Fig 1.
CCL2 acts on CXCR4-positive cells during inflammation in the bone marrow.
(Inset) Transgenic reporter mice used in this study. 1) CCL2::CCL2-mRFP (CCL2 protein level reporter), 2) CCL2::mRFP (CCL2 transcriptional reporter), and 3) CCR2::CCR2-EGFP (CCR2 protein level reporter) (A-G) Bone marrow of CCL2::CCL2-mRFP; CCR2::CCR2-EGFP double transgenic mice. A differential interference contrast (A) and an overlaid fluorescent image (B) show CCL2-mRFP and CCR2-EGFP cells in close contact (arrows). (C-G) Under normal conditions, CCR2-EGFP mostly localizes to cell surface (green arrow). After LPS injection, most CCR2-EGFP cells are activated by CCL2, which is evident in granular CCR2-EGFP signals and endocytic vesicles containing both CCL2-mRFP and CCR2-EGFP (yellow arrow). (H-I) Bone marrow of CCL2::mRFP; CCR2::CCR2-EGFP double transgenic mice. After LPS injection, most CCR2-EGFP cells are activated by endogenous CCL2, which is evident in granular CCR2-EGFP (white arrow). Scale bars, 10 μm.
Fig 2.
CCR2-positive cells are neighbored by CXCL12-positive cells in the bone marrow.
(inset) Transgenic reporter mice used in this study. (A-C) Bone marrow of CCL2::CCL2-mRFP; CXCR4::EGFP double transgenic mice. Note that CXCR4::EGFP is a transcriptional reporter, and therefore EGFP localizes to the entire cell in which CXCR4 is expressed (C). Most CCL2-mRFP cells are in close proximity to CXCR4::EGFP cells (green arrow) under normal conditions. After LPS injection, these CXCR4::EGFP cells now contain CCL2-mRFP granules (yellow arrow), indicating endocytosis of CCL2-mRFP/CCR2 (unlabeled endogenous CCR2) (B’). (D-F) Bone marrow of CXCL12::CXCL12-mRFP; CCR2::CCR2-EGFP mice. CCR2-postive cells (green arrow) are often in contact with CXCL12-positive cells (red arrow). Upon LPS injection, CCR2 is activated (white arrow: granular CCR2-EGFP signal) independently from CXCL12 signaling (because CXCL12-mRFP is not endocytosed by CCR2 positive cells). (G-I) Bone marrow of CXCL12::CXCL12-mRFP; CXCR4:EGFP mice. CXCR4 activation, which could have been visualized by CXCL12 (red arrow) endocytosis by CXCR4-positive cells (green arrow), is not increased by LPS injections. Scale bars, 10 μm.
Fig 3.
CCR2 and CXCR4 are co-expressed by bone marrow cells.
(A) Acutely isolated bone marrow cells were plated on a cover slip and their responses to CCL2 (10 nM) and CXCL12 (10 nM) were measured by Fura 2-based calcium imaging. The response to ATP, which increases intracellular [Ca2+] by purinergic receptors, indicates that the recorded cell was alive. (B) Venn diagram showing percentage of CCL2- and/or CXCL12-responsive cells. (C) More than 80% of CCL2-responsive cells also responded to CXCL12.
Fig 4.
(A) Calcium imaging on acutely isolated bone marrow cells. A low (10 nM) or high (40 nM) of CCL2 was treated for 5 min before a CXCL12 application. ATP-responsiveness at the end of experiment shows cell viability. As shown in Fig 3, most CCL2-responsive cells also responded to CXCL12 (yellow). When a higher dose of CCL2 was pre-treated, however, most cells did not respond to CXCL12 (red). (B) Quantification of calcium imaging. n = 50, 95, and 30, respectively; *p<0.05 vs. 10 nM, ***p<0.001 vs. 10 nM or 20 nM; Fisher’s exact test.
Fig 5.
CCR2-positive cells can be mobilized by CXCR4 inhibition.
CCR2::CCR2-EGFP mice were injected with AMD3100 or saline, and the percentage of EGFP-positive cells in the bone marrow or in the blood was measured by flow cytometry. AMD3100 injections decreased CCR2-EGFP cells in the bone marrow (A), and increased them in the bloodstream (B). n = 4 each; **p<0.01; Mann-Whitney test.
Fig 6.
Proposed model: monocyte egress by CCR2-mediated cross-desensitization of CXCR4.
Monocytes in the bone marrow co-express CXCR4 and CCR2. These cells are juxtaposed to bone marrow-resident cells, which express their respective ligands, CXCL12 and CCL2. (A) Under normal conditions, monocytes are retained by CXCR4 signaling, which is activated by constitutively secreted CXCL12. CCR2 on these monocytes is not active as its ligand CCL2 is stored but not released. (B) During inflammation, stored CCL2 is rapidly released and activates CCR2, which desensitizes CXCR4 and promotes cell migration.