% FCS at 37°C for 30 min. Wells were washed two times with PBS, then incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Southern Biotechnology Associates) in PBS plus 5% FCS at 37°C for 30 min. The wells were washed four times with PBS, then incubated with 0.003% hydrogen peroxide plus 0.1 mg/ml of 3,3',5,5'-tetramethylbenzidine (Sigma) for 30 min in the dark. The color reaction was stopped by adding 75 µl of 8 N H2SO4, and the samples were transferred to 96-well plates. The plates were read on a microplate reader at 450 nm, blanking on wells stained with only secondary antibody. All data points were performed in triplicate.

Effect of TNF and LPS on Expression of VCAM-1 Protein by Immunoblotting

Endothelial cell monolayers were treated with TNF (1 ng/ml) or LPS (1 µg/ml) with and without PDTC pretreatment as described in Effect of TNF, LPS, and Interleukin-1 on U-937 Cell Adhesion to Endothelial Cell Monolayers. The monolayers were then washed once with cold PBS, then incubated for 30 min on ice with extraction buffer [150 mM NaCl, 50 mM Tris, pH 8.0, 1% Triton X-100, 0.05% SDS, 1 µg/ml of aprotinin, 1 µg/ml of leupeptin, 1 µg/ml of pepstatin A, 1 µg/ml of chymostatin, and 50 µg/ml phenylmethylsulfonyl fluoride (PMSF)] with intermittent mixing. The monolayer was then scraped from the plate, transferred to a microfuge tube, and sonicated on ice. The lysates were centrifuged at 12,000 g for 5 min at 4°C, and the cytosolic extracts were snap-frozen in liquid N2 and stored at -80°C until analysis. The protein content was measured with a modified Lowry procedure (Bio-Rad DC Protein Assay, Hercules, CA). Equal amounts (60 µg) of protein were subjected to SDS-PAGE on 7% gels, followed by electrotransfer to nitrocellulose paper; equivalent loading and transfer was confirmed by staining of the blot with Ponceau-S before blocking. The blots were blocked for 45 min in PBS plus 5% milk plus 0.05% Tween 20, then incubated for 45 min with goat anti-human VCAM-1 antiserum (1.G11B1, Santa Cruz Biotechnology) 1:100 in PBS plus 0.1% milk plus 0.05% Tween 20. After being washed in PBS plus 0.05% Tween 20, the blots were incubated for 30 min with HRP-conjugated rabbit anti-goat IgG (DAKO, Carpinteria, CA) 1:1,000 in PBS plus 0.1% milk plus 0.05% Tween 20. After being washed in PBS plus 0.05% Tween 20, the blots were developed with a diaminobenzidine-hydrogen peroxide chromogenic system. Relative levels of VCAM-1 immunoreactive material were determined by densitometry.

Effect of TNF on VCAM-1 mRNA Expression

Endothelial cells were treated with TNF or vehicle (HBSS plus 0.5% BSA) for 4 h with and without PDTC pretreatment. The cells were then lysed, and total RNA was isolated with the RNeasy kit (Qiagen, Chatsworth, CA). Subsequent procedures were modified from those previously described (18). Briefly, total cellular RNA (5 µg) was resolved on a 1.5% agarose gel in 1× Tris-acetate-EDTA, transferred electrophoretically to nitrocellulose membrane (Zeta Probe-GT, Bio-Rad), and ultraviolet cross-linked for 5 min. The blots were then incubated for 30 min in prehybridization buffer (0.25 M Na2HPO4 and 7% SDS, pH 7.2) at 60°C. Hybridizations were performed at 55°C overnight with 1.9 × 106 cpm/ml of denatured [32P]CTP-labeled VCAM-1 cDNA probe (specific activity = 1.9 × 108 cpm/µg DNA; Random Primer Oligo Kit, Pharmacia, Piscataway, NJ). The blots were washed for 15 min each with 2× saline-sodium citrate (SSC; 1× SSC is 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0)-0.1% SDS (25°C), 0.5× SSC-0.1% SDS (25°C), and 0.1× SSC-0.1% SDS (55°C) and then exposed to photographic film (X-OMAT, Kodak) for 24 h at -70°C. After the blot was probed for VCAM, it was stripped by boiling before rehybridization with [32P]CTP-labeled 18S probe (pT7 RNA 18S control template; Ambion, Austin, TX). Relative amounts of VCAM-1 mRNA from each sample were estimated by densitometry of the autoradiographs and normalized to the density of the respective 18S signal.

