Characterization of hematopoietic progenitor mobilization in protease-deficient mice

Abstract

Recent evidence suggests that protease release by neutrophils in the bone marrow may contribute to hematopoietic progenitor cell (HPC) mobilization. Matrix metalloproteinase-9 (MMP-9), neutrophil elastase (NE), and cathepsin G (CG) accumulate in the bone marrow during granulocyte colony-stimulating factor (G-CSF) treatment, where they are thought to degrade key substrates including vascular cell adhesion molecule-1 (VCAM-1) and CXCL12. To test this hypothesis, HPC mobilization was characterized in transgenic mice deficient in one or more hematopoietic proteases. Surprisingly, HPC mobilization by G-CSF was normal in MMP-9-deficient mice, NE ؋ CG-deficient mice, or mice lacking dipeptidyl peptidase I, an enzyme required for the functional activation of many hematopoietic serine proteases. Moreover, combined inhibition of neutrophil serine proteases and metalloproteinases had no significant effect on HPC mobilization. VCAM-1 expression on bone marrow stromal cells decreased during G-CSF treatment of wild-type mice but not NE ؋ CG-deficient mice, indicating that VCAM-1 cleavage is not required for efficient HPC mobilization. G-CSF induced a significant decrease in CXCL12␣ protein expression in the bone marrow of Ne ؋ CG-deficient mice, indicating that these proteases are not required to down-regulate CXCL12 expression. Collectively, these data suggest a complex model in which both protease-dependent and -independent pathways may contribute to HPC mobilization. IntroductionThe use of hematopoietic progenitor cells (HPCs) to reconstitute hematopoiesis following myeloablative therapy has significantly improved the clinical outcome for patients with a variety of diseases. Recently, mobilized peripheral blood HPCs instead of bone marrow-derived HPCs have been used because of reduced engraftment times and relative ease of collection. Although the great majority of HPCs reside within the bone marrow, a small number of HPCs also circulate in the peripheral blood. The number of circulating HPCs can be dramatically increased, or mobilized, by a wide variety of stimuli including hematopoietic growth factors, chemotherapy, and chemokines. 1,2 Currently, granulocyte colony-stimulating factor (G-CSF) is the most commonly used agent to mobilize HPCs because of its potency and lack of serious toxicity. However, the mechanisms that mediate G-CSF-induced HPC mobilization are incompletely understood.We previously showed that expression of the G-CSF receptor (G-CSFR) on HPCs is not required for their mobilization into the blood in response to G-CSF. 3 This observation suggests that G-CSF induces HPC mobilization indirectly through the generation of trans-acting signals. Recent studies suggest that hematopoietic proteases released by neutrophils into the bone marrow microenvironment may represent such a signal. A highly proteolytic microenvironment is induced in the bone marrow during HPC mobilization by G-CSF. 4 In particular, matrix metalloproteinase-9 (MMP-9 or gelatinase B), neutrophil elastase (NE), and cathepsin G (CG) accumulate...

Hematopoietic stem cell mobilizing agents G-CSF, cyclophosphamide or AMD3100 have distinct mechanisms of action on bone marrow HSC niches and bone formation

“…Therefore, our results show that the downregulation of CXCL12 in response to CYP is mediated both at the transcriptional level as well as by proteolytic cleavage of the protein, similar to what we have previously found for G-CSF. 6,10,14,15,31 The more pronounced CXCL12 transcriptional downregulation induced by CYP may explain why a single dose of CYP is more effective to mobilize HSPC than G-CSF.…”

“…Furthermore, genetically modified C57BL/6 mice lacking some of these proteases mobilize normally in response to G-CSF. 10 In the past 5 years, it has emerged that G-CSF mobilizes in part by (1) blocking proliferation and osteogenic differentiation of mesenchymal stem cells (MSC), 11 (2) inducing osteoblast apoptosis 12 and (3) blocking bone formation. 13,14 This results in a dramatic reduction in the expression of chemokines and cytokines necessary to maintain and retain HSC in their endosteal and perivascular niches such as CXCL12, kit ligand (KL) and angiopoietin-1.…”

Neutrophils in cancer: neutral no more

“…Several adhesion molecules, such as integrin subunit α4 (ITGA4) and vascular cell adhesion molecule 1 (VCAM1), as well as some proteases are also important in neutrophil retention [39][40][41] . In addition to its positive influence on granulopoiesis, G-CSF is a well-known disruptor of neutrophil retention 42 .…”

“…Mature cells are retained in the bone morrow by an interplay between two C-X-C chemokine receptors, CXCR4 and CXCR2. Constitutive CXCL12 expression by 4 osteoblasts and other bone marrow stromal cells tether CXCR4 + neutrophils in the bone marrow, whereas secretion of CXCL1 and CXCL2 by endothelial cells and megakaryocytes encourage the release of neutrophils into the circulation via CXCR2 signaling [33][34][35][36][37][38] (Figure 1).Several adhesion molecules, such as integrin subunit α4 (ITGA4) and vascular cell adhesion molecule 1 (VCAM1), as well as some proteases are also important in neutrophil retention [39][40][41] . In addition to its positive influence on granulopoiesis, G-CSF is a well-known disruptor of neutrophil retention 42 .…”

AMD3100 synergizes with G-CSF to mobilize repopulating stem cells in Fanconi anemia knockout mice

“…However, in preliminary studies, we have found that the responsiveness of myeloid progenitors to a number of cytokines and chemokines (including stem cell factor, G-CSF and stromal cell-derived factor-1) have failed to discern such a phenotype (unpublished data, 2007). Alternatively, it has recently been shown that hematopoietic progenitor and stem cell mobilization is dependent on a complex model involving both protease dependent and independent mechanisms [41][42][43][44]. Given our observations in these studies and the fidelity with which the murine models recapitulate the human mobilization defects, the FA murine models may be useful in future studies to discern the basic mechanisms underlying this clinical phenotype.…”