IL-1β signaling is required for mechanical allodynia induced by nerve injury and for the ensuing reduction in spinal cord neuronal GRK2

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Abstract

Many neurotransmitters involved in pain perception transmit signals via G protein-coupled receptors (GPCRs). GPCR kinase 2 (GRK2) regulates agonist-induced desensitization and signaling of multiple GPCRs and interacts with downstream molecules with consequences for signaling. In general, low GRK2 levels are associated with increased responses to agonist stimulation of GPCRs. Recently, we reported that in mice with reduced GRK2 levels, inflammation-induced mechanical allodynia was increased. In addition, mice with impaired interleukin (IL)-1β signaling did not develop mechanical allodynia after L5 spinal nerve transection (SNT). We hypothesized that in the L5 SNT model mechanical allodynia would be associated with reduced neuronal GRK2 levels in the spinal cord dorsal horn and that IL-1β signaling would be required to induce both the decrease in GRK2 and mechanical allodynia. We show here that in wild type (WT) mice L5 SNT induces a bilateral decrease in neuronal GRK2 expression in the lumbar spinal cord dorsal horn, 1 and 2 weeks after L5 SNT. No changes in GRK2 were observed in the thoracic segments. Moreover, spinal cord GRK2 expression was not decreased in IL-1R−/− mice after L5 SNT. These data show that IL-1β signaling is not only required for the development of mechanical allodynia, but also to reduce neuronal GRK2 expression. These results suggest a functional relation between the L5 SNT-induced IL-1β-mediated decrease in GRK2 and development of mechanical allodynia.

Introduction

Nerve injury caused by trauma, surgery, or inflammation often results in the development of mechanical allodynia and/or hyperalgesia. The increased pain responsiveness during mechanical allodynia is mediated at least in part by increased excitability of neurons in the spinal cord (Hains et al., 2004). The intracellular mechanisms of increased neuronal excitability are thought to include changes in expression and responsiveness of receptors, activation of several kinases, release of neuropeptides and neurotrophins, and local production of inflammatory mediators (Kim et al., 2002, Milligan et al., 2005, Obata et al., 2004, Woolf and Salter, 2000, Yashpal et al., 2001).

Many mediators involved in pain perception and allodynia (e.g. substance P, glutamate, chemokines, prostaglandins) signal via G protein-coupled receptors (GPCRs). GPCR kinase 2 (GRK2) regulates the responsiveness of multiple GPCRs. GRK2 phosphorylates agonist-occupied GPCRs, which facilitates binding of arrestins, uncoupling from the Gα protein, and receptor internalization (Luttrell and Lefkowitz, 2002). More recent studies described an important additional mechanism via which GRK2 can regulate signaling, i.e. via a direct interaction with intracellular signaling molecules (e.g. MEK1/2, Akt, RKIP, p38 MAPK) (Reiter and Lefkowitz, 2006, Ribas et al., 2006). Changes in GRK2 levels modulate the responsiveness of GPCRs including C-C chemokine receptor (CCR)1/5, CCR2, β2-adrenergic receptors, and metabotropic glutamate receptors (Dhami et al., 2004, Jiménez-Sainz et al., 2006, Vroon et al., 2004, Vroon et al., 2007). In general, low cellular GRK2 expression is associated with increased receptor signaling (Fan and Malik, 2003, Vroon et al., 2004, Vroon et al., 2007), whereas GRK2 overexpression is related to decreased receptor signaling (Lombardi et al., 2002).