Activation of NF-kappa B

Isolation of nuclear extracts. The methods used were a modification of Schreiber et al. (32). HUVECs and HUAECs were incubated for 4 h with either vehicle or TNF (10 ng/ml) after pretreatment with and without PDTC (100 µM) or MG-132 (40 µM). The incubation medium was aspirated, and the cells were scraped from the plate in PBS. The cells were pelleted (1,500 rpm, 5 min) at 4°C; the PBS was decanted, and the pellets were resuspended in 0.4 ml of buffer A [10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol (DTT), 0.5 mM PMSF, 1 µg/ml of leupeptin, and 1 µg/ml of aprotinin] by gentle pipetting through a large-bore pipette tip. The cell suspension was allowed to swell on ice for 15 min, after which 25 µl of 10% Nonidet P-40 were added, and the suspension was vortexed for 10 s. The homogenates were centrifuged at 10,000 g for 30 s at 25°C; the nuclear pellet was washed once with and resuspended in 50 µl of buffer C (20 mM HEPES, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.5 mM PMSF, 1 µg/ml of leupeptin, and 1 µg/ml of aprotinin). The resuspended nuclear fraction was sonicated on ice, then centrifuged at 12,000 g for 1 min at 4°C. The supernatant from this spin (nuclear extract), as well as the cytosolic extract, was assayed for protein concentration with the Bio-Rad DC Protein Assay, then immediately used for gel shift assay.

Electrophoretic mobility shift assay. NF-kappa B consensus oligonucleotide 5'-AGTTGAGGGGACTTTCCCAGGC-3' (Promega, Madison, WI) was end labeled with [32P]ATP with T4 polynucleotide kinase (Promega gel shift assay system) according to the manufacturer's instructions. Nuclear extracts (10 µg of protein) were preincubated for 10 min at 25°C in 1 mM MgCl2, 2.5 mM EDTA, 2.5 mM DTT, 250 mM NaCl, 50 mM Tris · HCl (pH 7.5), and 0.25 µg/µl of poly(dI-dC) in 20% glycerol. Labeled oligonucleotide (0.07 pmol) was then added (total assay volume 20 µl), and binding reactions were incubated for 30 min at 25°C. The reactions were stopped by the addition of 4 µl of 10× gel loading buffer. Specificity of the binding was verified by including a 100-fold molar excess of unlabeled oligonucleotide in some reactions and by performing supershift analysis with antibodies to NF-kappa B subunits p50 and p65 (incubated with nuclear extracts on ice for 1 h before the addition of labeled oligonucleotide). The reaction mixtures were applied to a nondenaturing, 4% polyacrylamide gel and electrophoresed at 125 V (constant voltage) for 4 h. The gels were fixed in 10% acetic acid-40% methanol for 15 min, washed in distilled water, dried, and then exposed to X-ray film (Kodak X-OMAT) for 4-16 h at -70°C.

Western blotting of nuclear p50 and p65. Nuclear extracts from control and TNF (with and without MG-132)-treated HUVECs and HUAECs were prepared as described in Isolation of nuclear extracts. Equal amounts of nuclear protein (30 µg) from each were subjected to SDS-PAGE, then transferred to nitrocellulose membrane. The blots were blocked overnight with 5% milk powder in PBS and then incubated for 2 h with primary rabbit anti-human antibodies to p65 or p50 and for 1 h with HRP-coupled goat anti-rabbit secondary antibody, each in 0.1% milk in PBS. The blots were developed in imidazole-diaminobenzidine-H2O2 (1 mg/ml, 1 mg/ml, and 1 µl/ml, respectively) in 0.1% milk in PBS.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

