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a Division of Medical Oncology, Netherlands Cancer Institute, Amsterdam, The Netherlands;
b CLB, Sanquin Blood Supply Foundation, and Laboratory for Experimental and Clinical Immunology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands;
c Gene Therapy Program, Department of Medical Oncology, Academic Hospital of the Free University, Amsterdam, The Netherlands;
d Department of Hematology, Academic Medical Center, Amsterdam, The Netherlands
Key Words. Migration • SDF-1 • Hematopoietic progenitor cells • Bone marrow endothelial cells • Adhesion molecules • Homing
C.E. van der Schoot, M.D., Ph.D., Department of Immunohematology, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands. Telephone: 31-20-5123377; Fax: 31-20-5123474; e-mail: Schoot{at}clb.nl
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ABSTRACT |
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Migration of CD34+ cells is enhanced across interleukin 1ß prestimulated bone marrow endothelium, suggesting an important role for the endothelium in adhesion and formation of the chemotactic gradient. Under these conditions, 30-100 ng/ml SDF-1 induced a rapid and efficient migration of CD34+ cells (± 46% migration in 4 h). In contrast, 600-1,000 ng/ml SDF-1 were required for optimal migration across fibronectin-coated filters. Subsequent studies revealed that transendothelial migration of CD34+ cells is mediated by ß1- and ß2-integrins and PECAM-1 (CD31) but not by CD34 or E-selectin. Whereas these antibodies individually blocked migration for 25%-35%, migration was reduced by 68% when the antibodies were combined. Thus, these adhesion molecules play specific and independent roles in the transmigration process. Finally, O-glycosylated proteins appeared to play a role, since SDF-1-induced migration of CD34+ cells (treated with a glycoprotease from Pasteurella haemolytica) across endothelial cells was clearly inhibited.
In conclusion, we show that efficient SDF-1-induced migration of primary human CD34+ cells across bone marrow endothelium is mediated by ß1-integrins, ß2-integrins, CD31 and O-glycosylated proteins.
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Studies in murine models have indicated that E-selectin [2] and P-selectin [2, 3] are involved in the initial adhesion and rolling of HPC on the BM endothelial cells (BMEC). Studying the rolling of HPC along perfused microvessels in murine BM also revealed an important role for the ß1-integrin very late acting antigen (VLA)-4. The role of VLA-4 was confirmed in larger animals [4, 5]. A small proportion of human CD34+ cells migrates spontaneously across unstimulated human BMEC. Möhle et al. showed that the low spontaneous migration of HPC was reduced by blocking with a monoclonal antibody (mAb) against leukocyte function-associated antigen-1, but not by mAbs against L-selectin or VLA-4 [6]. In contrast, Yong et al. could not detect spontaneous migration of HPC across BMEC [7]. Only after stimulation of the HPC with the hematopoietic growth factors interleukin 3 (IL-3), IL-6, and stem cell factor was transmigration observed. The migration of these growth factor-activated HPC was inhibited by mAbs against CD18 and CD31 [7]. In vivo microvascular BMEC express constitutively the adhesion molecules E-selectin and VCAM-1 [8]. Moreover, migration of CD34+ cells in vivo is induced by chemokines produced by BM stromal cells. At present, the only known chemokine with strong chemotactic effects on CD34+ HPC is stromal cell-derived factor-1 (SDF-1) [9]. The dominant role of SDF-1 and its receptor in migration of CD34+ cells was demonstrated in knock-out mice which had impaired BM myelopoiesis [10-13]. Peled et al. recently described that SDF-1 and its receptor CXCR-4 are critical for BM engraftment in nonobese diabetic/severe combined immunodeficient mice [14]. These two observations indicate that migration of CD34+ cells in vitro should be studied using BMEC with upregulated adhesion molecules E-selectin and VCAM-1, and migration should be analysed using the chemokine SDF-1. Using such experimental conditions, Imai et al. reported that the VLA-4 and VCAM-1 molecules are involved in migration of murine HPC [15]. However, their murine endothelial lines express only low levels of E-selectin, while no expression of ICAM-1 could be detected [15]. In contrast, a recent study indicated that migration of human CD34+ cells is mediated by E-selectin and not by VCAM-1 or ICAM-1 [16].
