Adhesion molecules involved in transendothelial migration of human hematopoietic progenitor cells.

In the process of homing, CD34(+) hematopoietic progenitor cells migrate across the bone marrow endothelium in response to stromal cell-derived factor (SDF)-1. To develop more efficient stem cell transplantation procedures, it is important to define the adhesion molecules involved in the homing process. Here, we identified the adhesion molecules that control the migration of primary human CD34(+) cells across human bone marrow endothelial cells. Migration of CD34(+) cells is enhanced across interleukin 1beta 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 beta1- and beta2-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 beta1-integrins, beta2-integrins, CD31 and O-glycosylated proteins.

E-selectin and very late activation antigen-4 mediate adhesion of hematopoietic progenitor cells to bone marrow endothelium.

Adhesion of CD34+ hematopoietic progenitor cells (HPCs) to sinusoidal endothelium probably plays a key role in homing of transplanted CD34+ HPCs to the bone marrow (BM). We have investigated the role of various adhesion molecules in the interaction of purified CD34+ HPCs derived from BM or peripheral blood (PB) and a human BM-derived endothelial cell line. Adhesion of CD34+ HPCs to endothelial cells was measured with the use of a double-color flow microfluorimetric adhesion assay. In this assay, adhesion is measured under stirring conditions, simulating blood flow in sinusoidal marrow vessels. Adhesion of PB CD34+ cells to human BM endothelial cells (HBMECs) was observed only after interleukin (IL)-1beta prestimulation of the endothelial cells. This adhesion was strongly increased after addition of phorbol-myristate acetate (PMA). Adhesion of PB CD34+ cells to IL-1beta-prestimulated HBMECs was inhibited by blocking monoclonal antibodies (mAbs) against E-selectin and by neuraminidase treatment of the PB CD34+ cells. mAbs against very late activation antigen (VLA)-4 inhibited adhesion only when the E-selectin-mediated interaction was prevented. No clear inhibiting effect was found with blocking mAbs against beta2-integrins. Stimulation with the beta1-integrin-activating mAb, 8A2, induced adhesion of CD34+ cells to endothelial cells. In conclusion, stimulation of both endothelial cells and CD34+ HPCs is necessary for adhesion of CD34+ HPCs to endothelial cells. We furthermore demonstrated that E-selectin and VLA-4 mediated this adhesion.

Immortalisation of human bone marrow endothelial cells: characterisation of new cell lines.

BACKGROUND: Adhesion of haematopoietic progenitor cells (HPC) to human bone marrow endothelial cells (HBMEC) plays a key role in homing of HPC to bone marrow. Here we describe four new HBMEC cell lines that can be used to study the (specific) adhesion of HPC to HBMEC. DESIGN: HBMEC were immortalised with a retroviral construct containing the human papilloma virus 16 E6/E7 genes. Four cell lines were characterised. RESULTS: The cell lines showed their endothelial nature by the expression of von Willebrand Factor and VE-cadherin (CD144). Electron microscopic analysis revealed normal endothelial-cell characteristics, including the presence of Weibel-Palade bodies and intercellular junction structures. An extensive phenotypic analysis of the cell-lines was performed, they were found to resemble primary HBMEC. The only difference found was the absence of expression of E-selectin (CD62e) and VCAM-1 (CD106) on resting HBMEC cell lines. Upon stimulation with IL-1beta the expression of E-selectin, VCAM-1 and ICAM-1 (CD54) was upregulated. All resting cell lines bound CD34+ HPC. Adhesion was increased by addition of the phorbol ester PMA. Two cell lines showed increased binding upon IL-1beta prestimulation. Highest adhesion was observed after the combination of IL-1beta prestimulation of the endothelial cells and addition of PMA. Binding of CD34+ HPC to HBMEC was compared with the binding to human umbilical vein endothelial cell lines and to a human dermal microvascular endothelial cell line (HMEC-1). So far, we have only found relatively less binding of HPC to IL-1beta prestimulated HMEC-1 cells, which could be explained by a reduced induction of E-selectin and VCAM-1 upon IL-1beta stimulation of these cells. CONCLUSION: The immortalised HBMEC cell lines have maintained their normal phenotype for the majority of characteristics examined. The expression of E-selectin and VCAM-1, which are not constitutively expressed on the cell lines, can be induced by stimulation of the endothelial cells with IL-1beta. The cell lines have furthermore maintained their capability to bind HPC. They will therefore be useful to investigate the interactions between HPC and HBMEC involved in homing of HPC.

Adhesion of hematopoietic progenitor cells to human bone marrow or umbilical vein derived endothelial cell lines: a comparison.

