CCR2 chemokine receptor signaling mediates pain in experimental osteoarthritis

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.

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.

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.

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.

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.