The process of hematopoiesis is strictly regulated by intimate adhesive interactions between the hematopoietic stem cells and progenitor cells (HPCs) with the bone marrow microenvironment. HPCs can be mobilized into the peripheral blood, e.g. by treatment with chemotherapeutic drugs and/or hematopoietic growth factors. Intravenously reinfused HPCs migrate from the peripheral blood to the bone marrow, a process referred to as homing. Despite the clinical and biological significance, the adhesive mechanism(s) by which these processes of anchoring of HPCs within, mobilization from and homing to the bone marrow microenvironment are mediated, are still not completely understood.
CD34+ HPCs, bone marrow stromal cells and bone marrow endothelial cells are known to express a wide range of adhesion molecules, some of which have been shown previously to play a role in these processes. These include adhesion molecules from all known families, i.e. selectins, integrins, immunoglobulin-like adhesion molecules, CD44 and sialomucins. The sialomucin CD34 is expressed on HPC and on vascular endothelial cells. Although its precise function is unclear, several studies show that this mucin-like protein can act as an adhesion molecule.
To further study the role of the CD34 antigen, we have produced a CD34Fc-chimera, consisting of the extracellular domain of CD34 and the Fc domain of human IgG1. For this purpose a DNA construct encoding the chimera was transfected into SV40-transformed monkey kidney COS-7 cells and into Chinese hamster ovary (CHO) cells. The protein was isolated from supernatants of these cells by means of protein-A-Sepharose. The presence of both the CD34 and the Fc domain was demonstrated with the use of an ELISA and with Western blot analysis, using monoclonal antibodies against CD34 and monoclonal antibodies against the Fc domain. Furthermore, after preincubation of monoclonal antibodies against CD34 with the CD34Fc-chimera, the binding of this monoclonal antibody to the CD34 antigen present on KG1a cells was blocked. This blocking was dependent on the concentration of the chimera.
To study the role of CD34 in the binding of HPC to human bone marrow endothelial cells (HBMEC) or stromal cells, we used two adhesion assays. We first examined the binding of HPC to HBMEC cell lines in a solid-phase adhesion assay, and to frozen sections of rat bone marrow in a modified Woodruff and Stamper assay. Binding of the HPC to the HBMEC cell lines was not blocked by either the CD34Fc-chimera or the monoclonal antibody MD34.1 against CD34, whereas it was blocked by a combination of a monoclonal antibody against VLA-4 and a monoclonal antibody against CD18. In contrast, binding of HPC to the frozen section was blocked with the use of a monoclonal antibody against CD34, but preliminary results show that it was not blocked by the CD34Fc-chimera.
In conclusion, CD34 present on HPC mediates binding of HPC to bone marrow stromal cells. We could not show a role for CD34 expressed by HPC in the binding of HPC to HBMEC cell lines. |