U-937 Cell Adhesion

In response to TNF, HUVECs showed a dose-dependent increase in U-937 cell adhesion, with a threshold effective dose between 0.001 and 0.01 ng/ml (Fig. 1A). Over the same dose range, TNF did not stimulate adhesion in HUAECs (Fig. 1B). Incubation of HUAECs with TNF for a longer time period (24 h) also did not produce increases in U-937 cell adhesion (data not shown). In separate studies, after 4 h of incubation with graded doses of TNF, HUAECs were trypsinized, resuspended in endothelial cell growth medium, and mixed 1:1 with 0.4% trypan blue. There was no effect of TNF on viability, indicating that a toxic effect of TNF could not explain the lack of an adhesion response in HUAECs.


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Fig. 1.   Effect of treatment of endothelial cells with graded doses of human recombinant tumor necrosis factor (TNF) on adhesion to U-937 cells. Human umbilical venous endothelial cells (HUVECs; A) or human umbilical arterial endothelial cells (HUAECs; B) were incubated for 4 h with either vehicle control [0.5% BSA in Hanks' balanced salt solution (HBSS)] or TNF at the indicated doses. Endothelial cells were then gently but thoroughly washed free of TNF with fresh medium and then incubated for 10 min with 51Cr-labeled U-937 cells, and adhesion was measured as described in MATERIALS AND METHODS. cpm, Counts/min. Values are means ± SD for triplicate or quadruplicate replicates each in 3 (HUVEC) or 4 (HUAEC) separate experiments. * Significantly different from control (TNF = 0), P < 0.001.

Because a previous study (3) indicated that exposure of HUAECs to IL-1 (5 ng/ml) stimulated monocyte adhesion, it was important to test whether the lack of response of HUAECs to TNF in the present study was an artifact of our experimental conditions. Therefore, we compared the effects of IL-1 on U-937 cell adhesion in HUVECs and HUAECs. Incubation of HUVECs and HUAECs with IL-1 (5.0 ng/ml) stimulated U-937 cell adhesion in both cell types (HUVECs: 6.8 ± 1.76% for basal adhesion, 82.9 ± 1.72% for IL-1-stimulated adhesion; HUAECs: 19.7 ± 5.25% for basal adhesion, 43.7 ± 3.38% for IL-1-stimulated adhesion).

In HUVECs, U-937 cell adhesion in response to TNF (1.0 ng/ml) or LPS (1 µg/ml) was inhibited by the antioxidant PDTC and the proteasome inhibitor MG-132 (Fig. 2). To evaluate the role of VCAM-1 in the U-937 cell adhesion response, we examined the effect of a blocking antibody to monocyte VLA-4 (HP1/2) on TNF-stimulated adhesion. Preincubation of labeled U-937 cells with HP1/2 (10 µg/ml) for 30 min decreased TNF-stimulated adhesion to HUVECs by ~75% (Fig. 3). We next tested whether fibronectin (another ligand for VLA-4) might be involved in the adhesion of U-937 cells to HUVECs. Incubation of TNF-treated HUVEC monolayers with a fibronectin antibody did not prevent subsequent U-937 cell adhesion (Table 1); moreover, U-937 cells showed no higher adhesion to fibronectin-coated plates than to tissue culture plastic (data not shown). These results indicate that VCAM-VLA-4 interactions were likely responsible for most of the adhesion of U-937 cells to HUVECs.


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Fig. 2.   Effect of antioxidant pyrrolidinedithiocarbamate (PDTC; 100 µM) and proteasome inhibitor MG-132 (40 µM) on U-937 cell adhesion to TNF- or lipopolysaccharide (LPS)-treated HUVECs. Immediately or 1 h before control, TNF, or LPS treatment, PDTC or MG-132, respectively, was added to HUVEC monolayers. HUVECs were incubated for 4 h with TNF or LPS, and then adhesion of U-937 cells to monolayers was determined. Vehicles were HBSS for PDTC and dimethylformamide (DMF) for MG-132. Values are means ± SD for quadruplicate replicates each in 3 separate experiments. * Significantly different from control, P < 0.05.