In the present study, we have investigated the adhesion molecules involved in the SDF-1-induced migration of human cord-blood (CB)-derived CD34+ HPC across human BMEC. Our data demonstrate that HPC require ß1-integrins, ß2-integrins, CD31, and O-glycosylated proteins for efficient migration across the BM endothelium.
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MATERIALS AND METHODS |
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CD34+ HPC
Normal BM was aspirated from patients undergoing cardiac surgery (in the Academic Medical Centre; Amsterdam, The Netherlands) after informed consent. PB progenitor cells were obtained from patients treated with chemotherapy and G-CSF to induce stem cell mobilization. CB was collected after delivery, according to the guidelines of Eurocord Nederland. BM, PB, and CB mononuclear cells were enriched by density gradient centrifugation over Ficoll-paque (1.077 g/ml) (Pharmacia Biotech; Uppsala, Sweden; http://www.pnu.com). CB CD34+ cells were isolated with the VarioMacs system (Miltenyi Biotec GmbH; Gladbach, Germany). First, the mononuclear cells were incubated for 15 min at 4°C in PBE buffer (0.5% w/v bovine serum albumin [BSA] and 0.05 mM EDTA in phosphate-buffered saline [PBS]) with a hapten-labeled antibody directed against CD34 (QBEND10), in the presence of human IgG as a blocking reagent. The cells were washed in PBE and then incubated with antihapten microbeads for another 15 min at 4°C. The labeled cells were washed, resuspended in PBE and applied to a VS or RS separation column that was placed in the magnetic field of the VarioMacs. The column was washed four times to remove CD34– cells. Thereafter, the column was removed from the VarioMacs and the CD34+ cells were eluted with 1 ml PBE. The cells were further purified by means of a new RS column, after which at least 95% of the cells from CB expressed CD34 as determined by flowcytrometric analysis (FACScan, Becton and Dickinson [B&D] Immunocytometry Systems; San Jose, CA; http://www.bd.com).
Endothelial Cell Lines
All endothelial cell lines used were established by immortalization of primary endothelial cells with the replication-defective retroviral construct pLXSN16 E6/E7, containing the E6/E7 genes from the human papilloma virus 16 [24] as described previously [25]. It is necessary to use immortalized cells since it is not possible to obtain primary BMEC on a regular basis and in sufficient quantities. All endothelial cell lines were routinely cultured in fibronectin (FN)-coated (CLB) culture flasks and culture medium consisting of Medium 199 (GIBCO BRL), supplemented with 10% (v/v) pooled, heat-inactivated human serum (CLB), 10% (v/v) heat-inactivated fetal calf serum, 1 ng/ml basic fibroblast growth factor (Boehringer Mannheim; Mannheim, Germany), 5 U/ml heparin (Leo Pharmaceutical Products; Weesp, The Netherlands), 300 µg/ml glutamine (Sigma Chemical Co.; St. Louis, MO; http://www.sigma-aldrich.com), 100 U/ml penicillin, 100 µg/ml streptomycin and 100 µg/ml geneticin (G418), a neomycin analog (GIBCO BRL). After reaching confluency, the endothelial cells were passaged by treatment with trypsin/EDTA solution (GIBCO BRL). All cell lines were cultured at 37°C at 5% CO2.
Transendothelial Migration
Migration assays were performed in Transwell plates (Costar; Cambridge, MA) of 6.5 mm diameter, with 5-µm pore filters. Endothelial cells were plated at 20,000-30,000 cells/Transwell on FN-coated (CLB) filters. Nonadherent cells were removed after 18 h. The adherent cells were cultured for two to three days to obtain confluent endothelial monolayers. Confluency of the endothelial cell monolayers was confirmed by measuring permeability for fluorescein isothicyanate (FITC)-dextran 3000 (Molecular Probes; Leiden, The Netherlands). Monolayers of endothelial cells were used unstimulated or pretreated for 4 h with IL-1ß (10 U/ml Sanvertech Inc.; Heerhugowaard, The Netherlands).
