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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...

The functional role of ELR-positive CXC chemokines in host defense during acute viral-induced encephalomyelitis was determined. Inoculation of the neurotropic JHM strain of mouse hepatitis virus (JHMV) into the central nervous system (CNS) of mice resulted in the rapid mobilization of PMNs expressing the chemokine receptor CXCR2 into the blood. Migration of PMNs to the CNS coincided with increased expression of transcripts specific for the CXCR2 ELR-positive chemokine ligands CXCL1, CXCL2, and CXCL5 within the brain. Treatment of JHMV-infected mice with anti-CXCR2 blocking antibody reduced PMN trafficking into the CNS by >95%, dampened MMP-9 activity, and abrogated blood-brain-barrier (BBB) breakdown. Correspondingly, CXCR2 neutralization resulted in diminished infiltration of virus-specific T cells, an inability to control viral replication within the brain, and 100% mortality. Blocking CXCR2 signaling did not impair the generation of virus-specific T cells, indicating that CXCR2 is not required to tailor anti-JHMV T cell responses. Evaluation of mice in which CXCR2 is genetically silenced (CXCR2−/− mice) confirmed that PMNs neither expressed CXCR2 nor migrated in response to ligands CXCL1, CXCL2, or CXCL5 in an in vitro chemotaxis assay. Moreover, JHMV infection of CXCR2−/− mice resulted in an approximate 60% reduction of PMN migration into the CNS, yet these mice survived infection and controlled viral replication within the brain. Treatment of JHMV-infected CXCR2−/− mice with anti-CXCR2 antibody did not modulate PMN migration nor alter viral clearance or mortality, indicating the existence of compensatory mechanisms that facilitate sufficient migration of PMNs into the CNS in the absence of CXCR2. Collectively, these findings highlight a previously unappreciated role for ELR-positive chemokines in enhancing host defense during acute viral infections of the CNS.

Lymphocyte infiltration into the central nervous system (CNS) following viral infection represents an important component of host defense and is required for control of viral replication. However, the mechanisms governing inflammation in response to viral infection of the CNS are not well understood. Following intracranial (i.c.) infection of susceptible mice with mouse hepatitis virus (MHV), mice develop an acute encephalomyelitis followed by a chronic demyelinating disease. The CXC chemokine ligand 10 (CXCL10) is expressed following MHV infection and signals T cells to migrate into the CNS. The functional contribution of the CXCL10 receptor CXCR3 in host defense and disease in response to MHV infection was evaluated. The majority of CD4 + and CD8 + T cells infiltrating the CNS following MHV infection express CXCR3. Administration of anti-CXCR3 antibody reduced CD4 + T cell infiltration (p 0.05), while CD8 + T cell trafficking was not affected. Anti-CXCR3 treatment during chronic disease correlated with improved motor skills and reduced demyelination. The selective effect of anti-CXCR3 treatment on CD4 + T cells was not the result of either reduced proliferation or modulation in chemokine receptor gene expression. Therefore, CXCR3 signaling has a non-redundant role in T cell subset trafficking in response to viral infection.

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