Sixty percent of the cells migrated to the lower chamber within 20?h when nonprocessed CXCL12 was used as a stimulus. MMP-8 were observed on days 4 to 6 6 of the mobilization regimen, concomitantly with elevated MMP-9 serum levels and higher numbers of circulating CD34+ cells. Elevated Dantrolene sodium serum concentrations of both proteases were also found in umbilical cord blood serum. In functional assays, adhesion of HSPC Dantrolene sodium to osteoblasts as an essential component of the endosteal stem cell niche is negatively influenced by MMP-8. The chemokine CXCL12, which is critically involved in stem cell trafficking, can be proteolytically processed by MMP-8 treatment. This degradation has a strong inhibitory influence on HSPC migration. Taken together, our data strongly suggest that MMP-8 can be directly involved in hematopoietic stem cell mobilization and trafficking. Introduction Different cytokines, including hematopoietic growth factors and chemokines, are capable of mobilizing hematopoietic stem and progenitor cells (HSPC) from the bone marrow stem cell niches into the circulation [1C4]. Mobilization can be achieved at different kinetics: chemokines mobilize HSPC within minutes, as shown in mice or monkeys, whereas treatment of patients with colony-stimulating factors requires days for efficient mobilization [5C7]. Whether the different kinetics reflect different mechanisms is still unclear, since the underlying molecular mechanisms leading to HSPC mobilization are still poorly defined [8,9]. However, since clinical mobilization protocols rely almost completely on empirical findings, a better understanding of the mobilization mechanisms may allow improved HSPC mobilization strategies. Cytokine-induced HSPC mobilization can be regarded as a multistep process with a crosstalk between adhesive structures and cytokines [1,10]. During mobilization, adhesive interactions between HSPC and their microenvironmental niches have to be modulated, and proteolytic enzymes are ideal candidates for fulfilling this function. In fact, a highly proteolytic microenvironment characterized by the matrix metalloproteinase (MMP)-9, MT1-MMP, carboxypeptidase M, cathepsin G and K, and neutrophil elastase has Dantrolene sodium been found in the bone marrow after treatment with mobilizing providers [11]. MMP-9 has been suggested to be directly involved in mobilization, since antibodies against MMP-9 were capable of preventing the interleukin-8 (IL-8)-induced mobilization of HSPC in rhesus monkeys [12]. In humans, elevated serum concentrations of MMP-9 in the peripheral blood have been observed after granulocyte colony-stimulating element (G-CSF)-induced stem cell mobilization [13C16]. On the other hand, experiments in mice Dantrolene sodium yielded only low serum levels of MMP-9 after IL-8-induced mobilization [17], and MMP-9-deficient mice did not show any alterations in cytokine-induced mobilization [18]. It Cd44 is therefore likely that MMP-9 is not the only secreted MMP playing a role in stem cell mobilization. The MMPs comprise a large family of proteolytic enzymes that can degrade extracellular matrix (ECM) molecules, membrane-bound adhesion molecules, cytokines, and chemokines, therefore altering the cellCcell or cellCmatrix relationships [19C21]. The users of the MMP gene family can be divided into several subfamilies, including collagenases, gelatinases, stromelysins, matrilysins, and membrane-bound MMPs, which display different substrate specificities and cells distribution [22,23]. The two gelatinases, MMP-2 and MMP-9, are the most intensively analyzed MMPs, also in the bone marrow [24,25]. MMP-8 is definitely a major collagenase synthesized by polymorphonuclear cells. MMP-8 and MMP-9 share some substrate specificities, as both enzymes degrade na?ve collagen type I, VII, and X, although with different efficacies. Moreover, both enzymes break down denatured collagens or aggrecan [26]. Although it has been shown that neutrophils seem to be indispensable for hematopoietic stem cell mobilization [27], the release of MMP-8 during this process has yet to be analyzed in detail. In the present study, we analyzed the manifestation of MMP-8, in addition to MMP-9, in the human being bone marrow. A Dantrolene sodium potential launch of MMP-8 was analyzed in vitro via mobilizing providers, but also in vivo during G-CSF-induced mobilization. While only a very low quantity of circulating hematopoietic stem cells were found in normal peripheral blood, an enhanced quantity of stem cells can be observed in umbilical cord.