Endothelial Cell Culture

Bovine aortic endothelial cells (BAEC; provided by J.-A. Haeffliger, Department of Internal Medicine, University Hospital, Lausanne), isolated by collagenase treatment of bovine aorta, were established as primary culture and serially passaged in RPMI 1640 medium (Gibco Laboratories, Paisley, Scotland) supplemented with 10% FCS (Gibco Laboratories). For adhesion studies, BAEC (passages 3–5) were plated on 60 × 15-mm tissue culture dishes (Becton Dickinson, Basel, Switzerland) and grown within a 2-cm-diam circle delineated by a ring of 12 M polysiloxane as described elsewhere (72, 73). For use in the parallel flow chamber, BAEC were plated on 25-mm circular glass coverslips (Bellco Glass, Inc., Vineland, NJ). Because a lower reactivity of BAEC with L-selectin/μ chimera was observed with endothelial passages >8, only passages 3–5 were used to perform immunofluorescence analysis or adhesion assays.

Monocyte Isolation

Human monocytes were prepared from blood buffy coats obtained from healthy blood donors. Monocytes were isolated by centrifugation on Ficoll–Hypaque (Pharmacia, Uppsala, Sweden) and adhesion on gelatine 1% (Sigma Chemical Co., St. Louis, MO) at 37°C. Nonadherent cells were removed by three washes with HBSS (Gibco Laboratories). Adherent cells were then detached with PBS containing 5 mM EDTA and washed again in RPMI 1640 (Gibco Laboratories). The cell suspension obtained by this method contained >95% monocytes as determined by Giemsa stain and immunostaining with phycoerythrin-conjugated anti-CD14 mAb Leu-M3 (Becton Dickinson). L-selectin and CD14 expression by whole blood and isolated monocytes was evaluated by double immunofluorescence and flow cytometry (see below). Monocyte isolation caused a 40– 50% loss of L-selectin expression. Isolated monocytes were kept on ice and used immediately after isolation.

mAbs

Anti–L-selectin mAbs anti–LAM1-3, -4, and -11 and anti–VCAM-1 mAb HAE2 (all IgG1) were produced as described (72, 73). mAbs were purified from hybridoma culture supernatants on Affigel protein A (Bio-Rad, Glattbrugg, Switzerland). For cell adhesion–blocking experiments, purified mAb IgG was used at 10 μg/ml. For chimeric protein–binding inhibition experiments, purified mAb IgG was used at 50 μg/ml.

Monocyte–Endothelial Interactions under Rotation

Cell attachment assays were carried out under rotation as previously described (73, 76, 77). BAEC, grown to confluence on tissue culture dishes, were stimulated for 8 h with 100 U/ml TNF-α (Boehringer Mannheim, Mannheim, Germany). After washing, cytokine–activated endothelial cells were preincubated for 15 min with medium alone (RPMI 1640/5% FCS) or with medium supplemented with anti–VCAM-1 mAb, L-selectin/μ, or CD4/μ chimeric proteins. Monocytes (4 × 10⁶ cells) were preincubated for 15 min on ice in 120 μl of medium (RPMI 1640/5% FCS) or in medium supplemented with mAb. Endothelial cell monolayers were washed before adding monocytes. After 30 min of incubation at 4°C under rotation at 72 rpm, nonadherent cells were discarded. Petri dishes were then placed vertically in 2% glutaraldhehyde and fixed overnight. The number of adherent monocytes was counted in six to eight microscopic fields (0.5 mm² per field), and the results were expressed as mean ± 1 SD.

Monocyte–Endothelial Interactions under Flow

Well-defined laminar flow was produced over confluent endothelial cell monolayers on 25-mm circular glass coverslips introduced in a parallel plate flow chamber (70). Monocytes were suspended at 0.5 × 10⁶/ml in RPMI 1640 medium and perfused at room temperature (18°C) through the chamber at a shear stress of 1.8 dynes/cm² via a syringe pump (model 22; Harvard Apparatus, Indulab AG, Switzerland). Monocyte–endothelial interactions were visualized using a phase-contrast videomicroscope (Axiovert; Carl Zeiss, Lausanne, Switzerland) and CCD videocamera (model XC73CE; Sony, Japan) and videotaped (Panasonic s-VHS recorder; TSA Telecom, Lausanne, Switzerland). Endothelial cell monolayers were cultured for 8 h in medium or in medium containing 100 U/ml TNF-α and then treated for 20 min with saturating levels of chimeric proteins. To determine the involvement of endothelial glycosaminoglycans, endothelial monolayers were incubated for 45 min with heparinase I (1,200 mU/ml) or hyaluronidase (200 mU/ml) and then extensively washed with medium. Monocytes were pretreated for 15 min with saturating concentrations of anti–L-selectin mAb at 4°C and then suspended in medium. Stable adhesion was determined between 10 and 12 min of monocyte perfusion by analyzing 12–14 random fields (0.14 mm²/field, ×20 objective). Monocytes were considered as adherent after 20 s of stable contact. The rate of initial attachment was assessed by counting the number of monocytes that interacted with endothelial cell monolayers during the first 5 min of the experiments.