Before adding CD34+ cells to the upper compartment, the endothelial monolayers were washed three times with assay medium (Iscove's modified Dulbecco's medium with 0.25% [w/v] BSA [BSA, fraction V, Sigma]). Freshly isolated CD34+ cells (20,000-100,000) were added to the upper compartment in 0.1 ml of assay medium, and 0.6 ml of assay medium with or without the indicated concentrations of recombinant human SDF-1 (Strathmann Biotech GmbH; Hannover, Germany) was added to the lower compartment. In blocking experiments, the CD34+ cells were preincubated for 10 min at 37°C with mAbs. As a control we used mAbs CD13 and/or CD59. Both antigens are highly expressed on HPC and endothelial cells. A combination of the two antibodies was used to exclude aspecific inhibition by high concentrations of antibodies. An 0.1-ml sample containing cells in assay medium was diluted in 0.5 ml of assay medium and kept as input control for quantitation of the number of migrated cells (see below). The Transwell plates were incubated at 37°C, 5% CO2 for 4 h. Preliminary experiments showed that after 4 h, a substantial fraction of the CD34+ cells had migrated. Cells that had migrated to the lower compartment were collected in a fluorescence-activated cell sorter (FACS) tube to which a fixed number of control cell-line cells (HL-60) labeled with Calcein AM (Molecular Probes) was added. FACScan analysis was used to determine the ratio between labeled and unlabeled cells, with characteristic light scatter parameters, in the migrated fraction as described before [26]. By comparison of this ratio to that of the input control, the number of migrated cells was quantitated. Using this method, we were able to determine reliably a minimum number of 200 migrated cells.
Enzyme Treatment
The progenitor cell surface proteins that contain O-linked glycans were cleaved by pretreatment with glycoprotease [27] (derived from Pasteurella Haemolytica, a kind gift of Dr. Mellors, University of Guelph; Ontario, Canada) for 30 min at 37°C. As a control CD34+ cells without glycoprotease treatment were also incubated at 37°C. Thereafter, both treated and untreated CD34+ cells were washed extensively and resuspended in assay medium prior to the migration assay. The efficacy of the treatment with glycoprotease was established by the complete loss of reactivity of CD34 antibody MD34.2, belonging to epitope class I (data not shown).
Statistical Analysis
All results were expressed as the mean ± standard error (SE). Significance of differences was determined with a two-sided Student's t-test. Two-sided p values less than 0.05 were considered to be significant.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Spontaneous and SDF-1-induced migration across IL-1ß-prestimulated HBMEC-60 endothelial cells (4.2% and 46.2%, respectively) was significantly higher than across unstimulated cells (2.2% and 18.7%, respectively). Both in the unstimulated and IL-1ß-prestimulated situation, the SDF-1-induced migration over HBMEC-60 was significantly higher than the spontaneous migration.
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). In contrast, the SDF-1 dose-response curve for migration across FN-coated filters reached its optimum around 600 ng/ml SDF-1. Thus, CD34+ cells responded optimally to lower concentrations of SDF-1 when an endothelial layer was present. This observation was confirmed in paired experiments using the same source of CB CD34+ cells (data not shown).
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Inhibition of SDF-1-Induced Transendothelial Migration of CB CD34+ Cells
Blocking antibodies were used to investigate the role of various adhesion molecules in SDF-1-induced transendothelial migration. Most experiments were performed with IL-1ß-prestimulated HBMEC-60, because under this condition optimal migration was observed, and the enhanced expression of VCAM-1 and E-selectin mimics the situation in vivo. mAbs against ß2-integrins (CD18), ß1-integrins (CD29), or PECAM-1 (CD31) partially inhibited (23 ± 4%, 25 ± 7% and 38 ± 12% ([mean ± SE] respectively) the SDF-1-induced migration compared to a control mAb (p < 0.05 for all three mAbs) (Fig. 3). A stronger inhibition was observed when the antibodies against the different adhesion structures were combined. All combinations demonstrated a significantly stronger inhibition than mAbs to CD18 or CD29 alone. The combination of all three antibodies was not superior to the combination of two antibodies, and a maximal inhibition of 68 ± 6% was obtained (Fig. 3). A combination of two control antibodies (CD13 and CD59) was used to exclude aspecific inhibition by high concentrations of antibodies.
