CXCR2 and CXCR4 antagonistically regulate neutrophil trafficking from murine bone marrow

Abstract

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.

Figure 1. Cxcr2^–/– neutrophils are selectively retained in the bone marrow of mixed chimeras.

(A) Generation of mixed chimeras. Bone marrow cells from WT and Cxcr2^–/– mice (expressing Ly5.1 and Ly5.2, respectively; 1 × 10⁶ cells from each) were mixed in a 1:1 ratio and transplanted into lethally irradiated congenic WT recipients (expressing Ly5.1). Mice were analyzed 6–8 weeks after transplantation. (B) Representative dot plots showing the contribution of WT and Cxcr2^–/– cells (with and without Ly5.1, respectively) to neutrophils (Gr-1^hi) in the blood and bone marrow. (C) Quantitation of mature neutrophils (Gr-1^hiSSC^hi) in the blood, bone marrow, and spleen. (D) NDI was calculated as described in Methods to estimate the percentage of total body neutrophils in the blood. (E) Number of B lymphocytes (B220⁺) or T lymphocytes (CD3⁺) in the blood (left) and B lymphocytes in the bone marrow (right). T lymphocyte chimerism was assessed 6 months after transplantation (n = 3). (F) Number of WT or Cxcr2^–/– CFU in culture (CFU-C) or CFU-granulocyte (CFU-G) in the bone marrow (n = 3). n = 27 (blood); n = 6 (bone marrow and spleen) from at least 3 independent transplantations, unless otherwise indicated.

Figure 2. CXCR2 deficiency produces a myelokathexis-like phenotype.

(A) Representative dot plots of mixed chimera bone marrow showing the percentage of Gr-1^hiSSC^hi cells within the total Gr-1⁺ myeloid cell population for WT and Cxcr2^–/– cells. (B) Percent Gr-1^hiSSC^hi cells within the total Gr-1⁺ myeloid cell population for n = 7 chimeric mice from 2 independent transplants. (C) Representative photo­micrographs of sorted WT and Cxcr2^–/– Gr-1⁺ cells. Scale bars: 20 μm. (D) Manual leukocyte differentials of sorted cells from n = 5 mice from 2 transplants. Blast, myeloblast; Band, band neutrophil; Seg, segmented neutrophil. ***P < 0.001, 2-way ANOVA.

Figure 3. Mobilization of Cxcr2^–/– neutrophils by G-CSF is impaired.

(A) Mixed chimeras (n = 5) were given a single injection of G-CSF (125 μg/kg), and the absolute neutrophil count for each genotype was determined 1.5 hours after injection. (B) G-CSF (125 μg/kg/d, twice daily) was administered to a separate cohort of n = 5 chimeric mice for 5 days, and blood neutrophils were quantified. (C) Number of WT or Cxcr2^–/– Gr-1⁺SSC^hi cells in the bone marrow and spleen after 5 days of G-CSF administration. (D) The calculated NDI after 5 days of G-CSF. ^†P < 0.05, ^‡P < 0.01 versus time 0; **P < 0.01, ***P < 0.001 versus Cxcr2^–/– at the same time point; 2-way ANOVA.

Figure 4. CXCR2 and CXCR4 signals interact antagonistically to regulate neutrophil release.

(A) Representative dot plots show cell surface CXCR4 expression of WT and Cxcr2^–/– Gr-1⁺SSC^hi bone marrow cells from a Cxcr2^–/– mixed chimera (right), and cells treated with an isotype-matched antibody (left), shown as controls. Bar graphs show CXCR4 MFI and percent CXCR4⁺ cells from n = 5 mice. White bars, WT; black bars, Cxcr2^–/–. (B) Cxcr2^–/– mixed chimeras (n = 5) were given a single subcutaneous injection of AMD3100 (5 mg/kg), and neutrophils were quantified at the indicated times. (C and D) Number of neutrophils in the bone marrow and spleen (C) and NDI (D) at 1 hour after AMD3100 administration (n = 3). (E and F) MKO (n = 10) and DKO (n = 4) mixed chimeras were established as described in Figure 1. Blood, bone marrow, and spleen neutrophils (E) and NDI (F) were quantified 7 weeks after transplantation. ***P < 0.001, 1-way ANOVA. (G) MKO mixed chimeras (n = 3) were given a subcutaneous injection of GROβ (100 μg/kg), and the number of WT and Cxcr4^–/– neutrophils in the blood was measured after 30 minutes. (B and G) ^†P < 0.05, ^‡P < 0.01 versus time 0; **P < 0.01, ***P < 0.001 versus WT at the same time point; 2-way ANOVA.

Figure 5. CXCR2 ligands are produced by bone marrow stromal cells and regulated by G-CSF.

(A) Bone marrow endothelial cells (7AAD^–CD45^loTer119^loCD31⁺) or osteoblasts (7AAD^–CD45^loTer119^loGFP⁺) were isolated by cell sorting from Col2.3:GFP transgenic mice. Shown are representative dot plots depicting the sorting strategy. (B) Normalized gene chip signal at baseline for all chemokines with an average signal intensity of greater than 400 in at least 1 of the cell types. When more than 1 probe set existed, the highest signal was selected. Ppbp encodes for CXCL7, and Mif is a nonchemokine ligand for CXCR2 and CXCR4 (60). (C) Expression of CXCR2 and CXCR4 ligands in endothelial cells from WT mice at baseline or after G-CSF administration. (D) CXCL2 protein in bone marrow supernatant at baseline or after G-CSF, measured by ELISA (n = 4 mice per group). The dashed line represents the limit of detection for the assay. *P < 0.05; **P < 0.01; ***P < 0.001; 2-way ANOVA.

Figure 6. Tug-of-war model of neutrophil trafficking from the bone marrow.

See Discussion for details.