Based on the role of GRK2 in the regulation of GPCR signaling and the fact that GPCR signaling plays an important role in mechanical allodynia, we have suggested that changes in GRK2 would contribute to inflammation-associated mechanical allodynia. Indeed, we recently demonstrated that λ-carrageenan-induced acute mechanical allodynia was increased in GRK2+/− mice compared to WT mice, indicating that low GRK2 levels are associated with increased sensitivity for inflammatory allodynia (Kleibeuker et al., 2007). Conversely, we showed that neuronal GRK2 expression is diminished in the lumbar spinal cord dorsal horn of rats during chronic constriction injury (CCI)1 of the sciatic nerve, a model of mechanical allodynia (Kleibeuker et al., 2007). During CCI and other models of mechanical allodynia several pro-inflammatory cytokines such as interleukin (IL)-1β and tumor necrosis factor (TNF)-α are produced in the spinal cord that contribute to increased pain sensitivity (Milligan et al., 2005, Raghavendra et al., 2003, Winkelstein et al., 2001). Intrathecal administration of IL-1β can increase nociception, whereas intrathecal administration of IL-1 receptor antagonist (IL-1RA) can prevent the development of and/or reverse established mechanical allodynia (Milligan et al., 2005, Sung et al., 2004). Additional evidence for the role of IL-1β in the development of mechanical allodynia comes from studies using mice with deficiencies in IL-1β signaling. Mechanical allodynia induced by L5–6 spinal nerve ligation or CCI is decreased in IL-1α/β−/− mice (Honore et al., 2006). Moreover, Wolf et al. (2006) showed that mechanical allodynia is abrogated in mice with a targeted deletion of the IL-1 type I receptor (IL-1R−/−) and in mice overexpressing IL-1RA, indicating that IL-1β is crucial for the development of mechanical allodynia after L5 spinal nerve transection (SNT).

GRK2 expression is downregulated during various inflammatory diseases including rheumatoid arthritis, multiple sclerosis, adjuvant arthritis, and experimental allergic encephalomyelitis (Lombardi et al., 1999, Lombardi et al., 2001, Vroon et al., 2003, Vroon et al., 2005), but also after pro-inflammatory cytokine treatment. In vitro, chronic IL-1β treatment of cultured spinal cord slices decreases GRK2 levels (Kleibeuker et al., 2007). In addition, IL-1β and several other pro-inflammatory cytokines (e.g. IFN-γ, IL-6, TNF-α) downregulated GRK2 expression in human peripheral blood lymphocytes or in a smooth muscle cell line (Lombardi et al., 1999, Ramos-Ruiz et al., 2000).

In the present study, we investigated whether L5 SNT-induced mechanical allodynia in mice is associated with a reduced neuronal GRK2 expression in the spinal cord dorsal horn. This may answer the question whether downregulation of neuronal GRK2 is a general mechanism which may contribute to the development of mechanical allodynia. In addition, we hypothesized that IL-1β signaling is required to induce mechanical allodynia as well as a decrease in GRK2 levels. To test this hypothesis, mechanical sensitivity and expression of GRK2 in the spinal cord dorsal horn were determined in the L5 SNT model in mice with a deficiency of IL-1β signaling (IL-1R−/−) and in wild type (WT).

Section snippets

Animals

IL-1R−/− mice (Labow et al., 1997) and their C57BL/6 X 129/Sv WT control male mice, 10–14 weeks old, were employed in this study (Jackson Laboratory, Bar Harbor, ME, USA). All mice were housed in groups of 4–5 in 26.5 × 20 × 13.5 cm cages or groups of 7–10 in 37 × 30 × 15 cm cages. Food and water were available ad libitum. All measurements were performed during the dark phase of a reversed 12 h light-dark cycle (lights off at 08:00). The experiments were performed according to international guidelines and

GRK2 expression in the mouse spinal cord

We have previously shown that the GRK2 antibody used in this study can be used to detect differences in GRK2 expression in the rat spinal cord using immunohistochemistry. Moreover, the antibody recognizes a single band on Western blots of mouse spinal cord, and GRK2 expression, as determined by Western blotting, is significantly reduced in the spinal cord of GRK2+/− mice (Kleibeuker et al., 2007). As shown in Fig. 1A and B, the difference in GRK2 expression between WT and GRK2+/− mice can also

Discussion

This study demonstrates that neuronal GRK2 levels in the lumbar spinal cord dorsal horn were downregulated in a murine model of mechanical allodynia (L5 SNT). Interestingly, in IL-1R−/− mice, which do not develop mechanical allodynia after L5 SNT, no such reduction was observed. These results indicate that IL-1β signaling is required for the decrease in neuronal GRK2 levels, as well as for the development of L5 SNT-induced mechanical allodynia.

We previously demonstrated that CCI of the sciatic

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