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shows that the presence of the single mAbs against CD34 or E-selectin did not significantly inhibit the SDF-1-induced transendothelial migration. The combination of these two mAbs tended to inhibit migration, but this did not differ significantly (p = 0.06) from the controls. When both antibodies (or the mAb against E-selectin alone) were added to the combination of mAbs against CD18, CD29, and CD31, a slightly increased inhibition was observed, but again this increase did not reach significance.
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). However, the inhibition of migration obtained with the blocking mAbs against ß1-integrins, ß2-integrins and PECAM-1 was not augmented further by the glycoprotease treatment of the CD34+ cells (data not shown). CD34+ cells derived from CB, PB, or BM were used in these experiments.
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Although the SDF-1-induced migration across unstimulated HBMEC cells was low, we also investigated which molecules were involved under this condition. The results of these experiments were variable (Table 2). None of the single antibodies (CD18, CD29, and CD31) significantly inhibited the transmigration, and the combinations of the antibodies had no significant effect.
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DISCUSSION |
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In accordance with other reports, we found that the presence of SDF-1 greatly enhanced transendothelial migration of CB CD34+ cells [9, 15]. From our own previous studies it is known that CB CD34+ cells show maximal migration over FN-coated filters when concentrations between 600 and 1,000 ng/ml SDF-1 are used [26]. In the present work we demonstrate that maximal transendothelial migration is observed with considerably lower amounts of SDF-1. The presence of 30 or 100 ng/ml SDF-1 in the lower compartment of the Transwell system was already sufficient to induce maximal migration. Imai et al. previously showed that BMEC produce SDF-1 [31]. However, the low spontaneous migration of CB CD34+ cells over HBMEC excludes that the shift of the dose-response curve is due to high levels of endogenous SDF-1 production. Most likely, the observed differences in dose-response curves are related to an efficient presentation of SDF-1 by endothelial cells. Probably, the chemokine is bound to vascular proteoglycans because SDF-1 associates at high affinity with heparan sulfates [32-34]. Indeed, when SDF-1 was added to the lower compartment of the Transwell system and the free SDF-1 was subsequently washed away, increased migration was still observed (data not shown). This observation suggests that endothelial cells are able to build up a concentration gradient within the endothelial layer. Also in vivo SDF-1 is presented on the surface of BMEC [33]. The importance of the presentation of SDF-1 by endothelial cells was also shown by Peled et al. who recently demonstrated that only immobilized and not soluble SDF-1 could upregulate integrin adhesiveness of CD34+ cells [33].
IL-1ß prestimulation of endothelial cells further increased the SDF-1-induced migration. Also the spontaneous transendothelial migration of the CD34+ cells was significantly increased under these conditions, but remained below 5%. The fact that IL-1ß prestimulation increased transendothelial migration stresses the importance of integrin-mediated adhesion. This was confirmed by experiments with the endothelial cell line HBMEC-28S, which shows a lower expression of the adhesion molecules E-selectin and VCAM-1 upon IL-1ß prestimulation than does HBMEC-60, and a dramatically lower migration (data not shown). In this regard it is of interest that E-selectin and VCAM-1 are expressed on small endothelial vessels in hematopoietically active tissues in the in vivo situation, as shown by Schweitzer et al. [8]. Moreover, we have previously found that VCAM-1 and E-selectin are involved in the interaction between HPC and HBMEC [28]. Finally, IL-1ß prestimulation normally induces cytokine production (e.g., IL-8 or IL-6) by endothelial cells. HBMEC-60 and HBMEC-28S barely release IL-6 and IL-8 under resting conditions. After 4 h of prestimulation with IL-1ß, HBMEC-60 produced IL-6 and IL-8 in a nanogram/ml range, but the production by HBMEC-28S was significantly less (data not shown). Thus, besides the upregulation of important adhesion molecules, the release of various cytokines may also be important. Recently, Peled et al. showed that IL-6, which is also induced by IL-1ß prestimulation, can upregulate the expression of the CXCR-4 receptor on HPC [14]. This event did not occur in our assays, probably because of a shorter exposure to IL-6. Peled et al. stimulated HPC for 48 h with IL-6, while exposure in our assay is only 4 h.