Production of L-Selectin/μ Chimeric Protein

The L-selectin/μ chimeric protein was prepared by a method described in detail elsewhere (78). Briefly, sequences encoding the lectin domain, the EGF-like domain, and the first two short consensus repeats of L-selectin were amplified by PCR using synthetic oligonucleotides. An artificial splice donor site was introduced at the 3′ end of the PCR product. The PCR product was then subcloned in a plasmid containing the CH2, CH3, and CH4 domains of IgM heavy chain (μ) in genomic configuration (kindly provided by A. Traunecker, Basel Institute for Immunology, Basel, Switzerland). After digestion with NotI and XhoI, the pL-selectin/μ fragment was subcloned in the pcDNAI expression vector (Invitrogen, San Diego, CA). A CD4/μ chimera was constructed by substituting the L-selectin coding sequence in pcDNA I L-selectin/μ with a CD4 fragment encoding the first two NH₂-terminal domains of CD4. Chimeric molecules were produced in COS cells transiently transfected with appropriate cDNAs. Chimeras were used as concentrated COS cell conditioned media or after purification by immunoadsorption to immobilized anti–LAM1-3 mAb (77). The molecular characteristics of L-selectin/μ chimera were analyzed by SDS-PAGE. In reducing conditions, purified L-selectin/μ migrated with molecular masses ranging from 95,000 to 110,000 daltons. In nonreducing conditions, the decameric L-selectin/μ chimera migrated as a single band of very high molecular mass remaining at the end of the migration in the 3.75% SDS–polyacrylamide stacking gel. No additional band of lower molecular mass was observed in the 7.5% SDS–polyacrylamide running gel. The concentration of L-selectin/μ was measured by ELISA as previously described (75, 77). The concentration of CD4/μ chimera was determined by ELISA using goat anti–human IgM heavy chain polyclonal antibody as capture antibody (Vector Laboratories, Inc., Burlingame, CA). The chimeric protein was then detected with biotinylated polyclonal goat anti–human IgM heavy chain antibody (Vector Laboratories, Inc.), avidin–HRP (Pierce, Oud-Beijerland, The Netherlands), and O-phenylendiamine (0.125%, wt/ vol.; Sigma Chemical Co.) in 0.1 M citrate buffer, pH 4.5, as the substrate. E- or P-selectin/μ chimeric protein concentration was determined using purified L-selectin/μ chimera and purified human IgM as standards. Samples were run in triplicate at 1:500 to 1:5,000 dilutions. Under these conditions, a linear relationship was observed between signal intensity and protein concentration. Absorbance at 490 nm was measured using an ELISA reader (model MR 5000; Dynatech Laboratories, Inc., Chantilly, VA).

Immunofluorescence Analysis

Indirect immunofluorescence analysis was performed using suspended BAEC, which had been detached from plastic flasks with PBS/5 mM EDTA. After three washes in RPMI 1640/1% FCS medium, BAEC were incubated for 30 min at 4°C with L-selectin/μ or CD4/μ chimera. Chimeric protein binding to suspended endothelial cells was revealed using FITCconjugated rabbit anti–human IgM heavy chain (Dako, Glostrup, Denmark). Flow cytometry was performed using a cytofluorimeter (EPICS Profile; Coulter Corp., Hialeah, FL). Cells were gated by forward- and side-scatter signals. 5,000 cells were analyzed in each experiment.

