G-CSF–mediated thrombopoietin release triggers neutrophil motility and mobilization from bone marrow via induction of Cxcr2 ligands

Abstract

Emergency mobilization of neutrophil granulocytes (neutrophils) from the bone marrow (BM) is a key event of early cellular immunity. The hematopoietic cytokine granulocyte-colony stimulating factor (G-CSF) stimulates this process, but it is unknown how individual neutrophils respond in situ. We show by intravital 2-photon microscopy that a systemic dose of human clinical-grade G-CSF rapidly induces the motility and entry of neutrophils into blood vessels within the tibial BM of mice. Simultaneously, the neutrophil-attracting chemokine KC (Cxcl1) spikes in the blood. In mice lacking the KC receptor Cxcr2, G-CSF fails to mobilize neutrophils and antibody blockade of Cxcr2 inhibits the mobilization and induction of neutrophil motility in the BM. KC is expressed by megakaryocytes and endothelial cells in situ and is released in vitro by megakaryocytes isolated directly from BM. This production of KC is strongly increased by thrombopoietin (TPO). Systemic G-CSF rapidly induces the increased production of TPO in BM. Accordingly, a single injection of TPO mobilizes neutrophils with kinetics similar to G-CSF, and mice lacking the TPO receptor show impaired neutrophil mobilization after short-term G-CSF administration. Thus, a network of signaling molecules, chemokines, and cells controls neutrophil release from the BM, and their mobilization involves rapidly induced Cxcr2-mediated motility controlled by TPO as a pacemaker. IntroductionNeutrophils are the most abundant and, arguably, the most important leukocyte in the vertebrate immune system. Under normal conditions, human bone marrow (BM) produces ϳ 10 11 neutrophils per day. 1 In "danger situations" such as peripheral infections, the constant release of neutrophils can be dramatically increased within hours, a process termed danger or stress mobilization. 2 The hematopoietic cytokine granulocyte-colony stimulating factor (G-CSF) is central to the danger mobilization of neutrophils in both humans and mice. 3 However, although recombinant G-CSF has been used in clinical hematology for 20 years, the molecular mechanisms by which it mobilizes neutrophils are still not well understood.Neutrophils are restrained in the BM by the binding of their chemokine receptor Cxcr4 to the chemokine Cxcl12, which is expressed in a membrane-associated fashion by BM stromal cells. 4 There is evidence that G-CSF breaks the Cxcr4-Cxcl12 bond by activating neutrophil proteases, 5 thereby releasing neutrophils from the BM into the bloodstream. 3,6 However, several findings cannot be explained by the Cxcr4-Cxcl12 breakage concept alone. First, G-CSF can mobilize neutrophils in protease-deficient mice, arguing against the need for protease activation for this process. 7,8 Second, because neutrophil-specific deletion of Cxcr4 in mice results in much higher numbers of circulating neutrophils compared with wild-type animals, factors other than Cxcr4 must be involved in steering neutrophils into the BM blood sinuses (unless the process is passive). Finally, the specific Cxcr4-antagonist AMD3100 a...

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