Our data demonstrate that HPC require a set of adhesion molecules to migrate efficiently across IL-1ß-prestimulated BM endothelium. In this situation, the migration of HPC was only partially inhibited by mAbs against ß2-integrins, ß1-integrins or PECAM-1, and the effect of the single mAbs was much lower than when combinations of mAbs were used. Previous studies have only partially addressed the role of adhesion molecules in the regulation of HPC transendothelial migration. Möhle et al. [6] observed a role for ß2 integrins and Yong et al. [7] for CD31 in spontaneous transendothelial migration of HPC. VLA-4 antibodies did not have an inhibitory effect on this spontaneous migration [6]. Remarkably, a role for VLA-4 and VCAM-1 in SDF-1-induced migration of murine HPC across BMECs has been described by Imai et al. [15]. In contrast to these and our results, Naiyer et al. did not see any effect of VCAM-1 on SDF-1-induced transendothelial migration of CD34+ cells [16]. They also did not observe an effect of antibodies to ICAM-1. However, combinations of antibodies were not tested in their assay and these investigators tested migration after 24 h. The blocking of the single mAbs in our assay was around 25% after 4 h of migration, and it might well be that single antibodies only delay transmigration and that after 24 h of migration, these percentages of inhibition could no longer be detected. In our system, CD34 and E-selectin did not seem to play a role. We cannot exclude that these observations are due to the static condition of our assay. In contrast to our data, Naiyer et al. recently showed that E-selectin mediates SDF-1-induced transendothelial migration across BMEC [16]. Naiyer et al. used a similar static Transwell assay. Moreover, they prestimulated the endothelial cells with IL-1ß for 12-16 h. In our hands, maximal expression of E-selectin is observed after 4 h, and expression is much lower after 12 h [25].
Finally, we showed that treatment of CD34+ cells with glycoprotease from Pasteurella haemolytica caused an inhibition of migration across IL-1ß-prestimulated HBMEC-60 cells, indicating that O-glycosylated adhesion molecules such as CD34, CD43, CD44, or CD45 are probably also involved in the transmigration process. Pilarski et al. recently showed a potential role for CD44 in HPC adhesion studies [35]. However, the presence of mAbs against ß1-integrins, ß2-integrins and PECAM-1 in combination with glycoprotease-treated CD34+ cells did not give a stronger inhibition than the presence of the three mAbs alone. A possible explanation for this lack of additive inhibition could be cross-talk between the different adhesion molecules. For instance, cross-linking of CD34 or CD43 antigen by a putative ligand on BMEC might activate the ß2-integrins, as has been described [36, 37].
We observed about 20% migration across unstimulated HBMEC-60, which was not inhibited by mAbs against CD18, CD29, CD31, CD34, or E-selectin (neither single nor in combination). Apparently, HPC are able to use additional as yet unidentified (adhesion) molecules for their transmigration. These molecules might also be responsible for most of the transmigration across unstimulated HBMEC.
Because we quantified migration of total CD34+ cells in majority consisting of more committed progenitor cells, we cannot conclude whether similar adhesion molecules are required for the transmigration of more primitive HPC. However, there are currently no clear indications that CD34+ subsets differ in their migrational behavior. In a previous study we did not observe differences between phenotypically defined subsets in migration across an FN-coated filter [26]. Also Aiuti et al. showed no SDF-1-induced preferential migration of primitive precursors [9]. Finally, Jo et al. demonstrated that there was no difference between transendothelial migration of the week 2 CB cobblestone area-forming cells (CAFC) (which detects committed progenitors) and the week 5 CB CAFC (which detects candidate stem cells) [38].
In conclusion, we have shown that IL-1ß prestimulation of endothelial cells enhances SDF-1-induced migration of CB CD34+ cells. This SDF-1-induced transendothelial migration is more efficient than migration over FN-coated filters. ß2-integrins, ß1-integrins, PECAM-1, and O-linked structures are involved in the migration of HPC across HBMEC, but our data suggest the involvement of other as yet unidentified molecules as well.
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ACKNOWLEDGMENTS |
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References |
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