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. 2009 Jun 24;29(25):8051-62.
doi: 10.1523/JNEUROSCI.0485-09.2009.

Visualization of chemokine receptor activation in transgenic mice reveals peripheral activation of CCR2 receptors in states of neuropathic pain

Hosung Jung  1 , Sonia Bhangoo, Ghazal Banisadr, Caroline Freitag, Dongjun Ren, Fletcher A White, Richard J Miller
Affiliations

Visualization of chemokine receptor activation in transgenic mice reveals peripheral activation of CCR2 receptors in states of neuropathic pain

Hosung Jung et al. J Neurosci. .

Abstract

CCR2 chemokine receptor signaling has been implicated in the generation of diverse types of neuropathology, including neuropathic pain. For example, ccr2 knock-out mice are resistant to the establishment of neuropathic pain, and mice overexpressing its ligand, monocyte chemoattractant protein-1 (MCP1; also known as CCL2), show enhanced pain sensitivity. However, whether CCR2 receptor activation occurs in the central or peripheral nervous system in states of neuropathic pain has not been clear. We developed a novel method for visualizing CCR2 receptor activation in vivo by generating bitransgenic reporter mice in which the chemokine receptor CCR2 and its ligand MCP1 were labeled by the fluorescent proteins enhanced green fluorescent protein and monomeric red fluorescent protein-1, respectively. CCR2 receptor activation under conditions such as acute inflammation and experimental autoimmune encephalomyelitis could be faithfully visualized by using these mice. We examined the status of CCR2 receptor activation in a demyelination injury model of neuropathic pain and found that MCP1-induced CCR2 receptor activation mainly occurred in the peripheral nervous system, including the injured peripheral nerve and dorsal root ganglia. These data explain the rapid antinociceptive effects of peripherally administered CCR2 antagonists under these circumstances, suggesting that CCR2 antagonists may ameliorate pain by inhibiting CCR2 receptor activation in the periphery. The method developed here for visualizing CCR2 receptor activation in vivo may be extended to G-protein-coupled receptors (GPCRs) in general and will be valuable for studying intercellular GPCR-mediated communication in vivo.