Homing of hematopoietic progenitor cells (HPC) to the bone marrow may be mediated by adhesion molecules specifically expressed on human bone marrow endothelial cells (HBMEC). This hypothesis suggests that HPC would preferentially bind to HBMEC compared to endothelial cells from other origins. In this study, HPC were allowed to adhere either to HBMEC cell lines or to human umbilical vein endothelial cells (HUVEC) in two different experimental set-ups. First, adherence was measured using a flow cytometric assay with three different colors identifying each cell population (HPC, HBMEC, HUVEC). HPC could adhere (in a competitive way) to the two endothelial cell lines under stirring conditions, which simulated adhesion under shear stress, as present in blood vessels. Because this assay requires relatively firm adhesion and the endothelial cells don't form a monolayer, we studied the same interactions under less stringent conditions. HPC were allowed to adhere to endothelial monolayers under gently rocking conditions. Differential adhesion of HPC to a set of endothelial cell lines did not correlate with the origin of the endothelial cells. Adhesion of HPC to both types of endothelial cells was inhibited in the presence of various combinations of monoclonal antibodies against the adhesion molecules VLA-4, CD18, and/or E-selectin. No indications were obtained for qualitative differences in the role of these molecules in adhesion of HPC to either HBMEC or HUVEC cell lines. In conclusion, no preferential adhesion of HPC to HBMEC compared to HUVEC cells was observed. This may be due to a lack of origin-specific differences between endothelial cells, implying that the specificity of homing is not regulated at the entrance of the bone marrow. Otherwise, the origin-specific differences between endothelial cells of different origins may be microenvironment-induced, rather then intrinsic, implying that care should be exercised with the use of endothelial cell lines in studies investigating the specificity of homing of HPC.

Alteration of B-cell antigen receptor signaling by CD19 co-ligation. A study with bispecific antibodies.

The activation of B-cell antigen receptor-associated protein tyrosine kinases is an early and crucial event in B-cell signaling. Apart from the B-cell antigen receptor (BCR), the B-cell-specific transmembrane glycoprotein CD19 has also been shown to directly activate intracellular signaling cascades. In addition, because CD19 and the BCR are associated on the surface of activated B-cells, it has been proposed that close approximation between these two entities is crucial for optimal B-cell triggering. To test this hypothesis, bispecific antibodies were generated that bind membrane IgM and CD19 simultaneously. Although CD19 bispecific antibodies strongly induced tyrosine phosphorylation, they were, in contrast to muF(ab)2 fragments, unable to induce a proliferative response. Detailed analysis of the early signaling events showed that compared with muF(ab)2 fragments CD19 bispecific antibodies potently raised the intracellular [Ca2+], which was correlated with an efficient tyrosine phosphorylation of syk. Strikingly, the assembly of Grb2 complexes that may couple the BCR to p21(ras) was clearly altered by the CD19 bispecific antibody. In addition to the reported Shc and 145-kDa phosphoproteins, a prominent 90-95-kDa phosphoprotein resembling CD19 was detected in the Grb2 complexes. Thus, studies with CD19 bispecific antibodies show that CD19 co-ligation both quantitatively and qualitatively alters BCR signaling.

B cell antigen receptor cross-linking induces tyrosine phosphorylation and membrane translocation of a multimeric Shc complex that is augmented by CD19 co-ligation.

The SH2 domain-containing transforming Shc protein has been implicated in mitogenic signaling via several surface receptors through p21ras. Following tyrosine phosphorylation by either receptor or non-receptor tyrosine kinases, Shc may interact with the adaptor protein Grb2, which is linked to Sos1, a guanine nucleotide exchange factor for human ras. Ligation of the antigen receptor complex on B cells (BCR) is known to activate various intracellular signaling pathways, which may accumulate in mitogenic responses. With respect to the initial steps, the activation of BCR-associated non-receptor tyrosine kinases appears to be indispensible. In this report we show that Shc proteins become tyrosine phosphorylated after BCR ligation on both transformed and normal human B cells. This is accompanied by the association of Shc with Grb2 proteins and a yet unidentified 145-kDa tyrosine phosphorylated protein. Subcellular fractionation revealed that this activation-induced multimeric Shc complex rapidly translocates towards the plasma membrane. Co-ligation of the BCR with the CD19 molecule results in a marked increase of these events, whereas CD19 cross-linking alone does not induce Shc tyrosine phosphorylation or translocation. Thus, in B cells the Shc complex may represent a molecular junction between the BCR and the mitogenic p21ras cascade.