67LR is up-regulated by G-CSF and by exposure to BM-derived endothelial cells in human BM CD34⁺ cells and is involved in their transendothelial migration. (A) 67LR and α₆-integrin expression on BM CD34⁺ cells after in vitro G-CSF treatment, in one representative experiment. BMMNCs from healthy donors were cultured for 24 and 48 hours with medium alone or in presence of 200 ng/mL G-CSF and analyzed by 3-color flow cytometry on mononuclear cells gated for side light scatter (SSC-H) and CD45⁺, with anti-67LR and anti-α₆ polyclonal antibodies or nonimmune polyclonal antibodies, as a negative control, and detected by a FITC-conjugated antirabbit antibody. 67LR expression on human BM CD34⁺ cells is increased by in vitro G-CSF treatment, whereas α₆-integrin expression decreases. (B) 67LR expression on BM CD34⁺ cells after in vitro G-CSF treatment and exposure to BM-derived endothelial cells, in one representative experiment. BMMNCs from healthy donors were cultured for 24 hours with medium alone, in the presence of 200 ng/mL G-CSF, or on confluent endothelial-cell layers in the presence of 200 ng/mL G-CSF, and analyzed by 3-color flow cytometry, as described. 67LR expression of human G-CSF–stimulated BM CD34⁺ cells is further increased by exposure to BM-derived endothelial cells. (C) Transendothelial migration of G-CSF–stimulated BM CD34⁺ cells. After incubation with a polyclonal anti-67LR antibody or a nonimmune antibody, CD34⁺ cells, stimulated for 72 hours with 200 ng/mL G-CSF, were plated in Transwell plates coated with confluent endothelial monolayers and allowed to migrate toward 200 ng/mL SDF1 (dark gray bars) or medium alone for 4 hours (light gray bars). Cells migrating into the lower compartment were collected and analyzed for their CFC content by methylcellulose colony assay. Data are the mean ± SEM of 3 separate experiments, ***P < .001. G-CSF–stimulated BM CD34⁺ cell transendothelial migration is significantly inhibited by anti-67LR antibodies.