In rodents and primates, the inhibition of VLA-4/VCAM-1 interaction following administration of function-blocking anti-VLA-4 or anti-VCAM-1 mAbs results in mobilization of HPC. While providing a proof-of-principle that the perturbation of VLA-4/VCAM-1 results in mobilization of HPC, these data do not give clear insights into the physiological mechanisms of mobilization resulting from the administration of cytokines. By immunohistochemical staining, we have found that VCAM-1 expression is dramatically reduced in vivo in the bone marrow (BM) stroma of mice mobilized with either G-CSF, SCF, G-CSF in combination with SCF or cyclophosphamide and that the kinetics of the loss of VCAM-1 expression significantly correlates with those of HPC mobilization. In the serum of mobilized patients, the concentration of soluble VCAM-1 is increased and is correlated with the number of mobilized CFU-GM. This strongly suggests that the loss of VCAM-1 expression in the BM is due to shedding by proteolytic cleavage of VCAM-1 rather than its internalization from the cell surface. We then demonstrate that media conditioned by peripheral blood neutrophils and BM CD34- cells contain serine-proteases able to cleave VCAM-1 from the cell surface, release HPC adhered to immobilized rhuVCAM-1 or to BM stromal cell monolayers whereas media conditioned by BM stromal cells or bone cells do not. We demonstrate that neutrophil elastase (NE) and cathepsin G (catG) display the same ability to cleave VCAM-1 and release adhered HPC as media conditioned by neutrophils or BM CD34- cells. Moreover, NE generates a pattern of VCAM-1 fragments identical to those generated by neutrophil- and CD34cell-conditioned media showing that NE is the main protease cleaving VCAM-1 in both conditioned media. In accord with these observations, NE concentration is increased in the plasma of mobilized patients and this increase is correlated with both the mobilization of CFU-GM and the increase of sVCAM-1 concentration. Finally, we show that VLA-4 is expressed at the surface of mobilized CD34+ PBPC in a high affinity state detectable by the mAb 9EG7, and is bound to proteolytic fragments of VCAM-1 detectable by Westernblot. In conclusion, we propose that mobilization of HPC is the result of the disruption of the VLA-4/VCAM-1 adhesive interaction as a consequence of VCAM-1 cleavage by neutrophil proteases released by neutrophils accumulating in the extravascular compartment of the BM following cytokine administration. This model provides a unified mechanism encompassing previous observations demonstrating the importance of neutrophils in the phenomenon and that the disruption of VLA-4/VCAM-1 interaction is sufficient to promote mobilization.
|Publication status||Published - 2000|