CXCR4 Signals Regulate Neutrophil Release from the Bone Marrow but Not Clearance from the Circulation.
Authors: Eash Kyle J. , Means Jacquelyn M. , Woloszynek Jill R. , Liu Fulu , Link Daniel C. 
Published: October 2019

Abstract
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.