Localized CCR2 Activation in the Bone Marrow Niche Mobilizes Monocytes by Desensitizing CXCR4

<div><p>Inflammatory (classical) monocytes residing in the bone marrow must enter the bloodstream in order to combat microbe infection. These monocytes express high levels of CCR2, a chemokine receptor whose activation is required for them to exit the bone marrow. How CCR2 is locally activated in the bone marrow and how their activation promotes monocyte egress is not understood. Here, we have used double transgenic lines that can visualize CCR2 activation <i>in vivo</i> and show that its chemokine ligand CCL2 is acutely released by stromal cells in the bone marrow, which make direct contact with CCR2-expressing monocytes. These monocytes also express CXCR4, whose activation immobilizes cells in the bone marrow, and are in contact with stromal cells expressing CXCL12, the CXCR4 ligand. During the inflammatory response, CCL2 is released and activates the CCR2 on neighboring monocytes. We demonstrate that acutely isolated bone marrow cells co-express CCR2 and CXCR4, and CCR2 activation desensitizes CXCR4. Inhibiting CXCR4 by a specific receptor antagonist in mice causes CCR2-expressing cells to exit the bone marrow in absence of inflammatory insults. Taken together, these results suggest a novel mechanism whereby the local activation of CCR2 on monocytes in the bone marrow attenuates an anchoring signalling provided by CXCR4 expressed by the same cell and mobilizes the bone marrow monocyte to the blood stream. Our results also provide a generalizable model that cross-desensitization of chemokine receptors fine-tunes cell mobility by integrating multiple chemokine signals.</p></div

Proposed model: monocyte egress by CCR2-mediated cross-desensitization of CXCR4.

<p>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.</p

CCR2 and CXCR4 are co-expressed by bone marrow cells.

<p>(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.</p

CCR2-positive cells are neighbored by CXCL12-positive cells in the bone marrow.

<p>(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.</p

CCL2 acts on CXCR4-positive cells during inflammation in the bone marrow.

<p>(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.</p

Subcellular localization of the wild-type and mutant <i>EPHA2</i> receptors on transfected EphA2^−/− MEF (E13.5) cells.

<p>EphA2 knockout MEF cells expressing wild-type or mutant <i>EPHA2</i> were incubated with clustered ephrin-A5-Fc before fixation and treated with anti-Fc antibodies (red). After washing with PBS, cells were counterstained with anti-EphA2 antibody (green). Images were captured using a Nikon Eclipse C1 confocal microscope. Scale bar, 50 µm.</p

Ligand-stimulated EPHA2 activation regulates Akt and Erk activation.

<p>(<b>A, B</b>) Mutant EPHA2 proteins exhibit reduced activation of Akt and Erk by ephrin-A5. HEK293T cells were grown to confluence and serum-starved for 24 hours. 2 µg/mL cross-linked ephrin-A5-Fc was then added to the starvation media and cell lysates were immunoblotted with anti-phospho-Akt (Ser473) or anti-phospho-Erk (1/2), and then reprobed with anti-α-tubulin as a loading control. (<b>C</b>) Inactivation of EphA2 gene leads to reduction of Akt activity in mouse lenses. Each lens was prepared from 22 days old mice and extracted with lysis buffer. Total lens proteins were resolved by SDS-PAGE and western blot analysis was performed using indicated antibodies as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036564#s4" target="_blank">Materials and Methods</a>. The blot was reprobed with anti-α-tubulin as a loading control.</p

SAM domain of <i>EPHA2</i> is essential for ligand-independent promotion of cell migration.

