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Originally published online as doi:10.1189/jlb.1202593 on October 2, 2003

Published online before print October 2, 2003
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(Journal of Leukocyte Biology. 2003;74:1035-1044.)
© 2003 by Society for Leukocyte Biology

In vitro model for hematopoietic progenitor cell homing reveals endothelial heparan sulfate proteoglycans as direct adhesive ligands

Tanja Netelenbos*, Jacob van den Born{dagger}, Floortje L. Kessler*, Sonja Zweegman*, Peter C. Huijgens* and Angelika M. Dräger*,1

Departments of
* Hematology and
{dagger} Molecular Cell Biology, Vrije Universiteit Medical Center, Amsterdam, The Netherlands

1Correspondence: Department of Hematology, Vrije Universiteit Medical Center, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands. E-mail: A.Draeger{at}vumc.nl


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ABSTRACT
 
Proteoglycans (PGs) play a dominant role within the bone marrow (BM), but their role in homing of transplanted hematopoietic progenitor cells (HPC) is unknown. In this study, the role of heparan sulfate (HS) PGs on BM endothelium as adhesive structures was investigated. HPC (primary CD34+ cells and cell line KG-1a) were able to bind fractionated heparin, which could be competed by highly sulfated heparin/HS-glycosaminoglycans (GAGs). Under flow conditions, HPC adhered to immobilized heparin after rolling over E-selectin. Rolling of KG-1a on BM endothelial cell (EC) line 4LHBMEC was completely E selectin-dependent. Addition of heparin/HS-GAGs, endothelial treatment with chlorate, or anti-HS all partially inhibited firm adhesion. Moreover, enzymatic removal of endothelial HS-GAGs reduced initial adhesion. Finally, HPC-bound PGs isolated from 4LHBMEC, which was largely inhibited by enzymatic HS-degradation. In summary, we identified sulfated structures on BM endothelium, most likely HSPGs, as a novel class of glycoconjugates involved in the multistep homing cascade of HPC.

Key Words: glycosaminoglycans • adhesion • bone marrow endothelial cells


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INTRODUCTION
 
Homing of hematopoietic progenitor cells (HPC) to the bone marrow (BM) after peripheral blood stem-cell transplantations requires multiple, successive interactions between HPC and the vessel wall of the BM microvasculature [1 2 3 4 ]. Receptors and ligands on HPC and BM endothelial cells (BMEC), the cells lining the BM vessels, are thus the key players in the initial homing steps. In a comparable way as leukocyte extravasation in inflammation or lymphocyte homing, the main successive steps are thought to consist of rolling, activation, firm adhesion, and transendothelial migration [5 , 6 ]. A better knowledge of the molecular interactions responsible for this homing process could contribute to improved engraftment of intravenously injected HPC. Most likely, the endothelial participants in the tethering and rolling steps of HPC are E-selectin and vascular cell adhesion molecule-1 (VCAM-1), as these adhesion molecules are constitutively expressed on BMEC and have been shown together with P-selectin to be involved in in vivo rolling of HPC [7 , 8 ]. CD44 on HPC seems to be an important ligand for endothelial E-selectin [9 ]. The chemoattractant stromal cell-derived factor-1 (SDF-1), which up to date is the only chemokine described to be present on BMEC in vivo [10 , 11 ], is able to activate integrins and mediate arrest of HPC [11 , 12 ]. The firm adhesion step is mediated by ß1 and ß2 integrins on HPC via binding to their endothelial counter-receptors, respectively, VCAM-1, and intercellular adhesion molecule-1 (ICAM-1) [11 , 13 ].

Recently, we have reported the presence of new candidates on BMEC, which could participate in the adhesion cascade between HPC and BMEC, i.e., cell-surface heparan sulfate proteoglycans (HSPGs) [14 ]. PGs are expressed throughout the entire body, where they exist as membrane-associated molecules or as secreted components of the extracellular matrix (ECM) [15 ]. PGs are glycoconjugates consisting of a core protein with linear chains of glycosaminoglycans (GAGs) covalently attached. Of all GAGs, HS-GAGs show the largest variability in structure, and they can bind many proteins, such as chemokines, growth factors, proteases, protease inhibitors, lipoproteins, and adhesion molecules [16 ].

We have previously shown that cell-derived HS-GAGs from the BMEC cell line 4LHBMEC contain higher sulfated domains compared with similar GAGs from human umbilical vein EC [14 ]. These HS-GAGs as well as chondroitin sulfate/dermatan sulfate (CS/DS)-GAGs are able to bind and present SDF-1 [14 , 17 , 18 ]. Cell-surface HSPGs could act as direct recognition receptors for adhesive structures on HPC. HSPGs have been shown to be able to bind adhesion molecules such as L-selectin, P-selectin, CD45, CD11b, and neural cell adhesion molecule-1 [19 20 21 22 23 ]. With regard to the interaction between HPC and BM stroma, there is increasing evidence that stromal HS is involved in HPC binding and that this binding plays a role in hematopoiesis [22 , 24 25 26 27 ]. However, to date, it has not been reported whether sulfated structures on BMEC like HSPGs are involved in HPC binding.

This work was performed to study the role of cell-surface HSPGs on BMEC as mediators of direct adhesion toward HPC in a flow-adhesion model system mimicking the first homing step. Our results demonstrate a novel function for this type of glycoconjugate in HPC homing.


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MATERIALS AND METHODS
 
Cell cultures
4LHBMEC, a human BMEC cell line, was cultured as described [14 ]. The CD34+ myeloblastic cell line KG-1a (CCL-246.1, American Type Culture Collection, Manassas, VA) was used as a model HPC line. Cells were maintained in RPMI 1640 (Gibco, Grand Island, NY), supplemented with 10% fetal calf serum (Gibco).

