CCR2 chemokine receptor signaling mediates pain in experimental osteoarthritis - PubMed (original) (raw)

CCR2 chemokine receptor signaling mediates pain in experimental osteoarthritis

Rachel E Miller et al. Proc Natl Acad Sci U S A. 2012.

Abstract

Osteoarthritis is one of the leading causes of chronic pain, but almost nothing is known about the mechanisms and molecules that mediate osteoarthritis-associated joint pain. Consequently, treatment options remain inadequate and joint replacement is often inevitable. Here, we use a surgical mouse model that captures the long-term progression of knee osteoarthritis to longitudinally assess pain-related behaviors and concomitant changes in the innervating dorsal root ganglia (DRG). We demonstrate that monocyte chemoattractant protein (MCP)-1 (CCL2) and its high-affinity receptor, chemokine (C-C motif) receptor 2 (CCR2), are central to the development of pain associated with knee osteoarthritis. After destabilization of the medial meniscus, mice developed early-onset secondary mechanical allodynia that was maintained for 16 wk. MCP-1 and CCR2 mRNA, protein, and signaling activity were temporarily up-regulated in the innervating DRG at 8 wk after surgery. This result correlated with the presentation of movement-provoked pain behaviors, which were maintained up to 16 wk. Mice that lack Ccr2 also developed mechanical allodynia, but this started to resolve from 8 wk onwards. Despite severe allodynia and structural knee joint damage equal to wild-type mice, Ccr2-null mice did not develop movement-provoked pain behaviors at 8 wk. In wild-type mice, macrophages infiltrated the DRG by 8 wk and this was maintained through 16 wk after surgery. In contrast, macrophage infiltration was not observed in Ccr2-null mice. These observations suggest a key role for the MCP-1/CCR2 pathway in establishing osteoarthritis pain.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

MCP-1/CCR2 gene and protein expression in DRG is elevated 8 wk after DMM surgery. (A) Real-time RT-PCR of Mcp-1 and Ccr2 in sham and DMM wild-type mice normalized to age-matched naïve levels: at 4 wk postsurgery, n = 5, two-tailed t test, P > 0.05; at 8 wk postsurgery, n = 4 for naïve, n = 5 for sham and DMM, one-way ANOVA with Bonferroni posttest, MCP-1: *P < 0.001 vs. sham and naïve, CCR2: *P < 0.001 vs. naïve, and P < 0.01 vs. sham; at 16 wk postsurgery, n = 4 for naïve, n = 7 for DMM, two-tailed t test *P = 0.01 for MCP-1 vs. naïve, *P = 0.003 for CCR2 vs. naïve. Results show mean ± SEM. (B) Representative images of in situ hybridization using antisense probes for MCP-1 and CCR2 in DRG sections (L3–L5) taken from DMM wild-type mice at 8 wk postsurgery and age-matched naïve mice. Sense probe control is shown for the DMM condition. Magnification 10×. (Scale bars, 100 μm.) (C) Protein levels of MCP-1 in the supernatants of cells cultured from age-matched naïve, sham, and DMM mice at 4 and 8 wk postsurgery, n = 6 wells, representative of two independent experiments, one-way ANOVA with Bonferroni posttest, ***P < 0.001. Results show mean ± 95% confidence interval. (D) Representative traces of individual cells during calcium mobilization assay indicating a response to MCP-1. Response to 50 mM potassium solution, used as a positive control, is also shown.

Fig. 2.

Fig. 2.

Increases in macrophage DRG populations are seen at 8 and 16 wk after DMM surgery in wild-type mice. Doublecortin (DCX, neuron, green) and F4/80 (macrophage, red) staining of ipsilateral DRG in age-matched naïve and DMM wild-type (WT) mice at 8 wk postsurgery (Top), age-matched naïve and DMM wild-type mice at 16 wk postsurgery (Middle), and age-matched naïve and DMM _Ccr2_-null mice at 8 wk postsurgery (Bottom). White arrows indicate example macrophage staining. Magnification 20×. (Scale bars, 50 μm.)

Fig. 3.

Fig. 3.

Pain-related behaviors develop progressively in wild-type mice after DMM surgery. (A) Mechanical allodynia in the ipsilateral hindpaw of naïve (n = 7–10), sham (n = 9), and DMM mice (n = 9–13), Naïve time 0: n = 30, one-way ANOVA with Bonferroni’s multiple comparison test, **P < 0.01, ***_P_ < 0.001 vs. Time 0. Results show mean ± SEM. (_B_) Distance traveled, average number of climbs per hour, and average number of rears per hour during a 15-h period on a LABORAS platform. At 4 wk, _n_ = 8 naïve, _n_ = 10 DMM, _P_ > 0.05 by two-tailed t test. At 8 wk, n = 18 naïve, n = 10 sham, n = 15 DMM, *P < 0.05 and **P < 0.01 by one-way ANOVA with Bonferroni’s multiple comparison test. At 16 wk, n = 9 naïve and n = 8 DMM, *P = 0.03 for distance, **P = 0.003 for climbing, and *P = 0.0487 for rearing by two-tailed t test. For LABORAS results, data were log-transformed if necessary to ensure normality as determined by the D’Agostino–Pearson normality test before analysis. Results show mean ± SEM.

Fig. 4.

Fig. 4.

_Ccr2_-null mice present different pain-related behaviors after DMM surgery. (A) Mechanical allodynia in _Ccr2_-null naïve (n = 6–11) and DMM mice (n = 5–10), Naïve time 0: n = 17, one-way ANOVA with Bonferroni’s multiple comparison test, *P < 0.05, **_P_ < 0.01, ***_P_ < 0.001 vs. Time 0, Mean ± SEM. (_B_) Distance traveled, average number of climbs per hour, and average number of rears per hour during a 15-h period. At 8 wk, naïve _n_ = 6, DMM _n_ = 9, _P_ > 0.05 by two-tailed t test. At 16 wk, naïve n = 11, DMM n = 6, P > 0.05 by two-tailed t test. (C) Distance traveled during a 15-h period after administration of CCR2 receptor antagonist (CCR2 RA) (5 mg/kg, i.p.) or DMSO vehicle control to wild-type DMM mice at 9 wk postsurgery; n = 5, *P = 0.0109 vs. DMSO control by two-tailed t test. Dashed line indicates wild-type naïve level. For LABORAS results, data were log-transformed if necessary to ensure normality as determined by the D’Agostino–Pearson normality test before analysis. Results show mean ± SEM.

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