Glycosaminoglycan Characterization

Endothelial cells were incubated with various enzymes for 45 min at 37°C in 25 μl RPMI 1640. Concentration curves were done for each enzyme. Optimal inhibition of L-selectin/μ binding was observed at the chosen enzyme concentrations. Heparinase I (Sigma Chemical Co.) was used at 600 mU, and heparitinase II (Seikagaku Corporation, Tokyo, Japan) was used at 4 mU. In other experiments, BAEC were incubated with chondroitinase ABC (200–800 mU; Sigma Chemical Co.) or hyaluronidase (200 mU; Sigma Chemical Co.). In experiments investigating the role of sialic acid, BAEC were incubated with Vibrio cholerae neuraminidase (750 mU/ml; Boehringer Mannheim) or Arthrobacter ureafaciens neuraminidase (200 mU/ml; Oxford Glycosystems, Ltd., Abingdon, UK). At this concentration, neuraminidase completely inhibited CSLEX-1 mAb binding to KG-1 cells treated with this neuraminidase (100 U/ml). The role of sulfate was evaluated by culturing trypsinized BAEC (5 μg/ml trypsin for 30 min at 37°C) for 24 h in RPMI 1640 medium/10% FCS in the presence of 10 mM sodium chlorate. In additional experiments, BAEC were cultured with cycloheximide (10 μg/ml) for 30 min before and during TNF-α treatment.

Statistical Analysis

Analysis of variance (ANOVA) and the Bonferroni multiple comparisons test were used to assess statistical significance between the different treatments versus control when three or more groups were analyzed; the Mann-Whitney test was used to compare the median of two unpaired groups, and the Wilcoxon signed rank test was used for paired groups. P values <0.05 were considered significant.

Role of L-Selectin in Mediating Monocyte Adhesion to Cytokine-activated Aortic Endothelium

Monocyte adhesion assays were performed at 4°C under rotation. In these conditions, where L-selectin shedding is minimal and CD18-mediated adhesion is inactive (50, 73, 74, 76), few monocytes attached to unactivated BAEC monolayers (84 ± 20 monocytes/field, mean ± SD, n = 6). When BAEC were activated for 8 h with TNF-α (100 U/ ml), a significant increase in monocyte adhesion was observed (four- to ninefold, n = 6). Thus, in the experiment illustrated in Fig. 1, the number of monocytes attached to BAEC increased from 94 ± 10 to 425 ± 33/field upon endothelium activation with TNF-α (Fig. 1, medium). The mechanism responsible for this observation was investigated with mAbs against L-selectin or VCAM-1. Cell binding inhibition studies revealed that monocyte adhesion to cytokine-activated BAEC monolayers was inhibited by 64 ± 18% (mean ± SD, n = 6, P < 0.005) when monocytes were pretreated with the adhesion-blocking mAb anti–LAM1-3 (Fig. 1) (73, 76). Cell adhesion was not significantly inhibited in experiments with anti–LAM1-10 (not illustrated) or anti–LAM1-11 mAbs (Fig. 1), which recognize nonfunctional domains of L-selectin. A role for VCAM-1 in mediating monocyte attachment to activated BAEC was demonstrated by the capacity of the anti– VCAM-1 mAb HAE-2 to inhibit monocyte adhesion by 38 ± 6% (mean ± SD, n = 3, P < 0.01) (Fig. 1). However, the results with anti–LAM1-3 indicate that L-selectin plays a predominant role in monocyte attachment to cytokine-activated arterial endothelium under nonstatic conditions.

The notion that L-selectin could play a major role in the attachment of monocytes to cytokine-activated arterial endothelium was evaluated further in experiments comparing the effect of L-selectin/μ and CD4/μ chimera on the monocyte-binding capacity of BAEC monolayers. Whereas monocyte binding was not inhibited by pretreatment of BAEC monolayers with CD4/μ (30 μg/ml), strong inhibition (56 ± 9%, n = 3, P < 0.005) was observed when monolayers were preincubated with L-selectin/μ (50 μg/ml) (Fig. 2).