Isolation of CD34+ cells
Mobilized peripheral blood CD34+ cells were isolated from fresh or thawed leucapheresis material from patients with different hematological malignancies after informed consent. For isolation, the CD34 isolation kit with the Mini-Macs or the Auto-Macs system (Miltenyi, Bergisch Gladbach, Germany) was used. Purity was always more than 90%, as determined by flow cytometry using a CD34 antibody (HPCA2, Becton Dickinson, Mountain View, CA).

Monoclonal antibodies (mAb) and GAGs
mAb against HSPGs used were antisyndecan-1 (clone B-B4, Serotec, Oxford, UK), antisyndecan-3 (clone 1C7) [28 ], antisyndecan-4 (clone 8G3), and antiglypican-1 (clone S1) [29 ], all kindly provided by Guido David (Center of Human Genetics, Leuven, Belgium), and CD44v3 (clone 3G5, R&D Systems, Abingdon, UK). Anti-HS antibodies used were JM403 [30 ], 10E4 [31 ], and 3G10 [31 ] (Seikagaku, Tokyo, Japan). Antibodies against adhesion molecules used were anti-E-selectin [clones epithelial-derived neutrophil-activating factor 1 (ENA1) and ENA2, Sanbio, Uden, The Netherlands], anti-ICAM-1 (clone 6.5B5, Dako, Glostrup, Denmark), anti-VCAM-1 (clone 1g11, Immunotech, Marseille, France), and anti L-selectin (clone Dreg56, PharMingen, San Diego, CA). The following GAGs were used in the adhesion studies: heparin (Bufa Chemie, Uitgeest, The Netherlands, or Sigma Chemical Co., St. Louis, MO), low molecular weight heparin (LMWH), dalteparin (Fragmin, Pharmacia, Peapack, NJ), HS bovine intestinal mucosa (HSIM), HS bovine kidney (HSBK), DS, CS, hyaluronic acid (HA; all from Sigma Chemical Co.), HS from human aorta (generous gift from Dr. Marco Salmivirta, Dept. of Medical Biochemistry and Microbiology, University of Uppsala, Sweden), dextran (Dx; Pharmacia, Uppsala, Sweden), Dx sulfate (DxS; Sigma Chemical Co.), K5, and O-sulfated K5 (kindly provided by Dr. Gijs van Dedem, Organon Corp., Oss, The Netherlands). Modified heparins used were N-desulfated, N-acetylated (O-sulfated heparin), completely desulfated, N-sulfated (N-sulfated heparin), completely desulfated, and N-acetylated heparin (desulfated heparin; all from Seikagaku).

Binding experiments of HPC to LMWH-fluorescein isothiocyanate (FITC) and competition with GAGs
KG-1a or CD34+ cells were washed in phosphate-buffered saline (PBS)/bovine serum albumin (BSA) 0.1%, and 5 x 104 cells were incubated with 0.125 µg FITC-labeled LMWH-FITC [32 ] (Loxo, Dossenheim, Germany) in 50 µl HEPES buffer (20 mM HEPES, 132 mM NaCl, 6 mM KCl, 1.2 mM K2HPO4, 1 mM MgSO4, 5 mM glucose, 1 mM CaCl2 at pH 7.4)/BSA 0.1% for 20 min at 37°C. Cells were washed in HEPES/BSA 0.1%, and fluorescence was measured on a FACScalibur flow cytometer (Becton Dickinson, San Jose, CA). Cells were similarly stained with 10% 7-amino-actinomycin (Via-ProbeTM, PharMingen) to exclude dead cells. To compete for binding of LMWH-FITC, various soluble GAGs were present during the incubation at a concentration of 200 µg/ml. Data analysis was performed using Cellquest (Becton Dickinson, San Jose). The mean fluorescence index (MFI) was calculated as (mean fluorescence of bound LMWH-FITC) - (mean fluorescence of control)/(mean fluorescence of control). The percentage inhibition of binding was calculated by dividing the MFI of cells bound to LMWH-FITC in the presence of GAGs by the MFI of cells bound to LMWH-FITC without GAGs multiplied by 100.

Flow perfusion adhesion experiments
Rolling and adhesion of HPC were measured under conditions of flow in a parallel plate flow perfusion chamber with well-defined rheological characteristics [33 , 34 ]. Slides coated with adhesive components or EC were positioned in the flow chamber. Plastic coverslips (Thermanox, Nunc, Rochester, NY) were coated with PBS/BSA 1%, heparin-albumin (Sigma Chemical Co.), E-selectin chimeras (2 µg/ml), or both. E-selectin chimeras with a human Fc fragment from immunoglobulin G (IgG) were a kind gift of Dr. Susan Watson (NeXstar Pharmaceuticals, Boulder, CO) [35 ]. Before flow experiments, nonspecific binding sites on Thermanox slides were blocked for 2 h at room temperature with PBS/BSA 1%. EC were cultured to confluence in 4 days on gelatin-coated glass slides (18x18 mm) for use in the flow chamber. A day before perfusions, the culture medium was replaced by EC culture medium without EC growth factor and heparin (flow medium). EC were stimulated with tumor necrosis factor {alpha} (TNF-{alpha}; 500 U/ml; Boehringer, Mannheim, Germany) 4–6 h before perfusions. In most perfusion experiments, KG-1a cells were used, which have a very similar phenotype as HPC but lack expression of CXCR4, the high-affinity receptor of SDF-1 [36 , 37 ]. To study the possible role of chemokine receptors, KG-1a cells were pretreated with pertussis toxin (Sigma Chemical Co.) for 2 h at 37°C at 200 ng/ml. A similar incubation procedure with KG-1v (a myeloblastic cell line with high CXCR4 expression) inhibited almost all migration toward the chemoattractant SDF-1 (not shown). KG-1a cells were incubated with 50 µg/ml anti-L-selectin before perfusion experiments to study L-selectin interactions.

