CXCR2 Signals Act in Concert with CXCR4 to Regulate Neutrophil Release From the Bone Marrow.

Abstract 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 CXCR2 −/− neutrophils in the bone marrow (47 ± 9% of Gr-1 hi SSC hi cells in the bone marrow were derived from CXCR2 −/− cells; P CXCR2 −/− neutrophils in the blood was reduced to 0.4 ± 0.1% ( P 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 CXCR2 −/− neutrophils in the blood increased after treatment, but the percentage in the blood (2.5 ± 0.7%) was less than wild-type (P 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.