Interferon-y Is a Critical Modulator of CB2 Cannabinoid Receptor Signaling during Neuropathic Pain

• The present results revealed the crucial role of CB2 cannabinoid receptors in the development of neuropathic pain through an immune mechanism linked to modified IFN-activity. Hyperalgesia and allodynia induced by sciatic nerve injury were enhanced in CB2 / mice, as revealed by a mirror image of pain in the contralateral side. These behavioral manifestations of neuropathic pain matched the changes induced in microglial and astrocyte activation, astrocytic IFN-expression, and other biochemical parameters related to the immune response. Glial activators include chemokines that enhance pain sensation and are under the control of immune mediators, as well as several neuromodulators released by nearby neurons, such as prostaglandins (Tanga et al., 2006). Interferons represent crucial modulators of the central and peripheral immune responses (Bach et al., 1997) and the enhanced induction of IFN-genes in CB2 / mice could participate in their nociceptive hypersensitivity. Indeed, a prolonged spinal increase in IFN- levels in inflammatory responses in diseases such as viral infections and multiple sclerosis, is thought to contribute to the associated persistent pain states (Miyazaki et al., 2008). Furthermore, IFN-treatments in cancer therapy can result in spontaneous pain in humans (Quesada et al., 1986; Mahmoud et al., 1992). Intrathecal IFN- administration in mice can also cause pain-related behaviors in normal, but not in IFN-receptor knockout mice (Robertson et al., 1997). Although the molecular and cellular mechanism of interferon-induced pain remains mostly unclear, it has been demonstrated that IFN- can cause spontaneous firing of dorsal horn neurons in vitro and in vivo, as well as enhanced wind-up responses to electrical stimulation (Vikman et al., 2003, 2005, 2007). Interestingly, the pharmacological activation of CB2 cannabinoid receptors suppresses wind-up responses of spinal nociceptive neurons and this effect was more pronounced in the presence of pathological pain (Nackley et al., 2004). The enhanced IFN- response revealed by microarray experiments in CB2 / mice exposed to nerve injury has an important functional relevance in vivo. Thus, a direct relationship between the enhanced IFN- response and the neuropathic pain manifestations of CB2 / mice was demonstrated by using double knock-out animals deficient in CB2 receptors and IFN-. The behavioral phenotype of CB2 / mice showing an enhanced neuropathic pain was completely abolished in these double knock-out animals. IFN- is a crucial modulator of the central and peripheral immune responses suggesting that an immune alteration seems to underline the neuropathic pain responses in CB2 / mice. The manifestations of neuropathic pain observed in CB2 / mice and double knock-out mice deficient in CB2 and IFN- suggest that endocannabinoids play an important role in the control of the immune responses leading to the development of neuropathic pain. In support of this hypothesis, an enhancement in the levels of the two main endocannabinoids, anandamide and 2-arachidonoyl-glycerol, was revealed after sciatic nerve injury in the spinal cord and several brain areas involved in pain, such as the periaqueductal gray matter and the rostral ventral medulla (Petrosino et al., 2007). The endocannabinoid levels were also enhanced after sciatic nerve injury in the dorsal root ganglia (Mitrirattanakul et al., 2006) and the section of the sciatic nerve proximal to the lesion (Agarwal et al., 2007). These increased endocannabinoid levels are likely related to enhanced biosynthesis or decreased catabolism and transport because endocannabinoids are produced on demand without any substantial storage (Di Marzo, 1998). Therefore, endocannabinoids could produce a tonic activation of CB2 receptors after sciatic nerve injury that would limit the immune responses leading to the development of neuropathic pain. In agreement, both mechanical and thermal hyperalgesia produced after sciatic nerve injury were attenuated by the administration of N-arachidonoyl-serotonin, an inhibitor of fatty acid amide hydrolase, the enzyme responsible for the degradation of anandamide (Maione et al., 2007), which further support the role of endocannabinoids in the modulation of neuropathic pain. In contrast with the results here obtained in CB2 / mice, the genetic disruption of the CB1 receptor had no major consequences on the development of neuropathic pain (Castan˜e´ et al., 2006) despite the high expression of these receptors in the CNS (Tsou et al., 1998). However, CB1 receptors expressed in peripheral nociceptors but not in the CNS seem to be involved in the manifestations of neuropathic pain (Agarwal et al., 2007). In agreement, pharmacological activation of CB1 receptors have also been reported to reduce pain sensitivity in a variety of neuropathic pain models (Pertwee, 2005). Subsequent to nerve injury, gradients formed by CCL2 and CCL3 orchestrate the recruitment and activation of resident and monocyte-derived microglia via signaling through their respective receptors CCR2, CCR1, and CCR5 (Scholz and Woolf, 2007). In particular, CCR2 expression in either resident microglia or bone marrow-derived macrophages may be sufficient for the development of mechanical allodynia in a murine neuropathic pain model (Zhang et al., 2007). CB2 cannabinoid receptor activity may critically influence the induction of CCR2 expression by monocytes and thus inhibit their chemotaxis (Steffens et al., 2005). Moreover, endocannabinoids were found to abolish microglia activation by inhibiting NO release through a mechanism linked to the MAPK (mitogen-activated protein kinase) pathway (Eljaschewitsch et al., 2006). Our data revealed that IFN- treatment of mouse BV-2 microglial cells evoked marked microglial activation as indicated by induction of iNOS and CCR2 gene expression. CB2 signaling, however, interfered with the expression of these two IFN--inducible genes in BV-2 cells. These data suggest that CB2 receptor signaling exerts antiinflammatory effects in the neuropathic response by controlling IFN--mediated microglial activation and recruitment. Therefore, these data complement our in vivo observations in the neuropathic response in CB2 / mice. Mice genetically modified either by increasing or eliminating specific gene may be limited by the fact that this genetic change may be affecting not only the target gene but also other biological components, perhaps participating in the effects evaluated in these genetic models. Therefore, pharmacological studies using selective ligands of CB2 receptors would be useful to confirm the relevance of the present results. Nevertheless, these genetic manipulations have been considered a key approach to the identification of alterations associated to different pathological conditions and to the discovering of new potential therapeutic targets in a variety of neuropsychiatric disorders. In this study, the according results obtained in CB2 / mice and double knock-out mice deficient in CB2 and IFN-together with the pharmacological studies performed in vitro further support the relevance of the findings. Our immunofluorescence analysis revealed that IFN-was mainly expressed in neurons after sciatic nerve injury and this expression of IFN-matched the pattern of nociceptive hypersensitivity in all experiments. IFN- expression was also present in astrocytes, but absent in microglia. Previous studies have reported the presence of CB2 receptors in microglial cells (Romero- Sandoval et al., 2008) and neurons (Van Sickle et al., 2005). However, the possible presence of CB2 receptors in neurons and its possible functional role is still a controversial issue that requires additional investigation. Taken all these data into consideration, we can postulate a mechanism to explain the modulation of neuropathic pain through CB2 receptor activation (Fig. 8). Thus, the neuroinflammatory process leading to the development of neuropathic pain seems to be initiated by the microglial activation produced after nerve injury (Scholz and Woolf, 2007). This process requires a coactivation of astrocytes, which, together with neurons, release IFN- and promote consolidation and progression of the neuropathic pain state (Zhang et al., 2007). IFN-promotes microglia activation by the induction of several inflammatory pathways, including an enhancement in iNOS and CCR2 activity. Interestingly, previous studies have reported the expression of IFN- receptors in microglial cells and its modulation under pathological conditions (Cannella and Raine, 2004). CB2 receptors on microglial cells would play a crucial role to control and limit the spreading of this neuroinflammatory process. Thus, the activity of CB2 receptors in microglial cells would reduce the activation of these cells during neuropathic pain by regulating the expression of iNOS and CCR2. In the absence of CB2 receptors, IFN-would produce a more widespread activation of microglial cells, which would enhance the manifestations of neuropathic pain and would be responsible for the presence of a mirror image of pain in the contralateral side.