Elsevier

Brain Research Reviews

Volume 60, Issue 1, April 2009, Pages 125-134
Brain Research Reviews

Review
Chemokines and pain mechanisms

https://doi.org/10.1016/j.brainresrev.2008.12.002Get rights and content

Abstract

The development of new therapeutic approaches to the treatment of painful neuropathies requires a better understanding of the mechanisms that underlie the development of these chronic pain syndromes. It is now well established that astrocytic and microglial cells modulate the neuronal mechanisms of chronic pain in spinal cord and possibly in the brain. In animal models of neuropathic pain following peripheral nerve injury, several changes occur at the level of the first pain synapse between the central terminals of sensory neurons and second order neurons. These neuronal mechanisms can be modulated by pro-nociceptive mediators released by non neuronal cells such as microglia and astrocytes which become activated in the spinal cord following PNS injury. However, the signals that mediate the spread of nociceptive signaling from neurons to glial cells in the dorsal horn remain to be established. Herein we provide evidence for two emerging signaling pathways between injured sensory neurons and spinal microglia: chemotactic cytokine ligand 2 (CCL2)/CCR2 and cathepsin S/CX3CL1 (fractalkine)/CX3CR1. We discuss the plasticity of these two chemokine systems at the level of the dorsal root ganglia and spinal cord demonstrating that modulation of chemokines using selective antagonists decrease nociceptive behavior in rodent chronic pain models. Since up-regulation of chemokines and their receptors may be a mechanism that directly and/or indirectly contributes to the development and maintenance of chronic pain, these molecular molecules may represent novel targets for therapeutic intervention in sustained pain states.

Introduction

Neuropathic pain is experienced in association with many types of injury to the nervous system or as a consequence of diabetes, cancer or infectious agents. From the therapeutic point of view, neuropathic pain often proves refractory to existing therapies. A better understanding of the cellular and molecular processes involved in the development of neuropathic pain is essential for the development of novel therapies. Many pathophysiological mechanisms underlie the development of neuropathic pain states. The site of these mechanisms include not only the damaged nerve and dorsal root ganglia (DRG), but also changes in the central processing of sensory information, most notably at the level of the spinal cord. This phenomenon has traditionally been considered a neuronally mediated response. Recent findings have highlighted the active involvement of glial cells in the pathogenesis of nerve injury-induced neuropathic pain and uncover new targets for potential analgesics (Watkins and Maier, 2003, Marchand et al., 2005, Tsuda et al., 2005, Scholz and Woolf, 2007). Microglia release a variety of mediators including pro-inflammatory cytokines and chemokines that contribute to pain signaling. Chemokines are small proteins that were initially characterized as chemotactic peptides controlling the trafficking of leukocytes (for review see Charo and Ransohoff, 2006). Chemokine classification is based on the presence and position of the first cysteine residues. The CC group has two adjacent cysteines, the CXC group has one amino acid that separates the two cysteine residues, and the CX3X (three amino acids between two cysteines residues) chemokine CX3CL1 (also termed fractalkine) has only one member in its class. Chemokines are released locally from peripheral blood cells at sites of inflammation and are crucial during the inflammatory response since they are accountable for leukocyte recruitment to the site of damage. In addition, chemokines are also involved in pain processing as described in this chapter.

The understanding of the functional role of CCL2 in pain processing requires a precise knowledge of its distribution, and its co-localization with pain-related neuropeptides in DRG neurons and in their nerve terminals in the dorsal horn of the spinal cord. Within DRG, CCL2 is constitutively expressed in small and medium diameter size neurons under naïve conditions and in several chronic pain models (Fig. 1; Tanaka et al., 2004, White et al., 2005, Zhang and De Koninck, 2006). To identify the phenotype of CCL2-expressing cells, double-labeling immunohistochemistry combining CCL2 antibodies with classical neuronal markers revealed that CCL2-immunoreactivity was restricted to the cell bodies of DRG neurons. Among these, CCL2 labeling was detected in the subpopulation of peptidergic neurons immunoreactive for substance P and in primary sensory neurons expressing the neuropeptide CGRP (Dansereau et al., 2008). Moreover, CCL2-positive neurons also co-localized extensively with the capsaicin-heat and proton-gated ion channel, transient receptor potential vanilloid 1 (Dansereau et al., 2008). At higher magnification, confocal images of dually stained DRG neurons revealed that the intracellular pattern of staining for CCL2 only partially overlapped with those of substance P or CGRP (Fig. 1). Indeed, the neuropeptides substance P and CGRP were seen in punctate structures homogenously distributed throughout the cytoplasm whereas CCL2-ir formed large fluorescent puncta clustered around the nucleus within neuronal cell bodies (Dansereau et al., 2008). Confocal microscopy revealed the presence of CCL2 immunoreactivity in the dorsal horn of the spinal cord (Fig. 1). CCL2 dorsal horn staining was most intense within the substantia gelatinosa, as well as in a band spanning the boarder between inner lamina II and lamina III. Double-labeling experiments also showed that CCL2 co-localized with substance P and CGRP positive axon terminals in the outer portion of lamina II (Dansereau et al., 2008). In addition CCL2 staining was also concentrated in lamina IIi where both substance P and CGRP-immunoreactivities are sparse. CCL2 is only in primary afferent fibers, not in 2nd order neurons.