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Fig. 3.   Effect of a functional blocking antibody to very late activating antigen (VLA)-4 on U-937 cell adhesion in TNF-stimulated HUVECs. Endothelial cells were incubated with TNF (1 ng/ml) for 4 h. Radiolabeled, washed U-937 cells were preincubated for 30 min with either monoclonal antibody HP1/2 to VLA-4 or an isotype-matched control monoclonal antibody at a concentration such that on addition of U-937 cells to HUVECs, final antibody concentration would be 10 µg/ml. Adhesion of U-937 cells to HUVECs was then determined. Values are means ± SD for triplicate replicates each in 2 separate experiments. * Significantly different from control adhesion, P < 0.01. ** Significant difference between TNF and TNF+HP1/2 conditions, P < 0.0001.

                              
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Table 1.   Antibody to fibronectin does not block TNF-stimulated adhesion of U-937 cells to HUVEC

Surface Expression of ECAMs in TNF- and LPS-Treated Cells

In HUVECs, TNF produced a dose-dependent increase in surface expression of VCAM-1, with a threshold TNF dose between 0.001 and 0.01 ng/ml (data not shown). In contrast, there was no effect of TNF on VCAM-1 expression in HUAECs (Fig. 4) even after up to 24 h of incubation with TNF (data not shown). We also examined the effect of LPS on VCAM-1 expression; LPS treatment for 4 h produced about a 10-fold increase in VCAM-1 expression in HUVECs but had no effect in HUAECs (data not shown); PDTC and MG-132 pretreatment blocked the effects of both TNF and LPS on VCAM-1 surface expression (Fig. 5).


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Fig. 4.   Effect of TNF on vascular cell adhesion molecule (VCAM)-1 surface expression in HUVEC versus HUAEC monolayers. Endothelial cells were incubated with either control vehicle or TNF (1 ng/ml) for 4 h, and then surface expression was measured by ELISA as described in MATERIALS AND METHODS. OD450, absorbance units at 450 nm. Values are means ± SD from 2 separate experiments. * Significantly different from control expression.


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Fig. 5.   Effect of PDTC (100 µM) and MG-132 (40 µM) on cell surface VCAM-1 expression in TNF- or LPS-treated HUVECs. Immediately or 1 h before control, TNF, or LPS treatment, PDTC or MG-132, respectively, were added to HUVEC monolayers. Endothelial cells were incubated for 4 h with TNF or LPS, and then VCAM-1 surface expression was measured. Vehicles were HBSS for PDTC and DMF for MG-132. Values are means ± SD for triplicate replicates each in 2 separate experiments. * Significantly different from control, P < 0.001.

We examined surface expression of E-selectin and ICAM-1 in HUVECs and HUAECs in response to TNF (Table 2). E-selectin surface expression in HUVECs was stimulated from 29- to 33-fold in response to TNF from 0.01 to 1.0 ng/ml; at the highest dose of TNF, PDTC produced a significant but only partial (36%) decrease in E-selectin expression; however, MG-132 almost completely blocked the E-selectin response. Similar to what was observed for VCAM-1, there was no effect of TNF on E-selectin expression in HUAECs.

                              
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Table 2.   Surface expression of ICAM-1 and E-selectin in HUVEC and HUAEC

Basal ICAM-1 expression in HUAECs was about fourfold lower than that in HUVECs. However, TNF produced similar increases in ICAM-1 expression in both cell types (2.7- and 3.4-fold compared with basal expression in HUVECs and HUAECs, respectively). In both cell types, MG-132 but not PDTC pretreatment abolished the ICAM-1 response to TNF (1.0 ng/ml).

In response to 4 h of incubation with IL-1 (5 ng/ml), VCAM-1 surface expression in HUVECs increased significantly, from a basal value of 0.13 ± 0.02 to 1.21 ± 0.16 absorbance units at 450 nm (P < 0.0001). In HUAECs, IL-1 elicited a similar response (basal, 0.05 ± 0.03 units; IL-1, 0.71 ± 0.10 units; P < 0.0001).

