Neutrophils are a major component of the innate immune response. Their homeostasis is maintained, in part, by the regulated release of neutrophils from the bone marrow. Constitutive expression of the chemokine CXCL12 by bone marrow stromal cells provides a key retention signal for neutrophils in the bone marrow through activation of its receptor, CXCR4. Attenuation of CXCR4 signaling leads to entry of neutrophils into the circulation through unknown mechanisms. We investigated the role of CXCR2-binding ELR + chemokines in neutrophil trafficking using mouse mixed bone marrow chimeras reconstituted with Cxcr2 -/-and WT cells. In this context, neutrophils lacking CXCR2 were preferentially retained in the bone marrow, a phenotype resembling the congenital disorder myelokathexis, which is characterized by chronic neutropenia. Additionally, transient disruption of CXCR4 failed to mobilize Cxcr2 -/-neutrophils. However, neutrophils lacking both CXCR2 and CXCR4 displayed constitutive mobilization, showing that CXCR4 plays a dominant role in neutrophil trafficking. With regard to CXCR2 ligands, bone marrow endothelial cells and osteoblasts constitutively expressed the ELR + chemokines CXCL1 and CXCL2, and CXCL2 expression was induced in endothelial cells during G-CSF-induced neutrophil mobilization. Collectively, these data suggest that CXCR2 signaling is a second chemokine axis that interacts antagonistically with CXCR4 to regulate neutrophil release from the bone marrow.

The number of neutrophils in the blood is tightly regulated to ensure adequate protection against microbial pathogens while minimizing damage to host tissue. Neutrophil homeostasis in the blood is achieved through a balance of neutrophil production, release from the bone marrow, and clearance from the circulation. Accumulating evidence suggests that signaling by CXCL12, through its major receptor CXCR4, plays a key role in maintaining neutrophil homeostasis. Herein, we generated mice with a myeloid lineage-restricted deletion of CXCR4 to define the mechanisms by which CXCR4 signals regulate this process. We show that CXCR4 negatively regulates neutrophil release from the bone marrow in a cellautonomous fashion. However, CXCR4 is dispensable for neutrophil clearance from the circulation. Neutrophil mobilization responses to granulocyte colony-stimulating factor (G-CSF), CXCL2, or Listeria monocytogenes infection are absent or impaired, suggesting that disruption of CXCR4 signaling may be a common step mediating neutrophil release. Collectively, these data suggest that CXCR4 signaling maintains neutrophil homeostasis in the blood under both basal and stress granulopoiesis conditions primarily by regulating neutrophil release from the bone marrow. (Blood. 2009;

235 Truncation mutations of CXCR4 that cause increased receptor signaling are responsible for most cases of WHIM (warts, hypogammaglobulinemia, infections, myelokathexis) syndrome, which is characterized by the retention of mature neutrophils in the bone marrow despite peripheral neutropenia. This observation and others have established CXCR4 as a key regulator of neutrophil release from the bone marrow. However, it is unclear how modulation of neutrophil CXCR4 signaling is linked to their migration toward the vascular endothelium and subsequent entry into the circulation. Therefore, the question of whether neutrophil egress from the bone marrow is a passive, random process or actively directed and what (if any) signals regulate it remains unanswered. We recently analyzed a myelokathexis pedigree and discovered homozygous, loss-of-function mutations in CXCR2. Based on these observations, we developed a “tug-of-war” model in which opposing chemokine gradients, specifically release-inducing CXCR2 signals and retention-promoting CXCR4 signals, act antagonistically to regulate neutrophil release from the bone marrow. To test this model, we analyzed neutrophil trafficking in CXCR2−/− mice. These mice have a well-characterized defect in neutrophil emigration from the blood to sites of inflammation, leading to chronic subclinical infection and the systemic release of cytokines that stimulate granulopoiesis. To circumvent these potentially confounding effects, we generated mixed bone marrow chimeras reconstituted with a 1:1 ratio of wild-type and CXCR2−/− bone marrow. As expected, approximately 50% (46 ± 4%) of circulating B cells were derived from CXCR2−/− cells. In contrast, a significant decrease in the proportion of CXCR2−/− neutrophils in the blood was observed (23 ± 4%; P < 0.001). Consistent with a myelokathexis phenotype, there was a relative accumulation of mature CXCR2−/− neutrophils in the bone marrow (47 ± 9% of Gr-1hi SSChi cells in the bone marrow were derived from CXCR2−/− cells; P < 0.01). Neutrophil trafficking from the bone marrow was estimated by calculating the percentage of neutrophils in the blood out of the total amount in the blood and bone marrow (neutrophil distribution index or NDI). We estimated that 1.3 ± 0.2% of wild-type neutrophils were in the blood, but the percentage of CXCR2−/− neutrophils in the blood was reduced to 0.4 ± 0.1% (P < 0.01). These data provide genetic evidence that CXCR2 signals promote neutrophil release from the bone marrow in a cell-autonomous manner. To explore the epistatic relationship of CXCR2 and CXCR4 