Deog-Yeon Jo, MD, PhD, Associate Professor, Division of Hematology/Oncology, Department of Internal Medicine, Chungnam National University Hospital, 640 Daesa-dong, Jung-gu, Daejon 301–721, Korea. E-mail: email@example.com
Summary. This study investigated human bone marrow endothelial cells (BMEC) chemoattractive activity in relation to haematopoietic cell trafficking. BMEC-conditioned medium induced chemoattraction of haematopoietic progenitor cells. Migration was not inhibited by pretreating the cells with pertussis toxin (PTX) or 12G5, indicating that the chemoattractive activity was not dependent on stromal-cell-derived factor-1 (SDF-1). Spontaneous migration, but not SDF-1-mediated chemotaxis of haematopoietic progenitors, was better supported by BMEC as compared with umbilical vein endothelial cells. The superior migration was abolished by pretreating the cells with PTX, indicating that BMEC-derived SDF-1 favours bone marrow endothelium, with better transmigration of haematopoietic progenitors.
Bone marrow endothelial cells (BMEC) support the proliferation and differentiation of haematopoietic progenitors (Rafii et al, 1997). In addition, their anatomic location suggests that BMEC play an important role in haematopoietic cell trafficking. Therefore, it could be anticipated that BMEC differ from endothelial cells outside the bone marrow, and could be specialized for their expected role.
A concentration gradient of stromal-cell-derived factor-1 (SDF-1) across the endothelium in the bone marrow seems to be the major mechanism for homing of haematopoietic stem/progenitor cells (Peled et al, 1999a). It has been reported that murine BMEC express SDF-1 mRNA and that its conditioned medium has chemotactic activity, which is abrogated with anti-SDF-1 antibody, suggesting the release of SDF-1 from BMEC (Imai et al, 1999). However, Peled et al (1999b) failed to find any chemotactic activity of haematopoietic progenitors in murine BMEC-conditioned medium, although the cells strongly expressed SDF-1 mRNA. It is not clear whether human BMEC actively release chemoattractants, including SDF-1, and what role these chemoattractants might play in haematopoietic cell trafficking. We found that BMEC produced chemoattractive activity in haematopoietic progenitors that was not attributed to SDF-1, even though the cells release low level SDF-1, and that BMEC-derived SDF-1 favours bone marrow endothelium, with better transmigration of haematopoietic progenitors.
Materials and methods
Human bone marrow endothelial cells (BMEC-1) (Francisco et al, 1996), murine bone marrow stromal cells (MS-5) and MO7e cells were grown in the proper medium. Human umbilical endothelial cells (HUVEC), primary human BMEC and primary human marrow stromal cells were prepared and cultured as described previously (Jaffe et al, 1973; Verfaillie et al, 1990; Rafii et al, 1994). CD34+ cells were purified from bone marrow by magnetic-activated cell sorting (MACS) (Miltenyi Biotec, Auburn, CA, USA). Three-day conditioned medium (CM) was prepared from these cells using serum-free medium X-VIVO (Biowhittacker, Walkersville, MA, USA). The transmembrane and transendothelial migration of CD34+ cells and MO7e cells was measured using 24-well TranswellTM with a 3-micron microporous membrane (Corning-Costar, Cambridge, MA, USA), as described previously (Jo et al, 2000). For blocking experiments, the cells were preincubated with 200 μg/ml of pertussis toxin (PTX; Sigma, St Louis, MO, USA) at 37°C for 2 h or with 40 μg/ml of 12G5 (R & D Systems, Mckinley Place, NE, USA) blocking monoclonal antibody to CXC-chemokine receptor R4 (CXCR4) at 37°C for 30 min. The concentrations of SDF-1α and stem cell factor (SCF) in CM were measured using commercial enzyme-linked immunosorbent assay kits (R & D Systems), according to the manufacturer's instructions. The results are expressed as the means ± standard deviation (SD) of three or more independent experiments. Data were analysed using the Student's t-test for paired samples.