Expression of Total VCAM-1 Protein in TNF-Treated Cells

To determine whether the difference in VCAM-1 surface expression between HUVECs and HUAECs might be due to a difference in expression of VCAM-1 protein, we incubated the two different cell types with TNF (1.0 ng/ml) with and without pretreatment with PDTC and examined whole cell cytosolic homogenates by Western blot (Fig. 6). Consistent with the results from surface expression studies, PDTC had no effect on VCAM-1 expression in control HUVECs and HUAECs. TNF stimulated VCAM-1 protein 25-fold in HUVECs; this was lowered to a 5-fold increase with PDTC treatment. However, similar to the results of surface expression measurements, TNF had no apparent effect on VCAM-1 protein expression in HUAECs.


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Fig. 6.   Whole cell expression of VCAM-1 by Western blot (top) in HUVECs vs. HUAECs treated for 4 h with vehicle (0.5% HBSS) or TNF (1 ng/ml) in presence (+) and absence (-) of PDTC (100 µM). Equal amounts (60 µg) of total protein from cell lysates were loaded in each well. Relative expression was determined by densitometric analysis of stained blot (bottom).

Measurement of VCAM-1 mRNA Levels in TNF-Treated Cells

To begin to examine the basis for the difference in VCAM-1 protein expression between HUVECs and HUAECs, we tested the effect of TNF with and without PDTC on VCAM-1 mRNA levels (Fig. 7). There was negligible VCAM-1 mRNA expression in control cells (both HUVECs and HUAECs), and PDTC had no effect on the control expression of VCAM-1 message. In HUVECs, TNF produced a 44-fold increase in VCAM-1 mRNA levels; 72% of this increase was blocked by PDTC. In HUAECs, TNF stimulated VCAM-1 expression fourfold, and PDTC treatment blocked this increase by 50%. These results demonstrate that the most likely explanation for negligible VCAM-1 protein expression in TNF-stimulated HUAECs compared with HUVECs lies in a striking relative deficit in induction of VCAM-1 mRNA expression in HUAECs.


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Fig. 7.   Northern blot (top) of VCAM-1 mRNA in HUVECs vs. HUAECs treated with vehicle or TNF (as in Fig. 6) in presence and absence of PDTC. Equal amounts (1 µg) of RNA were loaded on gel. Relative expression was determined by densitometric analysis of autoradiogram (bottom). ODVCAM/OD18S, ratio of arbitrary absorbance units for VCAM-1 to those for 18S mRNA.

Activation of NF-kappa B

Using a combination of gel shift analysis and Western blotting, we examined whether the differences in TNF-elicited ECAM expression and U-937 cell adhesion between HUVECs and HUAECs were associated with differential activation of NF-kappa B. In HUVECs, TNF treatment produced two shifted bands, which were both decreased by about fourfold by pretreatment with PDTC (Fig. 8A, bands 1 and 2). In contrast, pretreatment with MG-132 completely eliminated band 2 and slightly attenuated band 1 (Fig. 8B). To identify the shifted complexes, we performed supershift analysis using antibodies to the p50 and p65 NF-kappa B subunits (Fig. 9). Band 1 was partially supershifted and band 2 was completely supershifted by the p50 antibody, whereas the p65 antibody appeared to preferentially effect a supershift of band 1. Results of Western blotting for these two proteins in HUVEC nuclear extracts in the presence and absence of MG-132 (Fig. 10) were consistent with these findings: increased presence of nuclear p50 after TNF treatment was virtually completely blocked by MG-132, whereas the proteasome inhibitor only slightly reduced the presence of nuclear p65. Taken together with the relative effects of MG-132 on the shifted complexes from the electrophoretic mobility shift assay and supershift analysis, these results indicate that band 2 contained mainly p50 (e.g., p50-p50 homodimer) and that p65 (e.g., p50-p65 heterodimer) was present in band 1. Results of these analyses with nuclear extracts from HUAECs were substantially the same (Figs. 8-10). Results of some studies suggested the presence of a third TNF-stimulated band in HUAECs that had a lower mobility than bands 1 and 2 (Fig. 8A) and could not be supershifted by either p50 or p65 antibodies (data not shown), but this band was not a consistent finding (Figs. 8B and 9). Overall, these results indicate similar effects of TNF on activation of NF-kappa B in both HUVECs and HUAECs.