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Figures

Figure 1.
Figure 1.
Visualization of MCP1–CCR2 interactions in vitro. A–F, In experiments to demonstrate this phenomenon in cell culture, HEK293 cells were transfected with either a chemokine ligand construct (MCP1–mRFP1 or SDF1–mRFP1) or a chemokine receptor construct (CCR2–EGFP or CXCR4–EYFP). After 24 h, ligand-expressing cells (red arrow) were cocultured with receptor-expressing cells (green arrow). A, Constitutively released MCP1–mRFP1 entered CCR2–EGFP-expressing cells, resulting in the formation of endocytic vesicles containing both MCP1–mRFP1 and CCR2–EGFP. B, The interactions between MCP1–mRFP1- and CCR2–EGFP-expressing cells were completely blocked by a CCR2 receptor antagonist (CCR2–RA; 300 nm). C, D, SDF1–mRFP1 did not interact with CCR2–EGFP (C), and MCP1–mRFP1 did not interact with CXCR4–EYFP (D). E, F, SDF1–mRFP1 did induce the endocytosis of CXCR4–EYFP (E), and this was blocked by a specific CXCR4–RA (AMD3100; 30 nm) (F). Scale bars, 10 μm. G, Proposed model for the assessment of MCP1–CCR2 interactions. MCP1–mRFP1 is localized to secretory vesicles (top space) and CCR2–EGFP to the plasma membrane (bottom space). When signaling occurs, released MCP1–mRFP1 (1) binds to CCR2–EGFP expressed on CCR2-expressing cells (2). Binding induces endocytosis of the MCP1–mRFP1/CCR2–EGFP complexes (3). Indications of CCR2 activation include the loss of membrane-localized EGFP signal and an increase in yellow/orange intracellular vesicles containing both MCP1–mRFP1 and CCR2–EGFP (bottom diagrams).
Figure 2.
Figure 2.
Generation of BAC transgenic reporter mice. A, BAC clones were modified by recombineering. EGFP or mRFP1 was inserted in place of the start codon to generate a transcriptional reporter (1) or the stop codon to generate a protein level reporter (2). CXCR4∷EGFP transcriptional reporter mice were obtained from the GENSAT project. The diagrams indicate expected subcellular localization of reporter proteins. pA, Poly-A signal. B, C, MCP1 reporter genes were expressed by microglia during an inflammatory response elicited by LPS injection (24 h after injection). Brain sections of LPS-injected mice are shown. D, E, The CXCR4 and CCR2 reporter genes were expressed by a subset of leukocytes under normal conditions. Blood smear samples were prepared from naive animals, and nucleated cells were identified by DAPI staining (blue). The reporter gene products (i.e., EGFP or mRFP1) of the transcriptional reporter mice (B, D) diffusely filled entire cells including the nuclei, whereas those of the protein level reporter mice (C, E) were limited to the appropriate subcellular localizations (i.e., MCP1–mRFP1 to vesicles, and CCR2–sEGFP mostly to the plasma membrane). Scale bars, 20 μm.
Figure 3.
Figure 3.
Interactions between MCP1- and CCR2-expressing cells can be visualized using BAC transgenic reporter mice. MCP1∷MCP1–mRFP1; CCR2∷CCR2–EGFP mice were injected with LPS. A, B, After the injection (24 h), MCP1 (red) was significantly upregulated across the brains of these animals (B) in contrast to naive animals which showed no MCP1 expression (A). C, MCP1 was upregulated mostly by microglia (purple arrows), which were identified by IBA-1-ir. D, GFAP-expressing astrocytes generally did not upregulate MCP1–mRFP1 (red arrows). A few cells which were closely associated with blood vessels and upregulated MCP1 did not express IBA-1 or GFAP (A, a white concave arrow; B–D, red concave arrows). The pictures were taken from the mediofrontal cortex. cc, Corpus callosum. E, F, After the injection (48 h), interactions between MCP1- and CCR2-expressing cells could be observed in the brain. In this example, two perivascular microglia (G; IBA-1-ir) are interacting with one circulating leukocyte (F; CCR2–EGFP+). Microglia not only upregulated MCP1 but also released MCP1 to activate the CCR2–EGFP-expressing leukocyte (E). H, The internalized vesicles contained both MCP1–mRFP1 and CCR2–EGFP, which indicates that the MCP1-expressing microglia had activated CCR2 receptors expressed by the leukocyte. X-Z and Y-Z cross-sectional images across the white lines are shown for H. bv, Blood vessel. Scale bars: A–D, 20 μm; E, F, 10 μm.
Figure 4.
Figure 4.
Activation of CCR2 receptors in bone marrow monocytes during an inflammatory response can be visualized in the BAC transgenic mice. A–D, MCP1∷mRFP1; CCR2∷CCR2–EGFP mice (A, B) and MCP1∷MCP1–mRFP1; CCR2∷CCR2–EGFP (C, D) mice were injected with LPS (B, D). After 24 h, bone marrow sections were imaged using a confocal microscope. Compared with CCR2 receptor expression under naive conditions (A, C; green arrow), activation of CCR2 receptors was evident after LPS treatment as indicated by internalization and loss of membrane localized CCR2–EGFP (B, D; concave arrow). When MCP1 protein reporter mice were used, MCP1 uptake by CCR2-expressing cells could also be visualized in yellow vesicles (D; yellow concave arrow). E, The activation of CCR2 receptors in the bone marrow was reversed by the CCR2 receptor antagonist (CCR2–RA). MCP1∷MCP1–mRFP1; CCR2∷CCR2–EGFP mice were injected with LPS alone (D) or LPS and CCR2–RA (E). The CCR2–RA was injected intraperitoneally (50 mg/kg) three times at 4 h intervals during the last 12 h period of the inflammatory response. The CCR2–RA increased membrane localization and decreased intracellular localization of CCR2–EGFP (green arrow; E). In some cases, MCP1–mRFP1-containing vesicles remained after CCR2–EGFP-containing vesicles had disappeared (white arrow). Scale bars, 10 μm.