Cells were aspirated from a reservoir through the perfusion chamber with a withdrawal syringe pump (sp210iw, World Precision Instruments, Sarasota, FL) at shear stress as indicated. The perfusion chamber was mounted on a microscope stage (Axioskop, Zeiss, Germany) equipped with a video camera (Sony, Tokyo, Japan) coupled to a VHS video recorder (VCR SVT-S300P, Sony). After 1 min perfusion with HEPES flow buffer, cells entered the perfusion chamber and started to interact with the substrate or endothelium (t=0). Perfusion experiments were performed for 5 min. After 5 min perfusion, the cell supply was stopped and exchanged for HEPES flow buffer to remove nonbound or loosely attached cells. Video images were evaluated afterwards, and cell–cell interactions were analyzed at 2–3 min of perfusion for the number of rolling and adhered cells and the rolling velocity per cell, from three images of 20 s each. The adhesive interactions between 2 and 3 min perfusion were defined as initial adhesion. The total number of firmly adhered cells was measured after 5 min perfusion from a minimum of 25 randomized, high-power fields. Recorded images were digitalized and analyzed with the software program Optimas 6.1 (Media Cybernetics Systems, Silver Spring, MD). Experiments were performed in triplicate on at least three separate occasions.

Pretreatment of 4LHBMEC
Confluent layers of EC were treated for 24 h with 30 or 60 mM sodium chlorate (Sigma Chemical Co.) in flow medium. Sodium chlorate inhibits the formation of 3'-phosphoadenosine 5'-phosphosulfate, which is the sulfate donor needed for GAG sulfation [16 , 38 ]. Successful treatment was confirmed by flow cytometry using JM403 antibody, which recognizes N-unsubstituted glucosamine units.

Cleavage of HS-GAGs on 4LHBMEC was achieved by treatment with a cocktail of 10 mU/ml each of the following enzymes: heparinase, heparitinase I, and heparitinase II (Seikagaku) for 1 h in Medium 199 with 10 mM HEPES and 2 mM CaCl2. Enzyme activity of each batch was checked before experiments by measuring absorbance of dimethylmethylene blue at 510 nm after incubation with HSBK or heparin, with or without enzymes [39 ]. To check the efficiency of heparinase treatment on EC, an immunofluorescence staining was performed on confluent layers of 4LHBMEC with the 3G10 antibody to detect cleaved HS stubs [31 ] or with the 10E4 antibody to detect disappearance of staining (not shown). To cleave CS/DS chains or HA, EC were treated with 1 U/ml chondroitinase antibody-binding capacity (ABC; Sigma Chemical Co.) or 20 U/ml hyaluronidase (streptococcal hyaluronidase, ICN, Zoetermeer, the Netherlands) for 60 min at 37°C, respectively. Chondroitinase ABC activity was checked by measuring absorbance at 232 nm CS and DS in the presence or absence of the enzyme [40 ]. Hyaluronidase treatment was evidenced by complete loss of the binding of biotinylated hyaluronan-binding protein to primary brain EC. To digest sialic acids, 4LHBMEC were pretreated for 60 min with sialidase (0.2 U/ml; acylneuraminyl hydrolase, Vibrio Cholerae, Behring, Marburg, Germany). Enzyme activity of sialidase was confirmed by flow cytometry, showing disappearance of expression of the HECA-452 epitope on KG-1a cells after similar sialidase treatment (not shown).

E-selectin on 4LHBMEC was blocked with anti-E-selectin (ENA2, 50 µg/ml, 25 min), and ICAM-1 and VCAM-1 adhesion molecules were simultaneously blocked (clone 6.5B5 and 1 g11, 50 µg/ml each, 25 min). Furthermore, the 10E4 antibody (50 µg/ml) was used to block HSPGs.

Binding experiments using isolated 3H-PGs
Total radiolabeled PGs, derived from 4LHBMEC as described [14 ], were treated or not with a cocktail of heparinases (10 mU/ml) overnight at 37°C to degrade HS-GAGs. Conversely, total PGs were treated with chondroitinase ABC (1 U/ml) to degrade CS/DS-GAGs. The remaining CS/DSPGs and HSPGs, respectively (20,000 cpm per situation), were incubated in the presence of the degradation products, with 0.2 x 106 KG-1a and CD34+ cells in a total volume of 100 µl during 20 min at 37°C in HEPES buffer/BSA 0.1%. Before incubations, nonspecific-binding sites on the tubes were blocked with PBS/BSA 1%. After the incubation, cells were washed twice, and bound radioactivity was counted.

L-selectin enzyme-linked immunosorbent assay (ELISA)
Binding of L-selectin to HS was studied using an ELISA method. HS from human aorta (50 µg/ml) in 50 mM Tris buffer containing 5 mM Ca++ and 5 mM Mg++ was coated in maxisorp ELISA plates (Nunc). All washing steps occurred in the same Tris buffer. Blocking step was performed with a 5% milk-powder solution. An L-selectin-IgG1 chimera (5 µg/ml; generous gift from Dr. Steven Rosen, Dept. of Anatomy, University of California, San Francisco) together with a concentration range of HSIM (Sigma Chemical Co.) or heparin (0.04–10 µg/ml in a four-step dilution) were added. Biotin-labeled anti-human IgG1 and streptavidin-peroxidase and orthophenylenediamine as the substrate detected HS-bound L-selectin.