The role of L-selectin in mediating monocyte primary adhesion was further examined in a parallel flow chamber at a shear stress of 1.8 dynes/cm² (70). All monocytes interacting with endothelial monolayers during the first 5 min of the experiment were counted. Most of these cells were rolling before being abruptly halted and becoming stably adherent or detaching. The inhibition of L-selectin with the function-blocking mAb anti–LAM1-3 reduced monocyte primary adhesion to 8-h TNF-α–activated endothelium by 78 ± 12% (mean ± SD, n = 4). Thus, after pretreatment with anti–LAM1-3 mAb, only 209 ± 44 monocytes/mm² (mean ± SD, n = 4) interacted with activated endothelium, whereas 970 ± 84/mm² interacting monocytes/mm2 were observed after pretreatment with the nonblocking anti–LAM1-11 mAb (not illustrated). Pretreatment of endothelial cells with L-selectin/μ similarly reduced monocyte primary adhesion by 65 ± 14% (mean ± SD, n = 3). In contrast, no inhibition was observed when endothelium was pretreated with the control chimeric protein CD4/μ. The number of stably adherent monocytes at the end of the 12-min flow experiments was strongly reduced by pretreating monocytes with the anti– LAM1-3 mAb (83 ± 8%; mean ± SD, n = 4) or endothelial cell monolayers with L-selectin/μ chimera (71 ± 9%; mean ± SD, n = 3). In the experiment illustrated in Fig. 3, 97 ± 30 (mean ± SD, n = 13) adherent monocytes/mm² were observed after monocyte preincubation with anti– LAM1-3, and 413 ± 56 monocytes/mm² were observed after pretreatment with anti–LAM1-11 mAb; in the same experiment, 130 ± 31 (mean ± SD, n = 13) monocytes/mm² adhered to activated endothelium pretreated with L-selectin/μ, whereas 380 ± 45 monocytes adhered to monolayers pretreated with CD4/μ (50 μg/ml).

Monocyte Adhesion to Cytokine-activated Aortic Endothelial Cells: Kinetic Analysis

Monocyte adhesion to BAEC was determined under rotation before and after 2, 4, 6, and 8 h of endothelial cell incubation with TNF-α (100 U/ml). A time-dependent increase in monocyte binding was observed up to 6 h after the addition of TNF-α (Fig. 4, solid circles). At ⩾2 h of activation, monocyte binding to BAEC was inhibited by 48 to 68% with anti–LAM1-3 mAb (Fig. 4, open circles). With unstimulated BAEC, the inhibition observed with monocytes pretreated with anti–LAM1-3 did not reach statistical significance.

Unstimulated and Cytokine-activated BAEC Express L-selectin Ligands

L-selectin ligand expression by suspended BAEC was detected by flow cytometry, L-selectin/μ being the probe and CD4/μ being the control. L-selectin/μ was found to bind to both unstimulated and cytokine-activated BAEC (Fig. 5, top, solid lines) whereas CD4/μ did not (Fig. 5, top, dotted lines). L-selectin/μ binding to BAEC was completely inhibited by the presence of 5 mM EDTA (Fig. 5, middle) or 100 μg/ml of function-blocking mAb anti–LAM1-3 or anti– LAM1-4, which react with epitopes located on the lectin domain of L-selectin (Fig. 5, bottom). These latter results demonstrate the calcium dependence of L-selectin binding to aortic ligands and the involvement of the L-selectin lectin domain in this reaction.

Because activation of aortic endothelium with TNF-α induced a progressive increase in L-selectin–dependent monocyte adhesion (Fig. 4), L-selectin ligand expression by BAEC was followed over a 24-h period of time. Surprisingly, unstimulated BAEC or BAEC activated by TNF-α (100 U/ml) for 2, 4, 6, 8, or 24 h were found to bind L-selectin/μ in a similar fashion (Fig. 6).

L-selectin Binding to BAEC: Different Ligand Characteristics on Unstimulated and Cytokine-activated Endothelial Cells

The role of proteoglycans in supporting L-selectin–endothelial interactions was investigated in experiments examining the effect of glycosidase or trypsin treatment on L-selectin binding to aortic endothelium. As illustrated in Fig. 7, L-selectin binding to unstimulated BAEC was not affected by hyaluronidase (bottom left) or chondroitinase ABC (middle right), whereas it was strongly inhibited by incubation with heparinase I (top right), heparinase I or III (not illustrated), and heparitinase II (middle left), and abrogated by cell exposure to trypsin (bottom right). Importantly, a quite different pattern was observed with BAEC activated by 8 h of incubation with TNF-α (100 U/ml) (Fig. 8). Although trypsin treatment completely inhibited the reaction (Fig. 8, bottom right), activated BAEC exposure to heparinase I, heparitinase II, or heparitinase III only had moderate inhibitory effects on L-selectin binding (Fig. 8, top right and middle). Thus, heparinase treatment induced a significantly higher decrease in L-selectin/μ binding to unactivated BAEC (mean percentage of decrease ± SD 42 ± 17%, n = 22) than to BAEC exposed for 8 h to TNF-α (26 ± 14%, n = 14, P = 0.005). As observed with unstimulated cells, hyaluronidase and chondroitinase did not inhibit L-selectin binding to activated BAEC (bottom left and middle right).

