Abstract
Hematopoietic progenitor cell release to the circulation is the outcome of signals provided by cytokines, chemokines, adhesion molecules, and proteolytic enzymes. Clinical recruitment of immature CD34+ cells to the peripheral blood (PB) is achieved by repeated G-CSF stimulations. Yet, the mechanisms governing progenitor cell egress during steady state homeostasis and clinical mobilization are not fully understood. Membrane type-1 metalloproteinase (MT1-MMP) and its endogenous inhibitor, RECK, are established key regulators of tumor and endothelial cell motility. We detected higher MT1-MMP and lower RECK expression on circulating human CD34+ progenitors and maturing leukocytes as compared to immature bone-marrow (BM) cells. MT1-MMP expression was even more prominent on CD34+ cells obtained from PB of G-CSF-treated healthy donors whereas RECK labeling was barely detected. In addition, five daily injections of G-CSF to NOD/SCID mice, previously engrafted with human cells, increased MT1-MMP and decreased RECK expression on human CD45+ leukocytes, immature CD34+ and primitive CD34+/CD38−/low cells, in a PI3K/Akt1-dependent manner, resulting in elevated MT1-MMP activity. Inverse regulation of MT1-MMP and RECK by G-CSF mobilization was confirmed by in situ immuno-labeling of BM sections, as well as by human MT1-MMP and RECK mRNA expression analysis of leukocytes repopulating the BM of chimeric mice. Blocking MT1-MMP function impaired mobilization, while RECK neutralization promoted egress of human CD34+ progenitors in the functional pre-clinical model of NOD/SCID chimeric mice. Targeting MT1-MMP expression by SiRNA or blocking its function reduced the in-vitro chemotactic response to SDF-1 of human CD34+ progenitors via matrigel and impaired to a similar extent the BM homing capacity of transplanted human CD34+ cells in NOD/SCID mice. In accordance, neutralization of RECK function, thus abrogating RECK-mediated inhibition of MT1-MMP, facilitated SDF-1-induced migration of steady state human BM CD34+ cells in vitro. Furthermore, following G-CSF mobilization, we also observed a reduction in CD44 expression on human leukocytes and, specifically, on immature CD34+ progenitor cells in the BM of chimeric mice. This was accompanied by accumulation of CD44 cleaved products of molecular weights, expected for MT1-MMP activity, in the BM supernatants. In chimeric mice co-injected with MT1-MMP-neutralizing Ab, less cleavage of CD44 was detected upon G-CSF mobilization, whereas in the absence of a mobilizing signal, increasing MT1-MMP activity by anti RECK Ab injection facilitated CD44 proteolysis on the BM cells. Finally, MT1-MMP expression correlated with the number of CD34+ cells, collected on the first apheresis day in 29 consecutive patients with lymphoid malignancies and in 21 healthy donors treated with G-CSF. In conclusion, our results indicate that G-CSF inversely regulates MT1-MMP and RECK expression on CD34+ progenitors, resulting in net increase in MT1-MMP activity. MT1-MMP proteolysis of CD44 diminishes progenitor adhesion to BM components, leading to cell egress. These cell autonomous changes provide a previously undefined mechanism for G-CSF recruitment of CD34+ progenitors and might serve as target for new approaches to improve clinical stem cell mobilization.
Identification of Lymphomyeloid Primitive Progenitor Cells in Fresh Human Cord Blood and in the Marrow of Nonobese Diabetic–Severe Combined Immunodeficient (NOD-SCID) Mice Transplanted with Human CD34+ Cord Blood Cells