The detection of CCL2 in soma and processes of DRG neurons as well as its presence in neuropeptide positive vesicle-like structures in the dorsal horn strongly suggest that CCL2 may be transported and subsequently secreted from neuronal nerve terminals. To test this hypothesis, the release of CCL2 was examined in isolated dorsal horn preparation following KCl or capsaicin stimulation, which are known to induce neurotransmitter release. KCl or capsaicin induced a significant increase of CCL2 in the superfusate over basal outflow. Both KCl- and capsaicin-induced increases in CCL2 release were abolished by co-superfusion with a calcium chelating agent, demonstrating the calcium-dependent mechanism for the release of CCL2 (Dansereau et al., 2008). These results corroborate recent data obtained by Miller et al. in which it was demonstrated in vitro that CCL2 is located in secretory vesicles and released upon depolarization by CCL2-EGFP transfected DRG-F11 neuronal cell line (Jung et al., 2008).

The ability of CCL2 to stimulate nociceptive neurons suggested its involvement in pain sensitivity modulation (Sun et al., 2006). Intrathecal administration of exogenous CCL2 induces a rapid and sustained hyperalgesia in the hot-plate test provoking profound hindpaw mechanical allodynia (Dansereau et al., 2008). These changes in non-noxious tactile threshold, previously observed on a short period of time (Tanaka et al., 2004), were maintained over 4 days after a single CCL2 i.t. administration (Dansereau et al., 2008). Importantly, these pro-nociceptive effects of CCL2 were clearly mediated via CCR2, as concomitant injection of CCL2 with the CCR2-selective antagonist INCB-3344 (Brodmerkel et al., 2005) completely blocked CCL2-induced hyperalgesia and allodynia (Dansereau et al., 2008). These behavioral observations are further supported by genetic evidence, since mice overexpressing CCL2 exhibited enhanced nociceptive behavior in responses to both thermal and chemical stimulus modalities (Menetski et al., 2007). Altogether, these data demonstrate that CCL2 enhances sensitivity to pain by direct action on CCR2 expressed by nociceptive and/or spinal neurons and suggest that CCL2/CCR2 signaling plays an important role in the establishment and/or persistence of pain. Finally, a CCR2 antagonist blocks CCL2 nociceptive effects, suggesting that CCR2 represents a target for the development of new therapeutic drugs for pain relief.

In summary, CCL2 is constitutively expressed by uninjured rat DRG and provide evidence for a direct implication of CCR2 in CCL2-induced acute nociception at the spinal level. Based on these data, we would like to propose a neuronal mechanism by which CCL2 might modulate acute pain in healthy animals through its receptor CCR2. This mechanism entails synthesis of CCL2 in DRG neurons where it appears to be stored in both neuropeptide-containing and neuropeptide-free vesicles and may be released in a calcium-dependent and sustained manner from both DRG neuronal cell bodies and their projections in the dorsal horn of the spinal cord. In the DRG, CCL2 may directly excite primary nociceptive neurons by autocrine and/or paracrine processes, perhaps participating to the intraganglionic cross-excitation phenomenon. In addition to this mechanism, CCL2 synthesized in DRG neurons, is released at the spinal dorsal horn to modulate the activity of post-synaptic neurons and glial cells, therefore facilitating pain transmission.

Section snippets

Role of CCL2-CCR2 signaling in triggering spinal neuroimmune reaction to peripheral nerve injury

The fact that nerve injury can induce spinal microglial activation has been demonstrated in several models of pain hypersensitivity (Colburn et al., 1999, Fu et al., 1999, Tsuda et al., 2003, Zhuang et al., 2005, Scholz and Woolf, 2007). Activated microglia have been known to release a number of pro-nociceptive substances, making them a key player in the pathophysiology of neuropathic pain (Watkins and Maier, 2003, Marchand et al., 2005, Tsuda et al., 2005, Scholz and Woolf, 2007). Unlike

CCR2: transgenics vs. pharmacology

This section reviews and compares data obtained in CCR2 deficient mice, in mice overexpressing CCL2 and the effects of CCR2 antagonists in models of acute and chronic nociception (Fig. 2). Mice deficient for CCR2 (CCR 2 −/−) were generated by homologous recombination as described in Abbadie et al., (2003). Mice overexpressing the ligand CCL2 (CCL2 tg) have been designed to express CCL2 in an astrocyte/Schwann cell specific manner and relate to previous studies on glial interactions. Recent

Cathepsin S and fractalkine: a new modulatory pathway for neuropathic pain

It is well established that astrocytic and microglial cells modulate the neuronal mechanisms of chronic pain in spinal cord and possibly in the brain (Watkins and Maier, 2003, Scholz and Woolf, 2007). In models of neuropathic pain following peripheral nerve injury, several changes occur at the level of the first pain synapse between the central terminals of sensory neurons and second order neurons. Neuropathic hyperalgesia and allodynia are likely to be mediated by glutamate, substance P and

Conclusion

As reported here, chemokines play a key role in coordinating injury associated nociceptive events as they serve to regulate inflammatory responses and can simultaneously act on elements of the nervous system. Within both the central and peripheral nervous system, both spinal microglia and neurons, respectively, could be responsible for chemokine action. The ability of small molecule antagonists of CCR2 to ameliorate ongoing pain hypersensitivity in animal models clearly indicates the importance

Acknowledgments

C. A. is grateful to Chris Moyes and Lihue Yang, Merck Research Laboratories for providing a CRR2 antagonist. S. B. is supported by the National Institutes of Health Grants NS043095, DA013141, and MH040165. Y. De K. acknowledges support from the Canadian Institutes of Health Research (CIHR) and Neuroscience Canada (Brain Repair program). Y. De K. is a Chercheur National of the Fonds de la Recherche en Santé du Québec (FRSQ). S. M.-P. is grateful to Pfizer for providing a CCR2-specific

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