Statistics
All data are expressed as mean ± SEM. Differences between interventions compared with control situations were assessed with the Mann-Whitney U test with SPSS software (SPSS Inc., Chicago, IL).


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RESULTS
 
HPC binding to fractionated heparin is inhibited by high-sulfated GAGs
To study the presence of heparin-binding sites on HPC, KG-1a and CD34+ progenitor cells were incubated with a FITC-labeled LMWH. As can be seen in Figure 1A , both cell types bound LMWH-FITC. In most of the CD34+ samples studied (five different donors), a small shoulder with high fluorescence was observed, indicating a subpopulation with higher heparin-binding ability. Binding of LMWH-FITC to KG-1a cells was inhibited for more than 60% in the presence of heparin (Fig. 1B) . Fractionated heparin (dalteparin) and HSIM equally reduced binding of LMWH-FITC, comparable with the effect of heparin. HSBK and CS showed no significant inhibition, whereas DS showed some inhibition. The modified heparins, O-sulfated, N-sulfated, and desulfated heparin, did not exert any effect on the binding of LMWH-FITC to KG-1a. Competition experiments with CD34+ cells showed a comparable pattern. Less inhibition was found in the presence of heparin and dalteparin compared with KG-1a, however still significant and comparable with inhibition with HSIM. The entire CD34+ population showed a reduced fluorescence intensity after heparin addition, and no shift of subpopulations was observed (not shown). In contrast to the findings with KG-1a, inhibition of binding was obtained with HSBK and O-sulfated heparin. The sulfated Dx polysaccharide reduced binding to both cell types for more than 50%. Unsulfated Dx did not show any inhibition, indicating that sulfation played an important role in competing with LMWH-FITC. These results show that HPC have heparin-binding receptors and that high sulfated heparinoid structures are effective in competing with LMWH-FITC binding to HPC, suggesting preferential binding to high-sulfated, heparin-like structures. The differences found between KG-1a and CD34+ cells suggest variation in type, number, and/or affinity of heparin-binding receptors.



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  		Figure 1. Binding LMWH to HPC and competition with GAGs. (A) KG-1a or CD34+ cells were incubated with LMWH-FITC (solid lines), and fluorescence was measured by flow cytometry. Dotted lines represent control cells. Shown are representative histograms from five separate experiments. (B) Competition of binding with GAGs to KG-1a (open bars) or CD34+ cells (solid bars) was achieved by incubating LMWH-FITC in the presence of various GAGs (200 µg/ml): heparin, O-sulfated (O-S) heparin, N-sulfated (N-S) heparin, desulfated (De-S) heparin, dalteparin, HSIM, HSBK, DS, CS, DxS, and Dx. Depicted is percentage-binding compared with control situation (100% binding). Experiments were performed in quadruplicate at four separate occasions. *, P < 0.05, compared with control; #, P < 0.05, for differences between KG-1a and CD34+ cells.

HPC roll over E-selectin substrates and adhere to heparin under flow conditions
Having shown that HPC can bind soluble heparin, we wanted to know whether HPC were able to bind immobilized heparin under flow conditions. Therefore, plastic coverslips were coated with heparin–albumin, and adhesion of KG-1a cells was measured. At a physiological shear stress of 1.0 dyne/cm2 [4 ], hardly any adhesion of KG-1a cells to heparin was seen (Fig. 2A ). However, below 0.25 dyne/cm2, adhesion increased significantly. No rolling was observed over heparin (not shown). In vivo, HPC can be captured by tethering and rolling over E-selectin amongst other molecules [8 ]. Coated slides with E-selectin induced rolling of KG-1a and primary CD34+ cells (median velocity 17.7 and 15.5 µm/s, respectively; Fig. 2B ). Co-coating of heparin–albumin with E-selectin chimeras, however, reduced the velocity of cells interacting with the substrates and thereby increased the number of arrested cells (median velocity 0.4 and 0.6 µm/s, respectively; Fig. 2B ). Thus, HPC can roll over E-selectin and subsequently adhere to heparin.



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  		Figure 2. KG-1a and CD34+ cells roll on E-selectin and adhere to heparin at physiological shear stress. (A) KG-1a cells were perfused over plastic coverslips coated with PBS/BSA 1% ({triangleup}) or heparin–albumin ({circ}) at different shear stress. Depicted is the number of firmly adhered cells in cells/mm2 (n=6). (B) KG-1a and CD34+ cells were perfused over slides coated with E-selectin chimeras ({triangleup}) or E-selectin chimeras and heparin–albumin ({circ}) at a physiological shear stress of 1.0 dyne/cm2. Shown is the velocity of 98 cells interacting with the substrates between 2 and 3 min perfusion. Cells were randomly chosen from representative flow experiments (out of four separate experiments), ranked from lowest up to highest velocity. *, P < 0.0001, for differences between E-selectin and E-selectin and heparin co-coatings.

TNF-{alpha} stimulation induces up-regulation of adhesion molecules but does not change HS expression on 4LHBMEC
To study the interaction of HPC with HSPGs in a more physiological assay, we performed flow-adhesion experiments with slides overlaid with the BMEC cell line 4LHBMEC. As it has been shown that BMEC in vivo constitutively express E-selectin and VCAM-1 [7 ], we induced these adhesion molecules on 4LHBMEC by TNF-{alpha} to mimic this situation. To monitor whether HS expression would change after 4 h TNF-{alpha} stimulation, EDTA-detached 4LHBMEC were incubated with two anti-HS antibodies: 10E4, which is directed against an epitope in the intact polysaccharide HS chain, and the 3G10 antibody, which recognizes HS stubs exposed after cleavage of HS [31 ] and thus represents the total amount of HSPGs on the cell surface. Binding of both antibodies did not change after activation of the endothelium with TNF-{alpha} (not shown).

