CXCR2 and CXCR4 antagonistically regulate neutrophil trafficking from murine bone marrow (original) (raw)
Absence of CXCR2 results in abnormal retention of neutrophils in the bone marrow. Consistent with previous reports (34, 37), we observed marked neutrophilia in Cxcr2–/– mice maintained under SPF conditions (absolute count, Cxcr2–/–, 4.63 ± 1.58 × 106 neutrophils/ml; congenic WT, 0.69 ± 0.06 × 106 neutrophils/ml; P = 0.04). To determine whether this phenotype was due to a cell intrinsic effect of a loss of CXCR2 signaling, mixed bone marrow chimeras were generating by transplanting a 1:1 ratio of WT and Cxcr2–/– bone marrow cells into irradiated congenic mice (Figure 1A). Blood neutrophil counts in the Cxcr2–/– mixed chimeras were reduced in comparison with mice reconstituted with WT cells alone (1.08 ± 0.08 × 106 versus 1.81 ± 0.29 × 106 neutrophils/ml; P = 0.003), which suggests that the neutrophilia in Cxcr2–/– mice is the result of a cell-extrinsic mechanism.
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-1hi) in the blood and bone marrow. (C) Quantitation of mature neutrophils (Gr-1hiSSChi) 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.
In the mixed chimeras, the number of Cxcr2–/– neutrophils in the blood was reduced compared with that in the bone marrow (Figure 1, B and C). Whereas 65.3% ± 7.6% of neutrophils in the bone marrow were derived from Cxcr2–/– cells, only 25.0% ± 3.5% of neutrophils in the blood were from Cxcr2–/– cells (P < 0.0001). Of note, the number of neutrophils in the spleen, another reservoir for neutrophils, was comparable between Cxcr2–/– and WT cells (Figure 1C). Neutrophil trafficking from the bone marrow was estimated by calculating the percentage of neutrophils in the blood relative to the total number of neutrophils in the blood, bone marrow, and spleen (neutrophil distribution index; NDI; ref. 5). Consistent with previous studies (22, 38), under basal conditions, 1.84% ± 0.32% of WT neutrophils were present in the blood (Figure 1D). In contrast, the percentage of Cxcr2–/– neutrophils in the blood was 0.57% ± 0.18% (P = 0.02). No perturbation in other hematopoietic lineages was observed (Figure 1E), which indicates that the observed differences in neutrophil chimerism are not caused by altered engraftment of Cxcr2–/– hematopoietic stem cells. Consistent with this observation, the number and cytokine responsiveness of myeloid progenitors in the bone marrow were comparable between WT and Cxcr2–/– cells (Figure 1F).
Myelokathexis is characterized by the accumulation of mature, often hypersegmented or dysplastic, neutrophils in the bone marrow (10). Consistent with this phenotype, we observed that the percentage of Gr-1hiSSChi cells — representing the most mature neutrophils (39) — relative to the total Gr-1+ myeloid cell population was higher for Cxcr2–/– than WT cells (Figure 2, A and B). To confirm this finding, Cxcr2–/– and WT Gr-1+ myeloid cells were sorted from the bone marrow of the mixed chimeras, and manual leukocyte differentials were performed. Cxcr2–/– cells showed an increase in the proportion of highly segmented, occasionally dysplastic-appearing, neutrophils (Figure 2, C and D). Collectively, these data showed that CXCR2 deficiency results in a myelokathexis-like phenotype with a cell-intrinsic retention of neutrophils in the bone marrow.
CXCR2 deficiency produces a myelokathexis-like phenotype. (A) Representative dot plots of mixed chimera bone marrow showing the percentage of Gr-1hiSSChi cells within the total Gr-1+ myeloid cell population for WT and Cxcr2–/– cells. (B) Percent Gr-1hiSSChi 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.
Neutrophil mobilization by G-CSF is impaired in the absence of CXCR2. Because it is the principal cytokine regulating emergency granulopoiesis (40), we next measured the short-term (1–2 hours) and long-term (5 days) neutrophil responses to G-CSF in the Cxcr2–/– mixed chimeras. Consistent with previous reports (5, 22), administration of G-CSF resulted in a 2.3- ± 0.5-fold increase in the blood of WT neutrophils within 2 hours (Figure 3A). In contrast, there was no significant increase in Cxcr2–/– neutrophils. After the full 5-day course of G-CSF, a significant increase in total neutrophils (both WT and Cxcr2–/–) in the blood, bone marrow, and spleen was observed in the mixed chimeras (Figure 3, B and C, compare with Figure 1C). Note that there were fewer Cxcr2–/– than WT neutrophils in the bone marrow because this cohort of mice by chance had lower engraftment of _Cxcr2–/–_cells (as measured by B lymphocyte chimerism; data not shown). Neutrophil release, as measured by the NDI, increased in response to 5-day G-CSF treatment in both genotypes (Figure 3D, compare with Figure 1D). However, the percentage of Cxcr2–/– neutrophils in the blood after G-CSF administration was still significantly lower than that for WT cells. These data show that maximal blood neutrophil responses to G-CSF require CXCR2 signaling.
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+SSChi 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.
CXCR2 antagonistically regulates CXCR4-mediated neutrophil retention in the bone marrow. Previous studies have established a dominant role for CXCR4 signals in the retention of neutrophils in the bone marrow (20, 22, 41–45). Because CXCR2 signaling has previously been shown to regulate CXCR4 cell surface expression through heterologous desensitization and receptor internalization (43, 46), we first assessed CXCR4 expression on Cxcr2–/– neutrophils. However, cell surface expression of CXCR4 on bone marrow neutrophils was similar between WT and Cxcr2–/– cells (MFI, 107 ± 7 and 100 ± 12, respectively; P = 0.4; Figure 4A), arguing against a simple mechanism in which the absence of CXCR2 signals results in neutrophil retention through increased CXCR4 expression.
