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 < 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.


No relevant conflicts of interest to declare.

Author notes


Asterisk with author names denotes non-ASH members.