We found that supernatants of leukapheresis products (SLPs) of patients mobilized with granulocyte-colony-stimulating factor (G- CSF

Objective-Having previously demonstrated that the complement system modulates mobilization of hematopoietic stem/progenitor cells (HSPC) in mice, we investigated the involvement of C5 cleavage fragments (C5a/ desArg C5a) in human HSPC mobilization.Methods-C5 cleavage fragments in the plasma were evaluated by ELISA using human anti-desArg C5a antibody, and the expression of the C5a/ desArg C5a receptor (CD88) in hematopoietic cells by flow cytometry. We also examined the chemotactic responses of hematopoietic cells to C5 cleavage fragments and the expression of SDF-1-degrading proteases that perturb retention of HSPC in bone marrow (BM), namely matrix metalloproteinase (MMP)-9, membrane type (MT)1-MMP and carboxypeptidase M (CPM).Results-We found that plasma levels of desArg C5a are significantly higher in patients who are good mobilizers and correlate with CD34 + cell and WBC counts in mobilized peripheral blood (mPB). C5 cleavage fragments did not chemoattract myeloid progenitors (CFU-GM) but desArg C5a did strongly chemoattract mature nucleated cells. Consistently, CD88 was not detected on CD34 + cells, but appeared on more mature myeloid precursors, monocytes and granulocytes. Moreover, G-CSF-mobilized PB MNC and PMN had a significantly higher percentage of cells expressing CD88 than non-mobilized PB. Furthermore, C5a stimulation of granulocytes and monocytes (i) decreased CXCR4 expression and chemotaxis towards an SDF-1 gradient, and ii) increased secretion of MMP-9 and expression of MT1-MMP and CPM. Conclusion-C5cleavage fragments not only induce a highly proteolytic microenvironment in the human BM which perturbs retention through the CXCR4/SDF-1 axis but also strongly chemoattract

Carboxypeptidase M (CPM) is a membrane-bound zincdependent protease that cleaves C-terminal basic residues, such as arginine or lysine, from peptides/proteins. We examined whether CPM is expressed by hematopoietic and stromal cells and could degrade stromal cell-derived factor (SDF)-1␣, a potent chemoattractant for hematopoietic stem/ progenitor cells (HSPC). We found that (a) CPM transcript is expressed by bone marrow (BM) and mobilized peripheral blood CD34؉ cells, myeloid, erythroid, and megakaryocytic cell progenitors, mononuclear cells (MNC), polymorphonuclear cells (PMN), and stromal cells, including mesenchymal stem cells; and that (b) granulocyte-colonystimulating factor (G-CSF) significantly increases its expression at the gene and protein levels in MNC and PMN. Moreover, we found that recombinant CPM cleaves fulllength SDF-1␣ (1-68) rapidly, removing the C-terminal lysine and yielding des-lys SDF-1␣ (1-67).We demonstrated that such CPM treatment of SDF-1␣ reduced the in vitro chemotaxis of HSPC, which, however, was preserved when the CPM was exposed to the carboxypeptidase inhibitor DL-2-mercaptomethyl-3-guanidino-ethylthiopropanoic acid. Thus, we present evidence that CPM is expressed by cells occurring in the BM microenvironment and that the mobilizing agent G-CSF strongly upregulates it in MNC and PMN. We suggest that cleavage of the C-terminal lysine residue of SDF-1␣ by CPM leads to attenuated chemotactic responses and could facilitate G-CSF-induced mobilization of HSPC from BM to peripheral blood.