Impaired neuropathic pain responses in mice lacking the chemokine receptor CCR2

Abstract

Mice lacking the chemokine receptor chemotactic cytokine receptor 2 (CCR2) have a marked attenuation of monocyte recruitment in response to various inflammatory stimuli and a reduction of inflammatory lesions in models of demyelinating disease. In the present study, we compared nociceptive responses in inflammatory and neuropathic models of pain in CCR2 knockout and wild-type mice. In acute pain tests, responses were equivalent in CCR2 knockout and wild-type mice. In models of inflammatory pain, CCR2 knockout mice showed a 70% reduction in phase 2 of the intraplantar formalin-evoked pain response but only a modest (20-30%) and nonsignificant reduction of mechanical allodynia after intraplantar Freund's adjuvant (CFA). In a model of neuropathic pain, the development of mechanical allodynia was totally abrogated in CCR2 knockout mice. CFA administration induced marked up-regulation of CCR2 mRNA in the skin and a moderate increase in the sciatic nerve and dorsal root ganglia (DRG). In response to nerve ligation, persistent and marked up-regulation of CCR2 mRNA was evident in the nerve and DRG. Disruption of Schwann cells in response to nerve lesion resulted in infiltration of CCR2-positive monocytes/macrophages not only to the neuroma but also to the DRG. Chronic pain also resulted in the appearance of activated CCR2-positive microglia in the spinal cord. Collectively, these data suggest that the recruitment and activation of macrophages and microglia peripherally and in neural tissue may contribute to both inflammatory and neuropathic pain states. Accordingly, blockade of the CCR2 receptor may provide a novel therapeutic modality for the treatment of chronic pain.

Fig. 3.

CCR2 expression after chronic injury in the skin (A), sciatic nerve (B and C), DRG (D and E), and dorsal horn of the spinal cord (F–I). Numerous monocytes/macrophages express CCR2 in the skin dermis (d) and around blood vessels (arrows in A) 2 days after CFA injection into the mouse hindpaw. Fewer CCR2 monocytes/macrophages are found in the sciatic nerve (not shown) and the DRG (D) after CFA. Conversely, abundant CCR2 cells are found both in the perineurium (P) (B) and surrounding axons (as seen with CGRP-LI; C) in the sciatic nerve and the DRG (E). Nerve injury induced the activation of both astrocytes and microglia in the spinal cord. However, CCR2-expressing cells do not colocalize with astrocyte marker (glial fibrillary acidic protein; F) but colocalize with microglia marker (OX-42; arrowheads in G–I) in the dorsal horn of the spinal cord. Not all microglia express CCR2 (green arrow in I). (Bar in G = 100 μmin A and B;25 μmin D and E; and 10 μm in C and F–I.)

Fig. 1.

Nociceptive responses to thermal or chemical stimulations. (A) Licking or jumping latency in the hot-plate assay; no differences between the two groups of mice. (B) Duration of licking and lifting in response to intraplantar formalin injection is significantly reduced in the homozygous mutant as compared with wild-type mice. (C) Area under the curve in B showing slight reduction in phase 1 (0–10 min) but a significant decrease in phase 2 (15–50 min) in mutant as compared with wild-type mice. (D) Mechanical allodynia after intraplantar MCP-1: maximal allodynia is detected 90 min after the injection. ^*, P < 0.05.

Fig. 2.

Nociceptive responses after two types of chronic pain: inflammation (A and B) and nerve injury (C). (A) Withdrawal threshold to mechanical stimulation (von Frey): mutant mice show a nonsignificant reduction in mechanical allodynia after CFA. (B) Paw withdrawal latencies to noxious thermal stimuli: no differences between the two groups. (C) After nerve injury, mutant mice did not display mechanical allodynia. ^*, P < 0.05.

Fig. 4.

Activated glial cells in the ipsilateral dorsal horn of the spinal cord 1 wk after partial sciatic nerve ligation. (A and B) The number of glial fibrillary acidic protein-positive cells was decreased (see arrows) in the superficial laminae in the CCR2-/- (B) as compared with the CCR2+/+ mice (A). Similarly, the number of microglia expressing phospho p38 mitogen-activated protein kinase (pp38, C and D) is lower in the CCR2 knockout mice (D) than in wild type (C). (C and D Insets) Higher magnification of laminae I and II. (Bar in D = 200 μm.)