Modulation of VLA-4 and L-selectin expression on normal CD34+ cells during mobilization with G-CSF

We have evaluated the immunophenotype, functional activity and clonogenic potential of CD34(+) cells from peripheral blood (PB) of normal donors before and after 4 and 6 days of G-CSF administration. The percentage and absolute number of CD34(+) cells significantly increased at days 4 and 6 of G-CSF administration, compared to the steady-state level (P < 0.0001). Two-colour fluorescence analysis showed, at days 4 and 6, a lower proportion of CD34(+)/c-kit(+) compared to the steady-state level (P < 0.0001), but a similar expression of CD13, CD33, CD38, HLA-DR and Thy-1 antigens on CD34(+) cells. The expression of adhesion molecules on CD34(+) cells revealed a significant reduction of CD11a (P = 0.009), CD18, CD49d and CD62L (P < 0.0001) at days 4 and 6, compared to the baseline level. Three-colour staining showed a reduction of the more immature compartment (34(+)/DR-/13(-)) and an increase of the more differentiated compartment (34(+)/DR+/13(+)). Downregulation of VLA-4 during mobilisation was seen almost exclusively on more committed cells (34(+)/13(+)); downregulation of CD62L, on the contrary, was observed on both early progenitors (34(+)/13(-)) and more committed cells (34(+)/13(+)). The expression of 34'NLA4+ decreased on both c-kit(+) and c-kit(-) cells, while the expression of 34(+)/62L(+) decreased on the c-kit(+) cells only. In vivo administration of G-CSF reduced the adherence capacity of CD34(+) cells to normal BM stroma; in vitro incubation with SCF or IL-3 enhanced the expression of CD49d on CD34(+) cells, while GM-CSF reduced the expression of CD62L. SCF was the only cytokine able to induce a significant increase of CD34(+) cell adherence to preformed stroma. Pre-incubation with the blocking beta 2 integrin monoclonal antibody caused a reduction of CD34(+) cell adherence. In conclusion, the decrease of CD49d expression on mobilized CD34(+) cells correlates with a poor adhesion to BM stroma; CD34(+) cells incubated in vitro with SCF showed, conversely, a higher expression of CD49d and a greater adherence capacity on normal preformed stroma.