67LR mediates in vitro cell adhesion and migration to laminin of G-CSF–mobilized CD34⁺ cells and affects MAPK phosphorylation in these cells. (A) In vitro cell adhesion to laminin of KG1, unstimulated BM, and G-CSF–mobilized PB CD34⁺ cells. KG1 cells, highly purified BM CD34⁺ cells from 3 donors and highly purified G-CSF–mobilized PB CD34⁺ cells from 3 donors were plated in wells coated with human placental laminin (5 μg/well), after preincubation with nonimmune rabbit antibodies (▪), an anti-67LR polyclonal antibody (dark gray bars), an anti-α₆ antibody (light gray bars), and allowed to adhere for 16 hours at 37°C. As negative controls, heat-denatured BSA was used for coating. Attached cells were fixed and stained with crystal violet; the absorbance (OD) of the eluted stain was measured by a spectrophotometer at 540 nm. All experiments were performed in triplicate and reported as a percentage of controls. The 100% values represent cell adhesion to heat-inactivated BSA (□). Results are presented as mean ± SD. 67LR supports cell adhesion to laminin of KG1 and G-CSF–mobilized CD34⁺ cells, whereas adhesion to laminin of unstimulated marrow CD34⁺ cells is 67LR independent and mostly mediated by α₆ integrins. (B) MAPK activation in CD34⁺ cell adhered to laminin. KG1 cells, highly purified unstimulated BM CD34⁺ cells, and highly purified G-CSF–mobilized PB CD34⁺ cells were plated in 35-mm wells coated with human placental laminin and allowed to adhere for 16 hours at 37°C. As negative controls, heat-denatured BSA was used for coating. Cells were then lysed and subjected to Western blot with anti–phospho-ERKs and anti–ERK-2 (as a loading control) polyclonal antibodies. Adhesion to laminin increased MAPK phosphorylation in both KG1 and BM CD34⁺ cells, whereas PB CD34⁺ cell adhesion to laminin, mostly mediated by 67LR engagement, did not increase MAPK activation. (C) In vitro cell migration to laminin of wild-type (□) and 67LR-transfected (▥) KG1cells. After incubation with nonimmune antibody (---) and a polyclonal anti-67LR antibody (pAb anti-67LR), KG1 cells were plated in Boyden chambers and allowed to migrate toward human placental laminin (25 μg/mL). The 100% values represent cell migration in the absence of chemoattractant. Values are the mean ± SD of 3 experiments performed in triplicate. Laminin-induced migration is increased in 67LR-transfected KG1 cells and is strongly reduced by blocking 67LR. (D) In vitro cell migration to laminin of G-CSF–mobilized PB CD34⁺ cells (▪). After incubation with nonimmune antibody (---) and a polyclonal anti-67LR antibody (pAb anti-67LR), highly purified G-CSF–mobilized CD34⁺ cells were plated in Boyden chambers and allowed to migrate toward human placental laminin. 100% values represent cell migration in the absence of chemoattractant. Values are the mean ± SD of 3 experiments performed in triplicate. Laminin-induced migration of G-CSF–mobilized PB CD34⁺ cells is almost totally abolished by blocking 67LR.