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+SSChi 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.
To more directly assess the relationship between CXCR2 and CXCR4 signals in the regulation of neutrophil trafficking, we treated mixed chimeras with AMD3100, a small-molecule CXCR4 antagonist. At 1 hour after AMD3100 administration, a 3.8- ± 1.2-fold increase in WT neutrophils in the blood was observed (Figure 4B). In contrast, no increase in Cxcr2–/– neutrophils in the blood was observed, despite the fact that the majority of neutrophils in the bone marrow 1 hour after AMD3100 administration were of CXCR2–/– origin (Figure 4, B and C). Accordingly, the NDI for Cxcr2–/– cells after AMD3100 administration was dramatically lower than that of WT cells (0.53% ± 0.21% versus 13.0% ± 3.27%; P = 0.02; Figure 4D), which suggests that neutrophil mobilization in response to transient CXCR4 inhibition is dependent on CXCR2.
We previously reported that mice carrying a myeloid-specific KO of CXCR4 (LysMCre/+Cxcr4fl/–; referred to herein as MKO) displayed marked basal neutrophilia (22). To study the genetic interaction of the _Cxcr2_- and _Cxcr4_-null alleles, we crossed MKO mice with the Cxcr2–/– mice to generate double-KO mice (LysMCre/+Cxcr2–/–Cxcr4fl/–; referred to herein as DKO). Similar to the Cxcr2–/– and MKO mice, DKO mice displayed marked neutrophilia at baseline (data not shown). To examine the cell-intrinsic properties of neutrophils lacking both CXCR2 and CXCR4, mixed chimeras were generated as described above using DKO or, as a control, MKO bone marrow cells. Recipient mice showed the expected level of donor engraftment in the bone marrow, with 52.1% ± 4.8% (DKO) and 62.2% ± 2.3% (MKO) of B lymphocytes derived from mutant cells. As expected, mixed chimeras containing MKO cells showed a marked redistribution of Cxcr4–/– neutrophils into the blood (Figure 4, E and F). Surprisingly, a similar phenotype was observed in DKO chimeras, which showed that loss of CXCR2 signals cannot rescue the neutrophilic phenotype of CXCR4-deficient neutrophils.
To examine whether CXCR4 signals are required for mobilization by CXCR2 ligands, MKO mixed chimeras were given a single injection of the CXCR2 agonist GROβ. Whereas the number of WT neutrophils in the blood of MKO chimeras increased 3.8- ± 0.5-fold 30 minutes after GROβ administration, only a minimal increase in Cxcr4–/– neutrophils was observed, which suggests that neutrophil release induced by CXCR2 activation is at least partially dependent on CXCR4 (Figure 4G). Collectively, these data showed that CXCR4 and CXCR2 antagonistically regulate neutrophil release from the bone marrow, with CXCR4 playing a dominant role.
Expression of chemokines by osteoblasts and endothelial cells in the bone marrow. Previous studies have established that bone marrow stromal cells, in particular osteoblasts and endothelial cells, are the major source of CXCL12 in the bone marrow (21, 25, 27, 45, 47–50). However, the expression of other chemokines, specifically ELR+ CXCR2 ligands, in bone marrow stromal cells is unknown. To address this issue, we analyzed stromal cells from the bone marrow of transgenic mice expressing GFP in osteoblast lineage cells (Col2.3-GFP; refs. 50, 51). Specifically, CD45loTer119lo stromal cells were sorted into osteoblast and endothelial fractions (GFP+ and CD31+, respectively), which were then subjected to RNA expression profiling (Figure 5A). Of note, expression of endothelial- or osteoblast-specific genes was appropriately enriched in the relevant cell fraction, demonstrating the fidelity of our sorting strategy (Supplemental Tables 1 and 2; available online with this article; doi:10.1172/JCI41649DS1). As reported previously (27, 50), constitutively high expression of CXCL12 was observed in osteoblasts and endothelial cells, with higher expression in osteoblasts (Figure 5B). CXCR2 ligands CXCL1 and CXCL2 were also constitutively expressed in osteoblasts and endothelial cells, but with higher endothelial expression.
CXCR2 ligands are produced by bone marrow stromal cells and regulated by G-CSF. (A) Bone marrow endothelial cells (7AAD–CD45loTer119loCD31+) or osteoblasts (7AAD–CD45loTer119loGFP+) 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.
To examine the effect of G-CSF on chemokine expression in the bone marrow microenvironment, endothelial cells were isolated from the bone marrow after G-CSF administration. Osteoblasts were not sorted, since their number is markedly reduced by G-CSF (27, 48, 50, 52). Of note, there was no change in bone marrow endothelial cell number (D.C. Link, unpublished observation). RNA expression profiling showed that CXCL2 expression in bone marrow endothelial cells was induced 2.7- ± 0.3-fold by G-CSF, whereas CXCL12 mRNA was modestly reduced to 47% ± 3% of its basal level; other chemokines remained unchanged (Figure 5C). Consistent with the mRNA data, CXCL2 protein was detected in the bone marrow supernatant at baseline, with increased expression after G-CSF administration, although the difference was not statistically significant (Figure 5D). Since osteoblast number is markedly reduced after G-CSF administration, these data suggest that the balance of expression in the bone marrow from proretention (CXCL12) to mobilizing chemokines (CXCL1 and CXCL2) may contribute to neutrophil mobilization by G-CSF.