Very late ANTIGEN-5 and leukocyto functional-associated ANTIGEN-1 are critical for early stage hematopoietic progenitor cell homing

Abstract

Abstract Adhesion molecule interactions of hematopoietic stem cells (HSC) and stromal cells including bone marrow microvascular endothelial cells (BMMEC) are critical for hematopoiesis. However, most of the identified HSC receptors mediate interactions between HSC and stromal cells in the extravascular space. We studied the interaction between HSC and BMMEC in the early period of homing with blocking monoclonal antibodies. Splenectomized C57BL mice were given 100 cGy of total body irradiation (TBI) followed by infusion into the tail vein of the bone marrow mononuclear cells (BMMNC). For the study of seeding kinetics early after transplantation, 2 × 10 6 BMMNC were infused 2 hours after TBI. At 0.5, 1, 2, 12, 18 and 24 hours after infusion, the content of total nucleated cells and colony-forming units granulocyte/macrophage (CFU-GM) in each of two femurs were determined. For the study of posttransplant circulating progenitor cells early after transplantation, 0.5 to 1.0 ml peripheral blood (PB) was aspirated in a heparinized syringe from the inferior vena cava. Experiments were repeated eight to ten times to ensure the reproducibility of results. Homing assay was performed after pretreatment with an antibody specific for VLA-4, VLA-5, LFA-1, or L-selectin. Briefly, BMMNC were incubated with an antibody (5 μg/ml) for 30 minutes at room temperature in IMBM and used immediately for transplantation studies. Posttransplant kinetics of CFU-GM from BM were evaluated at 0.5, 2, 12, 18 and 24 hours after infusion of BM cells. CFU-GM was the highest at 30 minutes. The number of CFU-GM decreased at first, reached a nadir at 12 hours, and then increased rapidly. As for posttransplant kinetics of CFU-GM from PB, 50 colonies/ml of CFU-GM was found 30 minutes at a frequency of 50 colonies/ml after infusion. These declined rapidly in 2 hours, and were not detectable after 24 hours in PB. Effects of pretreatment of BMMNC with antibodies against adhesion molecules on seeding efficiency of CFU-GM after transplantation are shown in the following table. Seeding efficiency is a ratio of the number of CFU-GM in two femurs to the number of transplanted CFU-GM. Blockage by anti-VLA5 and anti-LFA-1 antibodies caused a significant decrease in CFU-GM seeding efficiency at the early period of homing. Therefore, VLA-5 and LFA-1 may be involved in the recognition of BMMEC and/or adhesion to BMMEC.