signals to neutrophil trafficking, the neutrophil response to AMD3100, a small molecule CXCR4 antagonist, was examined in the CXCR2−/− mixed chimeras. Consistent with previous reports, treatment with AMD3100 resulted in a 5.2 ± 0.7-fold increase in wild-type neutrophils in the blood one hour after administration. In contrast, AMD3100 induced release of CXCR2−/− neutrophils was impaired, with only a 2.8 ± 0.3-fold increase observed (P < 0.05). G-CSF treatment is thought to induce neutrophil release through disruption of CXCR4 signaling. Thus, we next characterized the neutrophil response to 5 days of G-CSF treatment. Wild-type neutrophils displayed a shift from the bone marrow to the blood, with an NDI of 5.0 ± 0.4%. The number of CXCR2−/−neutrophils in the blood increased after treatment, but the percentage in the blood (2.5 ± 0.7%) was less than wild-type (P < 0.05). These data show that maximal neutrophil release requires the coordinated regulation of CXCR2 and CXCR4 signals. Studies are underway to assess neutrophil trafficking of CXCR4−/− × CXCR2−/− neutrophils. The tug-of-war model of neutrophil trafficking in the bone marrow predicts that CXCR2 ligands will be highly expressed in bone marrow endothelial cells or other cells closely associated with the endothelium. To test this prediction, endothelial cells (CD45− Ter119− CD31+) were sorted from the bone marrow of wild-type mice at baseline or after 5 days of G-CSF treatment. RNA expression profiling showed constitutive high level expression of the CXCR2 ligands CXCL1 and CXCL2. Moreover, expression of CXCL2 was significantly induced after G-CSF treatment. Chemokine expression was confirmed by real time RT-PCR and ELISA. Taken together, our data suggest that CXCR2 signaling is a second chemokine pathway that, in coordination with CXCR4, controls neutrophil release from the bone marrow. Disclosures: No relevant conflicts of interest to declare.

The number of circulating neutrophils is tightly regulated in order to effectively protect against microbial pathogens while minimizing damage to host tissue. Homeostatic control of neutrophils in the blood is achieved through a balance of neutrophil production, release from the bone marrow, and clearance from the circulation. Accumulating evidence suggests that signaling by the chemokine CXCL12, through its major receptor CXCR4, may play a key role in controlling neutrophil homeostasis. Indeed, gain-of-function mutations of CXCR4 are responsible for most cases of WHIM syndrome, a syndrome that features impaired neutrophil release from the bone marrow. Conversely, we previously reported that mice carrying a myeloid-specific deletion of CXCR4 (CXCR4f/−LysM+/Cre mice) display constitutive neutrophil release. Moreover, we provided data suggesting that neutrophil mobilization by G-CSF or Groβ are dependent on CXCR4 signaling, as neutrophil mobilization by these agents was absent in CXCR4f/−LysM+/Cre mice. These data firmly establish CXCR4 signaling as a key regulator of neutrophil release from the bone marrow under basal and stress conditions. Though controversial, there also is evidence that CXCR4 may play a role in neutrophil clearance from the blood by selectively trapping and removing aged neutrophils in the bone marrow. In this study, we examine the role of CXCR4 in neutrophil clearance using CXCR4f/−LysM+/Cre mice. Strain-matched wild type or CXCR4f/−LysM+/Cre mice were treated with a single injection of BrdU to label newly synthesized neutrophils. A similar percentage of myeloid cells in the bone marrow were labeled in wild type and CXCR4f/−LysM+/Cre mice, suggesting that the loss of CXCR4 does not affect granulocytic cell proliferation. Consistent with its role in regulating neutrophil release, the transit time for labeled neutrophils to appear in the circulation was significantly reduced in CXCR4f/−LysM+/Cre mice (45 hours) compared with wild type mice (72 hours). The half-life (t1/2 ) of neutrophils in the blood was calculated using the formula N=N0e−λt where N0 = the peak number of labeled cells, N = the number of cells at time t and λ = the decay constant. Surprisingly, no difference in the circulating neutrophil half-life was observed in CXCR4f/−LysM+/Cre mice compared to wild type mice (18.3 ± 13.6 hours vs.12.7 ± 9.5 hours respectively, P=0.43). We next performed adoptive transfer experiments to determine the site of neutrophil clearance. Specifically, an equivalent number of bone marrow neutrophils from wild type or CXCR4f/−LysM+/Cre mice were injected intravenously into recipient mice. Donor neutrophils were identified based on differential Ly5 gene expression. By 3 hours post-infusion, the majority of donor neutrophils were cleared from the blood. Compared to wild type neutrophils, CXCR4−/− neutrophils showed reduced homing to the bone marrow [number of donor neutrophils per femur: 6.7 ± 0.3 x 104 (wild type) compared to 2.6 ± 0.8 x 104 (CXCR4−/−); P <0.05]. Conversely, an increased number of CXCR4−/− neutrophils were present in the spleen. These data confirm that CXCR4 expression on neutrophils plays a role in the homing of neutrophils back to the bone marrow. However, neutrophil removal in the bone marrow appears to play only a minor role in neutrophil clearance from the blood, as neutrophil half-life was not significantly affected by the loss of CXCR4.

