Effect of synthetic neoglycoproteins on the adhesion of hematopoietic progenitor cell lines to human bone marrow endotheliajl cells using a novel 96-well assay

Contact of hematopoietic stem and progenitor cells (HSC/HPC) with the endothelial cells lining the marrow sinuses is an initial step in their homing to the bone marrow following transplantation. The recognition of sinusoidal endothehum and homing of HPC to the bone marrow may be analogous to the tissue specific homing of lymphocytes. The specific adhesive mechanisms involved are still largely undefined, and expression of cell adhesion or homing molecules on endothelial cells may be fundamental in the regulation of stem eel] trafficking. We have used a new in vitro adhesion assay to study the role of potential homing molecules involved in this process. Studies using this assay with blocking monoclonal antibodies to adhesion molecules have previously shown us that anti-VLA-4 has an inhibitory effect on HL60 adhesion to bone marrow endothelium. Hematopoietic progenitor cell lines, KG la and HL60 were membrane labelled with a fluorescent dye, PKH2. The adhesion of labelled KG la and HL60 cells to TNFcc activated human bone marrow endothelial cell cultures (HBMEC) was examined in 96-well plates following premcubation of the cell lines with synthetic neoglycoproteins of galactosyl-, rnannosyl- and fticosyl-BSA. The extent of KG1a/HL60 adhesion to bone marrow endothelial cells was assayed after varying times by measuring the fluorescence of adherent cells in a multiwell fluorescence plate reader after removal of nonadherent cells by washing. KG 1 a cell adhesion to marrow endothelium was unaffected by the presence of the neoglycoproteins. Galactosyl- and mannosyl-BSA had no effect on HL60 adhesion. However, fucosyl-BSA was found to significantly inhibit the adhesion of HL60 cells to marrow endothelium. Preincubation of HBMEC with fucosyl-BSA did not inhibit HL60 adhesion. Further characterization is required to establish involvement of a lectin-carbohydrate interaction.