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Fig. 8.   Electrophoretic mobility shift assay for nuclear factor-kappa B (NF-kappa B) activation induced by TNF (1 ng/ml) in presence and absence of PDTC (100 µM; A) or MG-132 (40 µM; B) in HUVECs and HUAECs. Endothelial cells were treated with either control vehicle or TNF (with or without pretreatment with inhibitor) for 4 h. Cells were harvested, and nuclear extracts were obtained and assayed by gel shift assay for NF-kappa B binding as described in MATERIALS AND METHODS. Nos. at left and right, shifted bands.


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Fig. 9.   Supershift analysis of TNF-stimulated NF-kappa B complexes obtained via electrophoretic mobility shift assay in HUVECs (A) and HUAECs (B). Before binding assays were performed, nuclear extracts were preincubated with antibodies to p50 and p65 or with excess unlabeled NF-kappa B oligonucleotide (Cold Oligo) as described in MATERIALS AND METHODS. Nos. at left, shifted bands.


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Fig. 10.   Western blot detection of p50 and p65 in nuclear extracts from HUVECs (A) and HUAECs (B) treated with TNF with and without MG-132. Each lane represents 30 µg of nuclear protein. Extra bands in HUAEC p50 blot are nonspecific, appearing as a result of color overdevelopment.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The adhesion of monocytes to endothelium is well established as a major early step in the development of atherosclerosis (9, 31) and may also play important (17) but currently uncharacterized roles in other inflammatory pathologies. The mechanisms regulating differential ECAM expression and monocyte adhesion are likely determinants of both the extent and the distribution of vascular injury in inflammation, but they have not been well characterized. A fundamental, yet poorly understood, observation is that atherogenesis occurs mainly in arteries, not in veins. Conversely, other inflammatory disorders appear to selectively target venous rather than arterial endothelium, particularly postcapillary venules (14). The reasons for this are not clear. Although differences in shear stress were originally suggested as a possible explanation (10), other studies reported greater leukocyte adhesion and emigration in venules over arterioles and capillaries, even when shear stress (22) and flow (26) are matched. Therefore, the objective of the present study was to compare the adhesion of promonocytic U-937 cells and expression of ECAMs in arterial and venous cells in response to inflammatory mediators.

The major rationale for our use of the promonocytic U-937 cell line rather than primary isolated monocytes was the difficulty in obtaining sufficient quantities of primary isolated monocytes from plasma. Nevertheless, in preliminary studies, we found that adhesion of U-937 cells to TNF-treated HUVECs and HUAECs was similar to that observed with human monocytes isolated from plasma. Moreover, U-937 cells have been previously shown to be an adequate monocyte model for endothelial adhesion studies (9).

We observed no effect of TNF and LPS on the adhesion of U-937 cells to HUAECs, whereas the adhesion to HUVECs was increased significantly by TNF and LPS. Similarly, TNF and LPS failed to induce surface expression of VCAM-1 and E-selectin in HUAECs. TNF also failed to induce expression of VCAM-1 mRNA in HUAECs. Finally, because a VLA-4 blocking antibody abolished ~75% of TNF-stimulated U-937 cell adhesion in HUVECs, the lack of a VCAM-1 response in HUAECs may be responsible, at least in part, for its lack of an adhesion response. These results demonstrate a novel and striking difference in the sensitivity of venous and arterial endothelial responses to TNF. It is unknown whether this can be extended to neutrophil adhesion because VCAM-1-VLA-4 interactions do not mediate neutrophil adhesion to endothelial cells (20). Future studies will also need to examine whether differences between venous and arterial endothelial cells such as we observed help explain anatomic susceptibility of venous versus arterial endothelium to inflammation in vivo. Clearly, our results support the hypothesis (7) that differences in venous and arterial sensitivity to inflammation may be explained by their relative abilities to upregulate expression of ECAMs.