<p>(<b>A</b>) Mutant <i>EPHA2</i> genes fail to promote αTN4-1 cell migration. αTN4-1 cells were grown to confluency and serum-starved for 24 hours. A scratch wound was made with a micropipette tip and the edge of cells was marked. 2 µg/mL cross-linked ephrin-A5-Fc was then added to the starvation media, and cells were allowed to migrate toward the center of the wound and photographed at the indicated times (representative figure of three independent experiments). The position of the initial scratch is indicated by dotted lines. Scale bar, 500 µm. (<b>B</b>) Quantification of the effects of <i>EPHA2</i> genes on αTN4-1 cell migration. The graphs represent the measurement of migration distance from three independent experiments. Mean values are presented with S.D as indicated. Statistical differences were analyzed using one-way analysis of variance (ANOVA) or calculated by a two-tailed student t-test. <b><i>Black asterisks</i></b><b>,</b> comparison between time 0 and 24 hours and time 0 and 48 hours; <b><i>Blue asterisks</i></b><b>,</b> comparison between the mock groups and the listed wild-type or mutant <i>EPHA2</i> genes at 24 or 48 hours; <b><i>Red asterisks</i></b><b>,</b> comparison between untreated and treated conditions at 24 or 48 hours. ***, <i>P</i><0.001; **, <i>P</i><0.01; *, <i>P</i><0.05; and ns, not significant. Values of <i>P</i><0.05 were considered to be statistically significant.</p

<i>EPHA2</i> cataract mutations in the SAM domain.

<p>(<b>A</b>) Schematic diagram showing the domains of EPHA2 receptor and the locations of four SAM domain mutations found in human cataracts (c.2819C>T; c.2915_2916delTG; c.2826-9G>A; and c.2842G>T) in the EPHA2 gene. FN-III: fibronectin type-III domain; TM: transmembrane domain; Kinase: protein tyrosine kinase domain; SAM: sterile-α-motif domain; P: PDZ-binding motif. The SAM domain comprises 5 α-hecices (H1–5). (<b>B</b>) Reduction of mutant EPHA2 protein levels in transfected cells expressing <i>EPHA2</i> mutants. Protein levels of <i>EPHA2</i> mutants are decreased in both HEK293T and αTN4-1 cells. The blot was reprobed with anti-α-tubulin as a loading control. The graphs represent the quantification of relative band intensity of EphA2 as connected by the levels of α-tubulin from three independent experiments. Total EphA2 protein band intensity was determined using ImageJ software. Mean values are presented with S.D as indicated. Statistical differences between multiple groups were analyzed using one-way analysis of variance (ANOVA). ***, <i>P</i><0.001; **, <i>P</i><0.01; *, <i>P</i><0.05; and ns, not significant. Values of <i>P</i><0.05 were considered to be statistically significant. (<b>C, D</b>) No difference between wild-type and mutant <i>EPHA2</i> genes in transcription levels. (<b>C</b>) Semi-quantitative RT-PCR and (<b>D</b>) Real-time PCR for wild-type and mutant <i>EPHA2</i> genes were performed using total RNA, isolated from transfected HEK293T cells. GAPDH transcript levels are used as controls. The graphs represent the quantification of western blots from three independent experiments.</p

Tyrosine phosphorylation of EPHA2 receptor by ephrin-A5 is not affected by SAM domain mutations.

<p>(<b>A, B</b>) Ephrin-A5 ligand stimulates EPHA2 phosphorylation. HEK293T (<b>A</b>) and αTN4-1 (<b>B</b>) cells were grown to confluence and growth factor-starved for 24 hours. 2 µg/mL cross-linked ephrin-A5-Fc was then added to the starvation media and cell lysates were immunoblotted with indicated antibodies. Western blot analysis was performed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036564#s4" target="_blank">Materials and Methods</a>. The blot was reprobed with anti-α-tubulin as a loading control. (<b>C</b>) The ratios of levels of phospho-EphA2 to total EphA2 are similar between the wild-type and mutant EPHA2 proteins. The graphs show total band intensity of anti-phospho-EphA2 immunoblot to total EphA2 and represent the average of three independent experiments. Quantification of phospho-EphA2 protein/total EphA2 protein levels was performed using ImageJ software. Mean values are presented with S.D as indicated. Statistical differences between multiple groups were analyzed using one-way analysis of variance (ANOVA). Values of <i>P</i><0.05 were considered to be statistically significant. ns: No statistically significant difference between the two groups.</p