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

Abstract

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⁺/VLA-4⁺ 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 β2 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.