In HUVECs, TNF- and LPS-stimulated U-937 cell adhesion was correlated with increased expression of VCAM-1, E-selectin, and ICAM-1. Moreover, both the increased adhesion and ECAM expression were blocked by the proteasome inhibitor MG-132, strongly supporting a role for NF-kappa B in both of these processes. Our results with the antioxidant PDTC also support an important role for reactive oxygen metabolites in the adhesion response, as well as in the response of VCAM-1 (and, to a lesser extent, E-selectin but not of ICAM-1) to TNF. These results are consistent with those previously reported in HUVECs (23, 30, 37), and our findings with MG-132 extend previous studies (19, 27, 37) by demonstrating a direct link between NF-kappa B activation and monocyte adhesion.

Several lines of evidence indicate that the adhesion response in these studies was mainly a VCAM-dependent (and possibly an ICAM-independent) phenomenon. First, in HUVECs, we observed a parallel blockade of both U-937 cell adhesion and VCAM-1 surface expression by both PDTC and MG-132. Second, a blocking antibody to VLA-4, the beta 1-integrin ligand to VCAM-1, produced a 75% reduction in TNF-stimulated adhesion of U-937 cells to HUVECs. Third, in HUVECs, although MG-132 blocked E-selectin expression, PDTC had only a partial effect on E-selectin. Finally, PDTC had no effect on ICAM-1 expression, and despite a TNF-stimulated increase in surface ICAM-1 in HUAECs, TNF did not elicit U-937 cell adhesion in HUAECs. We conclude that the most likely explanation for the failure of TNF and LPS to stimulate U-937 cell adhesion in HUAECs was the failure to upregulate VCAM-1 surface expression in these cells.

Previous studies (19, 29, 37) implicating NF-kappa B in proinflammatory-stimulated ECAM expression and, presumably, in monocyte-endothelial cell adhesion, have all used venous cells (HUVECs). Similar analysis in arterial endothelial cells has not been available. Several studies have examined either monocyte adhesion (3, 4) or ECAM expression (19, 37) (but not both) in HUVECs and other endothelial cell types, e.g., aortic cells (19), HUAECs (3, 4, 37), and microvascular cells (4, 11, 15); several issues limit the scope of these previous reports. In some studies comparing venous and arterial cells, species differences (6, 28, 29) and differences in the tissue source (6, 19) and passage number (37) make it difficult to determine whether vessel type per se is a significant variable. Moreover, no studies have compared both monocyte adhesion and NF-kappa B-dependent ECAM expression in venous and arterial cells under identical experimental conditions. The only previous studies (19, 29, 37) directly linking NF-kappa B-mediated ECAM expression and monocyte adhesion have used HUVECs.

Few studies on leukocyte adhesion and ECAM expression have directly compared HUVECs and HUAECs, and when they have, they are limited in scope. Beekhuizen and colleagues compared cell growth (5) and monocyte adhesion under basal conditions (4) and in response to IL-1 (3) in HUVECs and HUAECs. They found that basal adhesion to HUAECs and HUVECs was similar (4) and that IL-1 doubled monocyte adhesion to HUAECs (3). Because of the earlier finding by Beekhuizen et al. (3), we compared the effects of IL-1 on U-937 cell adhesion in HUVECs and HUAECs to ensure that the lack of response of HUAECs to TNF was not an artifact of our particular experimental conditions. We saw similar results with IL-1 in HUAECs (2.2-fold increase in U-937 cell adhesion) and also observed a 12-fold stimulation of U-937 cell adhesion to IL-1-treated HUVECs. Thus, although our HUAECs did not respond as robustly to IL-1 as did HUVECs, they showed a similar response to that previously reported (3). Our results indicate a striking resistance of HUAECs to certain, inflammatory mediators depending on the mediator involved. Different responses to TNF in HUVECs and HUAECs have been previously reported (36): proliferation of HUVECs is significantly decreased by TNF, whereas TNF had no effect on HUAEC proliferation.