Maintenance of neutrophil homeostasis in the blood is vital to the proper functioning of the innate immune system. Neutrophil release from the bone marrow is a major regulated determinant of neutrophil homeostasis in the blood, yet the molecular signals that regulate this process are largely unknown. Accumulating evidence suggests that stromal derived factor-1 (SDF-1), through interaction with its major receptor CXCR4, provides a key retention signal for neutrophils in the bone marrow. Definitive proof of this hypothesis has been hampered by the embryonic lethality of CXCR4−/ − mice and the severe engraftment defect observed in recipients of CXCR4−/ − fetal liver hematopoietic stem cells. To circumvent this problem, in the present study we generated gene-targeted mice in which CXCR4 was selectively inactivated only in myeloid cells. The mice (CXCR4f/−LysM+/Cre) expressed Cre-recombinase under the control of the lysozyme-M promoter and contained one CXCR4-null allele while the other was flanked by loxP sites. Myeloid-specific loss of cell-surface CXCR4 expression was documented by flow cytometry. CXCR4 expression in other lineages was documented at levels comparable to controls. Peripheral blood counts in CXCR4f/−LysM+/Cre mice were normal except for marked neutrophilia. The absolute neutrophil count was 1.23 ± 0.76 and 8.05 ± 3.00 in wild type (wt) and CXCR4f/−LysM+/Cre mice, respectively (p &lt;0.001). Of note, no increase in circulating hematopoietic progenitors or immature myeloid cells was observed in the peripheral blood of CXCR4f/−LysM+/Cre mice. In addition, no perturbation in B or T lymphocytes was detected. In the bone marrow, the number of Gr-1+ myeloid cells was reduced to 69.2 ± 24.1% of control mice. No accumulation of granulocytic precursors was observed, suggesting that granulocytic differentiation in CXCR4f/−LysM+/Cre mice was normal. As a metric for quantifying neutrophil distribution, we calculated the percentage of total body neutrophils in the blood (neutrophil distribution index or NDI) as described previously (Immunity, Vol. 17, 413–423, 2002). Consistent with previous reports, 1.0 ± 0.5% of neutrophils were in the blood of wt mice compared with 9.5 ± 4.2% in CXCR4f/−LysM+/Cre mice (p&lt;0.001). Together, these data provide strong genetic evidence supporting the hypothesis that CXCR4 signals are key regulators of neutrophil release from the bone marrow. A broad range of chemokines and cytokines are known to induce neutrophil release from the bone marrow. A recent report (Blood, Vol. 104, 565–571, 2004) showed that treatment of neutrophils with KC resulted in heterologous desensitization of CXCR4. Another report (Blood, Vol. 108, 812–820, 2006) demonstrated that G-CSF downregulates CXCR4. These observations raise the possibility that disruption of CXCR4 signaling may be a common mechanism by which all mobilizing agents induce neutrophil release. To test this hypothesis, we measured neutrophil mobilization one hour after a single 125 ug/kg injection of G-CSF, a time before any detectable change in SDF-1 expression levels in the bone marrow. Neutrophil numbers increased 1.92 ± 0.16 fold over baseline in wt mice (p&lt;0.05). In contrast, no change was detected in CXCR4f/−LysM+/Cre mice (1.05 ± 0.39 fold over baseline, p=ns). These data show that neutrophil mobilization by G-CSF is dependent upon CXCR4. Studies are underway to characterize neutrophil mobilization by other mobilizing agents in CXCR4f/−LysM+/Cre mice.