A novel interaction between CX₃CR₁ and CCR₂ signalling in monocytes constitutes an underlying mechanism for...

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, CX₃CR₁ receptor-mediated signalling in monocytes/macrophages in the sciatic nerve orchestrates the development of mechanical hypersensitivity (allodynia). CX₃CR₁-deficient mice however still develop allodynia, albeit delayed; thus, additional underlying mechanisms emerge as VCR accumulates. Whilst both patrolling and inflammatory monocytes express CX₃CR₁, only inflammatory monocytes express CCR₂ receptors. We therefore assessed the role of CCR₂ in monocytes in later stages of VCR-induced allodynia.

Mechanically evoked hypersensitivity was assessed in VCR-treated CCR₂- or CX₃CR₁-deficient mice. In CX₃CR₁-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 CX₃CR₁-deficient mice. In addition, THP-1 cells were used to assess CX₃CR₁-CCR₂ receptor interactions in vitro, with Western blot analysis and ELISA being used to assess expression of CCR₂ and proinflammatory cytokines.

We show that CCR₂ signalling plays a mechanistic role in allodynia that develops in CX₃CR₁-deficient mice with increasing VCR exposure. Indeed, the CCR₂ antagonist, RS-102895, proves ineffective in mice possessing functional CX₃CR₁ receptors but reduces VCR-induced allodynia in CX₃CR₁-deficient mice, in which CCR₂⁺ monocytes are elevated by VCR. We suggest that a novel interaction between CX₃CR₁ and CCR₂ receptors in monocytes accounts for the therapeutic effect of RS-102895 in CX₃CR₁-deficient mice. Indeed, we observe that CCR₂, along with its ligand, CCL₂, is elevated in the sciatic nerve in CX₃CR₁-deficient mice, whilst in THP-1 cells (human monocytes), downregulating CX₃CR₁ upregulates CCR₂ expression via p38 MAP kinase signalling. We also show that the CX₃CR₁-CCR₂ interaction in vitro regulates the release of pronociceptive cytokines TNF-α and IL1β.

Our data suggests that CCL₂/CCR₂ signalling plays a crucial role in VCR-induced allodynia in CX₃CR₁-deficient mice, which arises as a result of an interaction between CX₃CR₁ and CCR₂ in monocytes.