Heparan sulfate proteoglycans are highly sulfated molecules, and sulfate residues are important for the function of several selectin ligands (30, 32, 63, 68, 78). The role of sulfate residues in L-selectin–BAEC interactions was assessed by experiments using unactivated or TNF-α–activated BAEC cultured for 24 h in the presence of 10 mM sodium chlorate, an inhibitor of sulfate synthesis (7). As shown in Fig. 9, inhibition of sulfation inhibited most L-selectin binding to both unstimulated and cytokine-activated BAEC (bottom).

Cycloheximide treatment also strongly inhibited L-selectin binding, indicating that protein synthesis is required for ligand(s) expression by both unactivated and cytokine- activated endothelium (Fig. 9, middle).

Intact sialic acid residues are required for interactions between L-selectin and mucinlike glycoproteins such as GlyCAM-1, CD34, or PSGL-1. To assess whether sialic acid residues are involved in L-selectin binding to aortic endothelium, BAEC were pretreated for 45 min with V. cholerae (750 mU/ml) or A. ureafaciens neuraminidase (200 mU/ ml) before incubation with L-selectin/μ chimera (Fig. 9, Vibrio Cholerae). Endothelial cell exposure to neuraminidase did not significantly affect L-selectin/μ binding to unactivated BAEC. Thus, 63 ± 15% (n = 8) of BAEC treated with V. cholerae neuraminidase bound L-selectin/ μ, whereas 47 ± 26% (n = 8) of untreated cells bound the chimera. Similarly, L-selectin/μ binding to activated BAEC was not affected by neuraminidase. L-selectin/μ bound to 57 ± 19% (n = 6) of untreated cells and to 59 ± 16% (n = 6) of neuraminidase-treated cells. In contrast, monocyte exposure to V. cholerae neuraminidase (100 mU/ml) abolished L-selectin/μ binding to monocyte PSGL-1 (not illustrated) (78).

Role of Heparan Sulfates in Monocyte Adhesion to Cytokine-activated Endothelium

The role of heparan sulfates in supporting monocyte attachment to TNF-α–activated BAEC was studied by preincubating endothelial monolayers with heparinase I before monocyte addition. Adhesion assays performed under rotation after the addition of heparinase I indicated that heparan sulfates support monocyte attachment to 8-h TNF-α–activated BAEC. Monocyte adhesion to cytokineactivated aortic endothelium was reduced by 36 ± 11% (mean ± SD, n = 4, P < 0.01) using BAEC monolayers preexposed to heparinase I; BAEC pretreatment with V. cholerae neuraminidase (750 mU/ml, 45 min at 37°C) did not significantly inhibit monocyte binding (inhibition of L-selectin/μ binding −8 ± 6%, n = 3) (not illustrated). In control experiments in which monocytes were preincubated with anti–LAM1-3 mAb, monocyte attachment to TNF-α–activated BAEC monolayers was inhibited by 64 ± 18% (P < 0.005).

Additional experiments were performed to examine the contribution of heparan sulfate proteoglycans in mediating primary monocyte adhesion to activated endothelial monolayers under flow. Monocyte attachment was very significantly affected by the pretreatment of endothelial monolayers with heparinase I. At 1.8 dynes/cm², the total number of interacting monocytes (primary adhesion) during the first 5 min of the videotaped experiments was significantly reduced (P < 0.001). Thus, 304 ± 43 monocytes/ mm² (mean ± SD, n = 3) interacted with activated endothelial monolayers during this time, whereas 854 ± 72 interacting monocytes/mm² (mean ± SD, n = 3) were observed with untreated endothelium. The number of stably adherent monocytes was also considerably inhibited by the pretreatment of activated endothelium with heparinase I. Adherent monocytes were counted during the last 2 min of the 12-min experiments. Stable monocyte adhesion was reduced by 88 ± 6% (mean ± SD, n = 4, P < 0.001) after the pretreatment of endothelial monolayers with heparinase I (Fig. 10). Similar inhibition was obtained by treating monocytes with the function-blocking mAb anti–LAM1-3 (83 ± 8%), whereas the control anti–L-selectin mAb anti–LAM1-11 had no significant inhibitory effect.