We next studied rolling and firm arrest of the HPC cell line KG-1a on 4LHBMEC under flow conditions. As 4LHBMEC produce SDF-1, and KG1a cells lack CXCR4 expression [17 ], we were able to directly study the interactions with endothelial HSPGs without interference of SDF-1-mediated arrest. At a shear stress of 1.0 dyne/cm2, no rolling (not shown) and hardly any adhesion were seen in the absence of E-selectin expression. However, stimulated with TNF-{alpha}, the percentage of rolling cells between 2 and 3 min perfusion increased up to 47 ± 2% (n=53; not shown), and a high amount of cells (327±28 cells/mm2) firmly arrested on 4LHBMEC (Fig. 3 ). Blocking E-selectin almost completely prevented rolling, resulting in an inhibition of firm adhesion of 91 ± 3%. The integrin ligands ICAM-1 and VCAM-1 accounted for 40% in firm arrest of HPC, as determined by combined antibody-blocking studies (Fig. 3) . These results clearly indicate the involvement of additional structures in HPC firm adhesion.



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  		Figure 3. Adhesion of KG-1a cells to 4LHBMEC is largely dependent on E-selectin and partially on ICAM-1/VCAM-1. KG-1a cells were allowed to roll and adhere under flow conditions on 4LHBMEC, stimulated or not with TNF-{alpha} (4 h). Adhesion molecules on 4LHBMEC were blocked with antibodies against E-selectin (E-sel) or against ICAM-1 and VCAM-1. Shown is the number of firmly adhered cells after 5 min perfusion (cells/mm2; n=3–5). *, P = 0.012; #, P = 0.045, compared with adhesion on stimulated 4LHBMEC without blocking.

High-sulfated HS-like GAGs inhibit the interaction between KG-1a and 4LHBMEC under flow conditions
To investigate the involvement of GAGs on 4LHBMEC in binding of KG1a, we performed competition studies. Incubation of KG-1a cells with heparin resulted in a dose-dependent, inhibitory effect on the number of firmly adhered KG-1a cells to 4LHBMEC (Fig. 4A ). At a concentration of 50 µg/ml heparin, 91% inhibition of adhesion compared with control perfusions was found. Other GAGs were less effective in inhibiting adhesion: dalteparin, 68%; HSIM, 53%; HSBK, 41%; and DS, 35% (Fig. 4B) . CS and HA had no effect. To further explore the role of sulfation, KG-1a cells were incubated with the Escherichia coli K5 capsular polysaccharide, which has an identical composition as unsulfated HS chains [41 ], or with chemically O-sulfated K5. Only the O-sulfated K5 caused inhibition of adhesion of KG-1a to 4LHBMEC (Fig. 4B) . Furthermore, the polysaccharide DxS resulted in strong inhibition, whereas unsulfated Dx did not. These experiments emphasize the importance of sulfate groups for the observed antiadhesive activity. The inhibition of binding of HPC to 4LHBMEC by competing with highly sulfated polysaccharides corresponds to the results of the LMWH-FITC-binding study, suggesting that sulfated structures on 4LHBMEC, probably HSPGs, mediate binding of HPC under flow conditions. To distinguish between the effects of GAGs on rolling interactions and arrest, rolling analyses were performed for KG-1a cells incubated with heparin, dalteparin, and HSIM. Heparin and dalteparin inhibited rolling of KG-1a cells with 56% and 33%, respectively (Fig. 4C) . However, HSIM did not significantly inhibit rolling. To exclude that GAGs caused the inhibition of rolling and adhesion by binding to the endothelium instead of the HPC, slides with 4LHBMEC were pretreated with heparin (250 µg/ml; 60 min) and after washing, subjected to flow experiments. Preincubation with heparin did not inhibit binding of KG-1a (114±15% adhesion compared with control; n=6; not shown). Therefore, it can be concluded that heparins inhibit rolling and firm adhesion, whereas HS-GAGs inhibit firm adhesion of KG-1a cells to 4LBMEC by binding to receptors on KG-1a cells.



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  		Figure 4. Inhibiting effect of high-sulfated GAGs on the adhesion between KG-1a and 4LHBMEC under flow conditions. (A) KG-1a cells were incubated with different concentrations of heparin (2–250 µg/ml) for 60 min and perfused over TNF-{alpha}-stimulated 4LHBMEC. Depicted is percentage inhibition of firm adhesion of KG-1a cells compared with control situation (n=3). (B) KG-1a cells were preincubated with various GAGs or polysaccharides for 1 h: heparin, dalteparin (50 µg/ml), HSIM, HSBK, DS, CS, HA, capsular polysaccharide (K5), O-sulfated K5 (K5-OS), Dx, and DxS (all 200 µg/ml). Depicted is percentage firm adhesion after 5 min perfusion compared with control situation (100% adhesion; n=3). *, P = 0.037, compared with control. (C). Depicted is the number of rolling KG-1a cells pretreated with heparin, dalteparin (50 µg/ml), or HSIM (200 µg/ml) compared with control situation (100% rolling; n=3). *, P = 0.037, compared with control.