With respect to ECAM expression, one report (37) demonstrated an increase in HUAEC VCAM-1 surface expression after a 24-h exposure to TNF that was blocked partially by PDTC. However, that study did not examine monocyte adhesion in HUAECs. We saw no effects of TNF on VCAM-1 mRNA, cell surface expression, or U-937 cell adhesion in HUAECs even after 24 h of exposure to TNF (data not shown). The reason(s) for the differences in VCAM-1 expression in that study and ours is unclear. Our HUAEC findings differ from reports with other arterial endothelial types (aortic endothelial cells) from human (19, 25) and other species (2, 9, 28). These reports mostly show either increased monocyte adhesion (2, 17, 25) or adhesion molecule expression (19) in response to cytokines, endotoxin, or modified low-density lipoprotein. Several of these studies (2, 9, 16, 25) examined only arterial cells and did not directly compare venous cell responses to identical conditions. Only one study (9), using rabbit aortic and vena caval endothelial cells, examined both monocyte and U-937 cell adhesion and ECAM expression under the same conditions, finding similar responses to LPS in both cell types. Thus, although it is possible that HUAECs are unique endothelial cells in their relative insensitivity to the effects of TNF, our data suggest that more detailed and systematic comparisons of the response of other arterial cell types with that of venous cells to certain proinflammatory stimuli under similar experimental conditions are needed.

Reasons for the insensitivity of HUAECs to TNF are unknown. It is unlikely that a TNF-receptor defect can explain our findings because TNF elicited the same multiple of increase in ICAM-1 expression as in HUVECs; moreover, this increase was inhibited by MG-132. Other explanations include differences in the signaling pathway between TNF-receptor interaction and NF-kappa B activation (12, 21, 33), differences in the relative abilities of these two cell types to activate NF-kappa B, or nuclear events taking place after translocation of NF-kappa B (1). As a first step in examining this issue, we compared the abilities of nuclear extracts from TNF-treated HUVECs and HUAECs to produce an electrophoretic mobility shift after incubation with a NF-kappa B consensus oligonucleotide. We observed two major shifted complexes in HUVECs, corresponding to previously identified complexes containing p50 and p65 (29). Similar complexes were also observed in TNF-treated HUAECs, and the inhibitory effects of PDTC and MG-132 on apparent activation of NF-kappa B were the same in both cell types. These results indicate that the difference in TNF-stimulated ECAM (especially VCAM) expression and U-937 cell adhesion between HUVECs and HUAECs cannot be explained by differences in ability of this cytokine to activate NF-kappa B in these two cell types and may involve other nuclear events required for VCAM-1 expression, including activity of other TNF-activated transcription factors known to be required in addition to NF-kappa B for full VCAM-1 expression (23), or possibly nuclear inhibitory factors that could prevent VCAM-1 transcription in HUAECs even in the presence of activation of NF-kappa B. Further work is required to examine these possibilities.

In summary, we report novel and striking differences in the response of venous versus arterial endothelial cells to TNF and LPS. These agents stimulate adhesion of promonocytic U-937 cells to venous cells, yet arterial cells are almost wholly refractory to the effects of TNF and LPS. Differences in U-937 adhesivity appear to be explained by radically different expression of ECAMs, especially VCAM-1, in these cell types. These findings were not accompanied by differences in the apparent activation of the inflammatory transcription factor NF-kappa B between these two cell types. Our observations provide support for the hypothesis that variations in ECAM expression (in response to inflammatory stress) might explain the differential leukocyte adhesivity between venous and arterial endothelium in vivo.

    ACKNOWLEDGEMENTS

We thank Dr. Frank Gelder for providing the fibronectin antibody and Dr. Matthew Grisham for helpful discussions.

    FOOTNOTES

This work was supported by funds from the Louisiana State University Medical Center (LSUMC) Division of Vascular Surgery and the LSUMC Center for Excellence in Arthritis and Rheumatology; National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-52148 (to T. J. Kalogeris) and DK-43785 (to J. S. Alexander); and National Heart, Lung, and Blood Institute Grant HL-47615 (to J. S. Alexander).

Address for reprint requests: J. S. Alexander, Dept. of Physiology and Biophysics, Louisiana State Univ. Medical Center, 1501 Kings Highway, Shreveport, LA 71130.

Received 21 July 1997; accepted in final form 23 September 1998.

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Results
Discussion
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