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. 2008 Jan;104(1):254-63.
doi: 10.1111/j.1471-4159.2007.04969.x. Epub 2007 Oct 18.

Monocyte chemoattractant protein-1 functions as a neuromodulator in dorsal root ganglia neurons

Hosung Jung  1 , Peter T Toth, Fletcher A White, Richard J Miller
Affiliations

Monocyte chemoattractant protein-1 functions as a neuromodulator in dorsal root ganglia neurons

Hosung Jung et al. J Neurochem. 2008 Jan.

Abstract

It has previously been observed that expression of chemokine monocyte chemoattractant protein-1 (MCP-1/CC chemokine ligand 2 (CCL2)) and its receptor CC chemokine receptor 2 (CCR2) is up-regulated by dorsal root ganglion (DRG) neurons in association with rodent models of neuropathic pain. MCP-1 increases the excitability of nociceptive neurons after a peripheral nerve injury, while disruption of MCP-1/CCR2 signaling blocks the development of neuropathic pain, suggesting MCP-1 signaling is responsible for heightened pain sensitivity. To define the mechanisms of MCP-1 signaling in DRG, we studied intracellular processing, release, and receptor-mediated signaling of MCP-1 in DRG neurons. We found that in a focal demyelination model of neuropathic pain both MCP-1 and CCR2 were up-regulated by the same neurons including transient receptor potential vanilloid receptor subtype 1 (TRPV1) expressing nociceptors. MCP-1 expressed by DRG neurons was packaged into large dense-core vesicles whose release could be induced from the soma by depolarization in a Ca2+-dependent manner. Activation of CCR2 by MCP-1 could sensitize nociceptors via transactivation of transient receptor potential channels. Our results suggest that MCP-1 and CCR2, up-regulated by sensory neurons following peripheral nerve injury, might participate in neural signal processing which contributes to sustained excitability of primary afferent neurons.

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Figures

Fig. 1
Fig. 1. DRG neurons express MCP-1 and CCR2 in association with peripheral neuropathy
Sciatic nerve demyelination was induced by lysophosphatidylcholine (LPC) in CCR2-EGFP BAC transgenic mice. DRG were isolated at post operation day (POD) 14, cryosectioned, and subjected to immunohistochemistry using a polyclonal anti-MCP-1 antibody. A-C) Sham-operated control. D-F) LPC-treated group. Note that many neuronal cell bodies express both MCP-1 and CCR2. In addition, MCP-1 is also observed in numerous axon processes throughout the ganglion. G-I) TRPV1 expressing nociceptors (red arrows) upregulated CCR2 expression (yellow arrow). Some of larger neurons that do not express TRPV1 also expressed CCR2 (green arrow). Scale bars, 100 μm.
Fig. 2
Fig. 2. MCP-1-EGFP localizes to large dense-core vesicles (LDCVs) in DRG neurons
Cultured DRG neurons were infected with an MCP-1-EGFP expressing adenovirus and stained for CGRP and SynI. A) MCP-1-EGFP was concentrated in the perinuclear area, and localized in a punctate pattern in the soma and along axons. Signals in the soma were saturated to allow appreciation of the punctate localization along axons. An unsaturated image of a soma is magnified in the inset. B-C) Magnified view of axonal localization of MCP-1-EGFP. B-B’’) MCP-1-EGFP co-localized with CGRP. C-C’’) MCP-1-EGFP did not co-localize with SynI. Scale bars: A, 20 μm; B’’, C’’, 1 μm.
Fig. 3
Fig. 3. MCP-1-EGFP is released from the somata of DRG neurons
DRG neurons infected with an MCP-1-EGFP expressing adenovirus were analyzed using a fluorescence release assay. A) Representative phase contrast (ph) and fluorescence (EPI) images of a DRG neuronal soma are shown. DRG neurons were identified by their round and phase-bright somata. The entire soma was selected as a region of interest (ROI: white circle). B) Depolarization by high potassium (50K) induced the release of MCP-1-EGFP (red trace) (n=8). When neurons were not stimulated, fluorescence intensity was not significantly changed for the duration of recording (dashed trace). No release was detected when extracellular Ca2+ was absent (0Ca) (blue trace) (n=4).
Fig. 4
Fig. 4. CCR2 activation sensitizes TRPV1
HEK293 cells were transfected with CCR2 and TRPV1 expressing vectors and the responses of TRPV1 to capsaicin (CAP) were measured by Ca2+ imaging. Cells were treated with CAP for 1 min at the beginning and end of the recording (30 min). Between two CAP treatments, cells were incubated with either 100 ng/ml MCP-1 or buffer solution for 15 min. CAP responses were compared before and after the MCP-1 treatment in each cell. Sensitization of TRPV1 was represented by the enhancement of the second CAP response compared to the first one. A) Without MCP-1, the amplitude of two CAP responses was similar (blue trace), whereas the second CAP responses increased after incubation with MCP-1 (red trace). B) The result of TRPV1 sensitization by MCP-1 is summarized. Sensitization was measured with varying concentration of CAP, and represented by a fold induction of the second CAP responses to the first responses in each cell. Sensitization is prominent in lower concentrations of CAP (**p<0.01 vs. mock application control of each concentration). C) The mechanism of TRPV1 sensitization was examined with inhibitors of PLC (U; 10 μM of U73122) and PKC (S; 10 nM of staurosporine). The inhibitors were added 5 min before the recording. Sensitization was completely blocked by PKC and PLC inhibitors. Moreover, PLC inhibitors desensitized TRPV1 (*p<0.05 and **p<0.01). Numbers of recorded cells are noted in parentheses.
Fig. 5
Fig. 5. CCR2 activation sensitizes TRPA1
HEK293 cells were transfected with CCR2 and/or TRPA1 expressing vectors, and the responses to MCP-1 (100 ng/ml) and allyl isothiocyanate (AITC) (10 μM), a TRPA1 agonist, were measured by Ca2+ imaging experiments. Ruthenium red (RR; 30 μM), a TRP channel blocker, was added to block TRPA1 channels. A) The response to MCP-1 consisted of a transient component when it was expressed alone. B) An additional sustained component appeared when it was co-expressed with TRPA1 (red trace). The sustained component was abolished by the TRP channel blocker ruthenium red (blue trace). At the end of recording, cells were treated with AITC after washing out of RR to confirm the expression of TRPA1.
Fig. 6
Fig. 6. MCP-1 sensitizes the capsaicin-responsiveness of cultured DRG neurons
Cultured DRG neurons transduced with a CCR2 expressing adenovirus were pre-treated with MCP-1 or buffer solution for 10 min. Subsequently, neurons were treated with a low concentration (0.1 μM) of capsaicin (CAP) (L), a high concentration (1.0 μM) of CAP (H), and high potassium (50K). Neurons were identified by 50K-responsiveness. A) In the control group, most neurons did not respond to 0.1 μM CAP (blue trace). In the MCP-1-treated group, most neurons that responded to 1.0 μM CAP also responded to 0.1 μM CAP (red). B) Results are summarized. There was no difference in the total number of CAP-responsive neurons. However, MCP-1 pre-treatment increased neurons that were responsive to 0.1 μM CAP (pooled result of four different experiments. In the control group 27 out of 66 neurons responded to 1.0 μM CAP, of which only one responded to 0.1 μM CAP. In MCP-1-treated group, 44 out of 89 responded to 1.0 μM CAP, of which 28 responded to 0.1 μM CAP).
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