Figure 5.
Figure 5.
Activation of CCR2 receptors in peripheral leukocytes during the pathogenesis of EAE can be visualized in the BAC transgenic mice. EAE was induced in MCP1:mRFP1; CCR2∷CCR2–EGFP mice (B) and MCP1∷MCP1–mRFP1; CCR2∷CCR2–EGFP mice (C). The pictures were taken from the white matter from coronal cerebellar sections. Unlike control conditions (A), there was a marked infiltration of leukocytes (B, C; CCR2–EGFP+) into the parenchyma of the cerebellum (examined on postoperative day 14). Magnified views of the boxed areas are shown below B and C. Although there were close interactions between MCP1 (red arrow, cell body; red arrowhead, cellular process)- and CCR2-expressing cells (green concave arrow) (B, C), only MCP1–mRFP1 (C), but not mRFP1 alone (B), was transferred to CCR2–EGFP-expressing leukocytes as indicated from the appearance of yellow internal vesicles (yellow arrow and arrowhead). Scale bars, 20 μm.
Figure 6.
Figure 6.
CCR2 signaling is activated in the peripheral nerve after demyelination. MCP1∷MCP1–mRFP1; CCR2∷CCR2–EGFP mice were treated with LPC to produce focal demyelination of the sciatic nerve. On POD14 animals were killed, and sciatic nerves were isolated. Longitudinal sections were taken at the level of the midthigh. A, Under control conditions, there were few MCP1- or CCR2-expressing cells in the sciatic nerve. B, At the injury site, MCP1-expressing cells increased significantly in the nerve (red arrow), and there was infiltration of leukocytes both in and around the nerve (green arrow). C, D, Endoneurial fibroblasts upregulated MCP1. Endoneurial fibroblasts were identified as cells not associated with axons, which expressed PDGFR-α (C; blue arrow) and did not express S100 (D), a marker for Schwann cells (blue arrow). Note that some PDGFR-α-expressing cells upregulated MCP1–mRFP1 (C; purple arrow). Expression of PDGFR-α and S100 was examined by immunohistochemistry. The green arrows indicate leukocytes. E, Many of the CCR2-expressing leukocytes were observed undergoing active CCR2 receptor-mediated signaling as evidenced by the internalized yellow vesicles (yellow concave arrow). F, Injection of the CCR2–RA reversed the CCR2 receptor activation. Cross-sectional images across the white lines are shown below E and F. Scale bars: A–D, 30 μm; E, F, 10 μm.
Figure 7.
Figure 7.
CCR2 signaling is activated in the DRG. MCP1∷MCP1–mRFP1; CCR2∷CCR2–EGFP mice were subjected to LPC-induced demyelination of the sciatic nerve. A–F, In the DRG ipsilateral to the injury, expression of both MCP1 and CCR2 increased (D–F), whereas there was little expression of MCP1 or CCR2 under naive conditions (A–C). MCP1–mRFP1 mainly localized to neurons (large red arrow) and, to some extent, to satellite glia (small red arrow; D). CCR2–EGFP localized to neurons (large green arrow) and satellite glia (small green arrow; E). Most CCR2–EGFP-expressing neurons and satellite glia also contained MCP1–mRFP1 (yellow arrow; F). Injection of the CCR2–RA eliminated MCP1–mRFP1 in satellite glia (G–I; small green arrow). Also, after CCR2–RA treatment, MCP1–mRFP1- and CCR2–EGFP-expressing cells existed as separate populations (G–I; large green and red arrows). Cross-sectional images across the white lines are shown right to F and I. J, K, Intensities of mRFP1 and EGFP in shorter white lines in F and I are expressed in arbitrary units to compare relative signal intensities among different cells. J, In the LPC group, most neurons which express CCR2–EGFP also contain MCP1–mRFP1. Also, the CCR2–EGFP signal in neurons is relatively weaker than the signal in satellite glia (J). K, In the LPC plus CCR2–RA group, however, most CCR2–EGFP-expressing cells do not contain significant amount of MCP1–mRFP1 signal. In addition, the CCR2–EGFP signal in neurons is now as strong as the signal in satellite glia (K). Scale bars, 15 μm.
Figure 8.
Figure 8.
After demyelination, CCR2 signaling was not activated in the spinal cord. A, MCP1∷MCP1–mRFP1; CCR2∷CCR2–EGFP mice were subjected to LPC-induced demyelination of the sciatic nerve. There was no expression of MCP1 or CCR2 at a detectable level. Leukocytes outside the spinal cord were clearly visible (green arrow). B–D, CCR2 expression was also examined at the mRNA level by in situ hybridization. The spinal cord does not contain significant CCR2-expressing cellular components (B), whereas many cells in the sciatic nerve (C) and DRG (D) express CCR2 receptors in the LPC group. DH, Dorsal horn; DC, dorsal column. Scale bars, 60 μm.
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