Sulfated structures on 4LHBMEC are involved in adhesion of HPC under flow conditions
To study whether sulfated structures on 4LHBMEC were involved in adhesion under flow conditions, confluent cultures of 4LHBMEC were pretreated with sulfate-inhibitor sodium chlorate. To check for successful chlorate treatment, cells were detached from culture plates with EDTA and stained with JM403. When sulfate is not allowed to incorporate into HS, copies of the JM403 epitope are increased [30 ]. Figure 5 shows that JM403 expression was dose-dependently increased upon chlorate treatment. At the concentrations used, cell growth and viability of the cells were not affected, as determined by trypan blue staining (not shown). Addition of 15 mM sodium sulfate (to sulfate already present in the serum-containing medium) together with 60 mM sodium chlorate prevented the chlorate-induced increase in JM403 expression (Fig. 5) , indicating that sodium sulfate efficiently competed with sodium chlorate. Sodium chlorate reduced rolling and initially adherent cells by 26 ± 17% (n=5; P=0.005) and 48 ± 11%, respectively (P=0.005). Treating 4LHBMEC with 30 mM sodium chlorate, firm adhesion of KG-1a was inhibited significantly with 14%; whereas after 60 mM sodium chlorate treatment, this inhibitory effect was enhanced to 41% inhibition (Fig. 5) . The reduction in firm adhesion by 60 mM sodium chlorate could be completely abolished by simultaneous addition of 15 mM sodium sulfate, indicating no nonspecific chlorate effect on the EC influencing adhesion of HPC. Chlorate treatment did not induce changes in TNF-{alpha}-mediated E-selectin, VCAM-1, or ICAM-1 expression as measured by flow cytometry (not shown). To study the role of Gi protein-coupled signaling downstream chemokine receptors, KG-1a cells were pretreated with pertussis toxin before adhesion experiments. This did not result in a significant change of firm adhesion (16±17%; n=4).



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  		Figure 5. Effect of sodium chlorate treatment of 4LHBMEC on JM403 expression and adhesion of KG-1a cells under flow conditions. Confluent cultures of 4LHBMEC were treated with 30 or 60 mM sodium chlorate for 24 h to inhibit incorporation of sulfate. Sodium sulfate was added to compete with sodium chlorate. EC were detached with EDTA and stained with the anti-HS antibody JM403, recognizing N-unsubstituted glucosamine or IgM isotype control. As secondary antibody, phycoerythrin-conjugated anti-IgM was used, and cells were measured by flow cytometry. MFI as explained in Materials and Methods (•; MFI, right, y-axis; n=4–6). *, P = 0.011; #, P = 0.006, compared with control situation. KG-1a cells were perfused over TNF-{alpha}-stimulated 4LHBMEC, treated or not with sodium chlorate and/or sodium sulfate. Bars depict percentage inhibition of firm adhesion after 5 min perfusion compared with control situation (no inhibition, left, y-axis; n=3–5). **, P = 0.014; ##, P= 0.005, compared with control situation.

Endothelial HS and not CS/DS-GAGs, HA, or sialic acids plays a role in adhesion of HPC to 4LHBMEC under flow conditions
To determine which kind of sulfated structures on 4LHBMEC were involved in adhesion of KG-1a cells, endothelial monolayers were pretreated with specific GAG-degrading enzymes. A cocktail of heparinases (10 mU/ml) inhibited the number of adhering cells interacting with 4LHBMEC by 22% between 2 and 3 min of perfusion (n=4–6; P=0.04), indicating involvement of HSPGs. Rolling was not inhibited significantly (not shown). After 5 min perfusion, no significant difference in firm adhesion compared with the control was found. To alternatively study the role of endothelial HSPGs, we covered the endothelium with anti-HS antibody 10E4 (50 µg/ml) and found an inhibition of firm adhesion of 26 ± 6% (n=4; P=0.014).

Chondroitinase ABC (1 U/ml) and hyaluronidase (20 U/ml) treatment did not inhibit rolling or firm adhesion (respectively, -10±31 and 5±3% inhibition for chondroitinase and -34±57 and 4±24% inhibition for hyaluronidase). Besides GAGs, other sulfated structures on EC could be involved in adhesive events, such as sulfated sialyl LewisX. To determine the role of selectin-sialyl LewisX interactions in our model system, 4LHBMEC layers were treated with sialidase. Surprisingly, this treatment resulted in a threefold increase of initial adhesion and an increase in firm adhesion compared with the control situation with 50 ± 7% (n=5; P=0.005). Therefore, the results indicate that endothelial HSPGs but not CS/DSPGs, HA, or sialylated structures were involved in adhesion of HPC.

HPC bind isolated HSPGs and not CS/DSPGs from 4LHBMEC
As our model system using whole EC layers under flow conditions is rather complex as a result of the presence of multiple ligand-receptor pairs, we also performed binding experiments of HPC with isolated endothelial PGs from [3H]glucosamine-labeled cultures of 4LHBMEC. These PGs consist of HSPGs and CS/DSPGs in a ratio of ~1:1 [14 ]. Isolated PGs were untreated or treated with HS or CS/DS-degrading enzymes and subsequently incubated with HPC. KG-1a cells and isolated CD34+ cells could bind PGs (1.3–15.3% and 3.6–30.3% of input, respectively; and Fig. 6 ). After chondroitinase ABC treatment, KG-1a cells were still able to bind; whereas after heparinases treatment, binding to the remaining PGs was reduced to 39% (Fig. 6) . These experiments show that HPC bind BM endothelial-derived HSPGs. Candidate HSPGs involved in the binding of HPC may be syndecan-1, glypican-1, or HS-bearing CD44 (CD44v3), as antibodies directed against these HS core proteins bound to 4LHBMEC, as determined by flow cytometry (Table 1 ).