Chemoattractive activities of endothelial cell CM
BMEC-1 CM attracted 14·9 ± 3·7% MO7e cells in 1 h, and the migration increased with time up to 24 h, reaching 58·7 ± 2·8%(Fig 1A). Transmigration of the cells over a 4 h period induced by BMEC-1 CM was 31·4 ± 12·5%, which was better than for HUVEC CM or 100 ng/ml SCF, but less than for MS-5 CM or 100 ng/ml SDF-1α (Fig 1B). Pretreatment of MO7e cells with PTX abrogated the chemoattraction induced by MS-5 or SDF-1α CM (38·0 ± 1·4%vs 11·0 ± 0·1%, 30·0 ± 1·4%vs 2·5 ± 0·7% respectively), but it did not affect the chemoattraction induced by BMEC-1 CM, HUVEC CM or SCF. BMEC-1 CM induced the migration of 2·5 ± 1·5% of CD34+ cells in 4 h. Pretreatment of CD34+ cells with PTX did not affect BMCE-1 CM- or HUVEC CM-mediated migration, but it markedly inhibited the migration of cells induced by MS-5 CM (Fig 1C). BMEC-1 CM induced the migration of week-2 and week-5 cobblestone area forming cells (CAFCs) by 7·1 ± 1·5% and 12·7 ± 2·0% in 4 h, respectively, and this was also not inhibited by pretreating the cells with 12G5 (Fig 1D).
Measuring SDF-1α and SCF in CM
SDF-1α was not detected in HUVEC CM, even after various cytokine treatments. BMEC-1 CM and primary human BMEC CM contained 1710·7 ± 204 and 1050 ± 153 pg/ml SDF-1α, respectively, which were much lower than with primary bone marrow stromal cell CM or MS-5 CM (29 536 ± 532 pg/ml, P < 0·001; 101 791 ± 9045 pg/ml, P < 0·001). BMEC-1 CM contained 324 ± 24 pg/ml SCF.
Transmigration of haematopoietic progenitors through BMEC-1 versus HUVEC monolayers
In a 6-h migration of MO7e cells, BMEC-1 monolayers supported spontaneous transendothelial migration of MO7e cells better than HUVEC monolayers (3·1 ± 0·3%vs 0·9 ± 0·2%, P < 0·05). On the other hand, no difference was observed in SDF-1-mediated transendothelial migration. This superior spontaneous transendothelial migration through BMEC-1 monolayers was abolished by pretreating MO7e with PTX (Fig 2).
In this study, we showed that BMEC secrete soluble chemoattractive factors for haematopoietic progenitors. BMEC-1 CM induced rapid migration of MO7e cells, which increased with time up to 24 h, indicating that both chemotactic and chemokinetic activities were present in the CM. The failure to block transmigration by pretreating the cells with PTX or 12G5 indicated that SDF-1 is not a predominant chemoattractive factor that is released from BMEC. In good agreement with this, much less SDF-1 was secreted from BMEC than from primary human bone marrow stromal cells or MS-5 cells. These results indicate that BMEC are not a major source of SDF-1 in the marrow microenvironment, and strengthens the possibility that SDF-1 produced by BMEC has additional roles. The chemoattractive activity produced by BMEC might be due to the additive or synergistic effects of some known chemoattractive factors (Dutt et al, 1998; Kim & Broxmeyer, 1998), but it is more likely to be attributable to unidentified chemoattractive substances.
The differences in how BMEC and HUVECs support spontaneous transendothelial migration and the fact that the difference disappears in SDF-1-induced migration need to be addressed. It has been suggested that SDF-1, especially the surface-bound form, activates the adhesion molecules of haematopoietic progenitors, thereby enhancing its adhesiveness to their endothelial ligands (Peled et al, 1999b, 2000). SDF-1 antibody decreased the adhesion of haematopoietic progenitors to BMEC (Imai et al, 1999), and pretreatment of HUVECs with SDF-1 enhanced the adhesion of haematopoietic progenitor cells (Peled et al, 1999b). We showed that pretreatment of MO7e cells with PTX, which blocked CXCR4 signalling, abrogated spontaneous migration through the BMEC monolayer, indicating that the superior migration was dependent on CXCR4. Taken together, it is evident that BMEC-derived SDF-1 enhances the transendothelial migration of haematopoietic progenitor cells.
This work was supported by grants from the Korea Science and Engineering Foundation (R01-2000–00150), and Chungnam National University Hospital.