A novel interaction between CX 3 CR 1 and CCR 2 signalling in monocytes constitutes an underlying mechanism for persistent vincristine-induced pain.

BACKGROUND: A dose-limiting side effect of chemotherapeutic agents such as vincristine (VCR) is neuropathic pain, which is poorly managed at present. Chemokine-mediated immune cell/neuron communication in preclinical VCR-induced pain forms an intriguing basis for the development of analgesics. In a murine VCR model, CX3 CR1 receptor-mediated signalling in monocytes/macrophages in the sciatic nerve orchestrates the development of mechanical hypersensitivity (allodynia). CX3 CR1 -deficient mice however still develop allodynia, albeit delayed; thus, additional underlying mechanisms emerge as VCR accumulates. Whilst both patrolling and inflammatory monocytes express CX3 CR1 , only inflammatory monocytes express CCR2 receptors. We therefore assessed the role of CCR2 in monocytes in later stages of VCR-induced allodynia.

METHODS: Mechanically evoked hypersensitivity was assessed in VCR-treated CCR2 - or CX3 CR1 -deficient mice. In CX3 CR1 -deficient mice, the CCR2 antagonist, RS-102895, was also administered. Immunohistochemistry and Western blot analysis were employed to determine monocyte/macrophage infiltration into the sciatic nerve as well as neuronal activation in lumbar DRG, whilst flow cytometry was used to characterise monocytes in CX3 CR1 -deficient mice. In addition, THP-1 cells were used to assess CX3 CR1 -CCR2 receptor interactions in vitro, with Western blot analysis and ELISA being used to assess expression of CCR2 and proinflammatory cytokines.

RESULTS: We show that CCR2 signalling plays a mechanistic role in allodynia that develops in CX3 CR1 -deficient mice with increasing VCR exposure. Indeed, the CCR2 antagonist, RS-102895, proves ineffective in mice possessing functional CX3 CR1 receptors but reduces VCR-induced allodynia in CX3 CR1 -deficient mice, in which CCR2 + monocytes are elevated by VCR. We suggest that a novel interaction between CX3 CR1 and CCR2 receptors in monocytes accounts for the therapeutic effect of RS-102895 in CX3 CR1 -deficient mice. Indeed, we observe that CCR2 , along with its ligand, CCL2 , is elevated in the sciatic nerve in CX3 CR1 -deficient mice, whilst in THP-1 cells (human monocytes), downregulating CX3 CR1 upregulates CCR2 expression via p38 MAP kinase signalling. We also show that the CX3 CR1 -CCR2 interaction in vitro regulates the release of pronociceptive cytokines TNF-α and IL1β.

CONCLUSIONS: Our data suggests that CCL2 /CCR2 signalling plays a crucial role in VCR-induced allodynia in CX3 CR1 -deficient mice, which arises as a result of an interaction between CX3 CR1 and CCR2 in monocytes.