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  		Figure 6. Binding of KG-1a to isolated BM endothelial PGs treated with GAG-degrading enzymes. PGs were isolated from 4LHBMEC cultures labeled with [3H]glucosamine and were incubated with buffer alone (untreated), with HS-degrading enzymes (heparinases), or with CS/DS-degrading enzymes (chondroitinase ABC; 20,000 cpm per situation). The remaining PGs with degraded GAGs were subsequently incubated with 0.2 x 106 KG-1a cells. After washing the cells twice, bound radioactivity was counted. Depicted is percentage of binding of PGs to KG-1a after the various pretreatments compared with untreated PGs (100% binding). Experiments were performed in duplicate at five separate occasions. *, P = 0.005, compared with control situation.


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  		Table 1. Expression of HSPGs on 4LHBMEC

L-selectin binds HS and is involved in firm adhesion of KG-1a to 4LHBMEC
To study the role of L-selectin as a possible ligand for endothelial HSPGs, binding of L-selectin to isolated HS was studied. Using an ELISA method, it was shown that L-selectin chimeras bound to vessel wall HS. At a concentration of 5 µg/ml L-selectin–IgG, an ELISA signal of ~1.5 was found, which was completely absent by omission of the HS coating. This binding could be inhibited with HSIM and even stronger with heparin (Fig. 7A ) as fluid-phase competitors. In the flow-adhesion model using TNF-{alpha}-stimulated 4LHBMEC layers and KG-1a cells incubated with anti-L-selectin, an inhibition of firm adhesion of 27% was found compared with isotype-treated controls (Fig. 7B) . These findings suggest L-selectin as a possible HPC ligand that mediates the interaction with endothelial HSPGs.



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  		Figure 7. L-selectin binds HS and is involved in firm adhesion of KG-1a to 4LHBMEC. (A) ELISA plates were coated with HS from aorta origin, and L-selectin chimeras were added together with HSIM or heparin. Binding was determined as described in Materials and Methods. Depicted is percentage of inhibition of binding for heparin ({circ}) and HSIM ({triangleup}). (B) KG-1a cells were incubated with anti-L-selectin antibodies or IgG1 as a control and perfused over TNF-{alpha}-stimulated 4LHBMEC. Depicted is percentage firm adhesion of KG-1a cells (n=6). *, P = 0.002, compared with control situation.


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DISCUSSION
 
In this article, we report that HPC have heparin/HS-binding receptors, which are involved in adhesion to BMEC via recognition of endothelial-sulfated structures, most likely HSPGs, under conditions mimicking in vivo blood flow in BM microvessels. This study identifies endothelial HSPGs as a novel class of glycoconjugates involved in the multistep homing cascade of HPC.

We found four lines of evidence that HPC contain heparin/HS-binding receptors, which are involved in attachment to BM endothelium. First, HPC (KG-1a and leucapheresis-derived CD34+ cells) were shown to bind a FITC-labeled, fractionated heparin. This binding could be competed with high-sulfated polysaccharides. Second, KG-1a and CD34+ cells could bind immobilized heparin albumin under flow conditions after cells were slowed down by reducing flow velocity or by rolling over E-selectin. Third, rolling and firm adhesion of KG-1a cells on BM endothelium (4LHBMEC) were inhibited if KG-1a cells were preincubated with various high-sulfated heparin/HS-like GAGs. HSIM, HSBK, DS, and CS and do not differ much in overall sulfation (as estimated by Mono Q ion exchange chromatography; unpublished results). Therefore, no clear correlation can be made between the number of sulfate groups per disaccharide and the amount of inhibition of adhesion. This indicates that negative charge alone is not sufficient to explain the results, but appropriate monosaccharide constituents and sulfation patterns are required for adhesion of GAGs to KG-1a cells. Finally, by specific enzyme degradations, we showed that adhesion of KG-1a cells to isolated PGs from 4LHBMEC was largely reduced after treatment with HS-degrading enzymes, indicating that HS-binding receptors on HPC interact with BM-derived endothelial HSPGs.

We also found multiple lines of evidence that endothelial HSPGs were involved in HPC attachment. First, when 4LHBMEC were pretreated with the sulfate inhibitor sodium chlorate, rolling and adhesion between 2 and 3 min as well as firm adhesion were inhibited. Second, cleavage of GAG chains from 4LHBMEC by HS-degrading enzymes resulted in a statistical, significant reduction of initial adhesion, whereas chondroitinase ABC and hyaluronidase had no effect. In addition, when endothelial HS was blocked with antibody 10E4, reduction of firm adhesion of KG-1a was obtained, providing additional evidence for the adhesive role of endothelial HSPGs. Finally, HPC could bind isolated HSPGs from 4LHBMEC.

It has been known that sulfated polysaccharides are involved in leukocyte adhesion [42 , 43 ]. A few adhesion molecules have been reported to be able to bind HS, such as L- and P-selectin [44 , 45 ] and integrin CD11b [21 , 22 ]. In the BM stroma, HPC are thought to interact directly with HSPGs on fibroblasts or in the ECM [26 , 27 ]. Reported ligands involved in this binding are CD11b, CD45, and a phosphatidyl-inositol-linked adhesion molecule [25 , 46 ]. As stromal HSPGs bind cytokines as well [47 , 48 ], the combination of direct adhesion to HPC and the colocalization of cytokines are thought to create the multimolecular stem-cell niche [27 , 49 ]. The heparin/HS-binding receptors on HPC involved in binding to BM endothelium under flow conditions are not known. The partial inhibition of rolling and initial adhesion by adding heparins or modulating endothelial PGs suggest that adhesion molecules responsible for tethering and rolling are involved. However, incubation of KG-1a cells with HS-GAGs exclusively resulted in inhibition of firm adhesion. Furthermore, HPC binding to heparin under flow in combination with rolling on E-selectin also suggested a role in firm adhesion. One of the candidates involved could be L-selectin, which is highly expressed on HPC [50 ], although its function in homing remains unclear [8 , 51 , 52 ]. We performed ELISA-binding studies in which we show that L-selectin is able to bind endothelial HS. Under conditions of flow using blocking antibodies against L-selectin, it was shown that firm adhesion of KG-1a cells was inhibited. This suggests that L-selectin is a candidate ligand involved in binding to BM endothelial HSPGs.

Besides functioning as direct adhesive ligands, HSPGs and CS/DSPGs on BMEC can bind and present the chemokine SDF-1 toward HPC under flow conditions [17 ], thereby increasing firm adhesion of HPC via signaling of CXCR4 and subsequent integrin activation. To study only the direct binding of HSPGs to HPC and not an indirect, adhesive effect induced via SDF-1, we chose CXCR4-negative KG-1a cells as a model for HPC. If other chemokines were involved in our experiments, removal or undersulfation of their PG carriers would result in a release of bound chemokines. This would lead to a higher percentage of rolling cells as a result of the absence of integrin activation and subsequent arrest and thus a decrease in firm adhesion. However, we observed a decrease in rolling after removal of HS-GAGs or chlorate treatment, suggesting no chemokine involvement. Furthermore, we competed with endothelial GAG-binding of chemokines by incubating the endothelial layers with heparin before perfusions to release bound chemokines [53 ], but no effect was observed. In addition, incubation of KG-1a cells with pertussis toxin, blocking Gi protein-coupled signaling downstream-chemokine receptors, did not result in a significant decrease of firm adhesion, although non-Gi protein-coupled signaling is not excluded. Altogether, we conclude that the results indicate a direct adhesive role for endothelial PGs and not an indirect chemokine-mediated effect. Other mechanisms leading to firm arrest, such as E-selectin-mediated integrin triggering or capture by endothelial fraktalkine, could be involved in our experiments [54 55 56 ].

We found that treatment of BM endothelium with sodium chlorate inhibited early attachment and firm adhesion of HPC. As sodium chlorate inhibits sulfation in all structures using the sulfate donor 3'-phosphoadenosine 5'-phosphosulfate, other sulfated, membrane-bound structures besides PGs could be involved in HPC adhesion. A well-known ligand for L-selectin is sulfated sialyl LewisX [57 ]. This tetrasaccharide combines sialylated, sulfated, and fucosylated sequences on a backbone and provides high-affinity, multivalent interactions. By removing one of these components, sialic acids on 4LHBMEC, we aimed to study the involvement of this structure in HPC adhesion. However, by sialidase treatment, no reduction but an increase in firm adhesion was found. This makes it unlikely that sialic acids on 4LHBMEC function as part of adhesive sialyl LewisX-like structures. It has been described that sialic acids can function as negative regulators of cell–cell interactions, probably occurring through steric effects, thereby masking adhesive structures, such as adhesion molecules [58 59 60 ].

Cell-surface HSPGs, like sialic acids, usually extend well into the glycocalix (the carbohydrate zone surrounding a cell). Removal of HS-GAGs by enzyme treatment reduced rolling and adhesion between 2 and 3 min; however, after 5 min perfusion, no difference in firm adhesion was found compared with the control situation. Although we do not have direct evidence, it could be hypothesized that similar to sialic acids, adhesive structures were exposed after cleavage of HS-GAGs. If this were true, it would be impossible to find any inhibiting effect of GAG cleavage. In a recent study by Mulivor and Lipowsky [61 ], it is described from in vivo experiments using mesenteric venules that anti-ICAM-1-coated beads adhered in larger numbers to vessel walls when these were pretreated by heparinases. They suggest that HSPGs form a barrier and mask ICAM-1 that is smaller in size. Others also found increased leukocyte adhesion upon HS degradation [62 ]. Studying the interaction between two complete cell types under flow conditions is more complex than using isolated components, as adhesion is mediated via multiple receptor-ligand pairs. The removal of one type of receptor-ligand interaction may not destabilize adhesion, and furthermore, this removal may expose other adhesive structures as mentioned above. Therefore, we also used isolated PGs to study HPC binding. By specific enzyme degradations, we showed that adhesion of KG-1a cells to PGs was largely reduced after treatment with HS-degrading enzymes, indicating that HS-binding receptors on HPC interact with endothelial HSPGs. HSPG candidates involved in HPC binding could be syndecan-1, glypican-1, and CD44v3 (Table 1) .

Mobilization of HPC is regarded as the opposite process of homing. Many adhesion molecules involved in homing, such as very late antigen-4 and ligand VCAM-1, also play a role in mobilization by disruption of adhesive bonds [63 ]. Recently, sulfated polysaccharides, such as fucoidan and DxS, have been introduced as mobilizing agents of HPC [64 , 65 ]. Their mode of action could be the release of mobilizing chemokines such as SDF-1 [66 ] but also competition with adhesive interactions between HPC and HSPGs.

To our knowledge, we are the first to show that HSPGs on BM endothelium are adhesive coreceptors toward HPC, together with other EC adhesion molecules, and therefore, HSPGs may contribute to homing of HPC to the BM. Future studies will be directed toward the identification of HS-binding receptors involved in homing of HPC.


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ACKNOWLEDGEMENTS
 
We are grateful to Maud van Haperen and Bert van het Hof for excellent technical assistance. We thank Jan van der Linden (Utrecht Medical Center, The Netherlands) for assisting with the Optimas 6.1 software and Guido David (Center of Human Genetics, Leuven, Belgium) for the generous gift of anti-HSPG antibodies.

Received December 2, 2002; revised July 20, 2003; accepted July 25, 2003.


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