Interferon-gamma is a critical modulator of CB(2) cannabinoid receptor signaling during neuropathic pain

Abstract

Nerve injuries often lead to neuropathic pain syndrome. The mechanisms contributing to this syndrome involve local inflammatory responses, activation of glia cells, and changes in the plasticity of neuronal nociceptive pathways. Cannabinoid CB(2) receptors contribute to the local containment of neuropathic pain by modulating glial activation in response to nerve injury. Thus, neuropathic pain spreads in mice lacking CB(2) receptors beyond the site of nerve injury. To further investigate the mechanisms leading to the enhanced manifestation of neuropathic pain, we have established expression profiles of spinal cord tissues from wild-type and CB(2)-deficient mice after nerve injury. An enhanced interferon-gamma (IFN-gamma) response was revealed in the absence of CB(2) signaling. Immunofluorescence stainings demonstrated an IFN-gamma production by astrocytes and neurons ispilateral to the nerve injury in wild-type animals. In contrast, CB(2)-deficient mice showed neuronal and astrocytic IFN-gamma immunoreactivity also in the contralateral region, thus matching the pattern of nociceptive hypersensitivity in these animals. Experiments in BV-2 microglia cells revealed that transcriptional changes induced by IFN-gamma in two key elements for neuropathic pain development, iNOS (inducible nitric oxide synthase) and CCR2, are modulated by CB(2) receptor signaling. The most direct support for a functional involvement of IFN-gamma as a mediator of CB(2) signaling was obtained with a double knock-out mouse strain deficient in CB(2) receptors and IFN-gamma. These animals no longer show the enhanced manifestations of neuropathic pain observed in CB(2) knock-outs. These data clearly demonstrate that the CB(2) receptor-mediated control of neuropathic pain is IFN-gamma dependent.

Figure 1.

Classification of genes showing significant changes in expression in the spinal cord after sciatic nerve injury. About one-third of upregulated genes are responsible for the inflammatory/immunological processes, whereas only a small amount of genes belonging to this category was downregulated. A large number of genes involved in signaling and metabolism were upregulated and downregulated. A fewer number of genes regulating cell structure, translation/transcription, and DNA repair functions are in rather equal number upregulated or downregulated. A large number of genes with unknown function have a different expression after sciatic nerve injury.

Figure 2.

Affymetrix gene array analysis of spinal cord tissues during neuropathic pain development in CB₂ ^−/− mice. A, The list of genes that were more strongly regulated in CB₂ ^−/− animals compared with CB₂ ^+/+ mice. B, Genes that were more strongly regulated in CB₂ ^+/+ animals compared with CB₂ ^−/− mice. The intensity and direction of gene regulation are represented in a heat map (red, downregulated; green, upregulated).

Figure 3.

Expression of genes regulated by IFN-γ or IFN-α after partial sciatic nerve ligation (PNL) was upregulated. A, The increase in expression level of IFN-γ-regulated genes was more intensive in CB₂ ^−/− than in CB₂ ^+/+ mice, whereas the upregulation in IFN-α-regulated genes was less intensive in CB₂ ^−/− animals. B, Taqman real-time PCR analysis of the expression of three GTPase regulated genes also showed a stronger expression change in CB₂ ^−/− than in CB₂ ^+/+ mice after neuropathy. wt-i, Ipsilateral paw of CB₂ ^+/+ mice; wt-c, contralateral paw of CB₂ ^+/+ mice; ko-i, ipsilateral paw of CB₂ ^−/− mice; ko-c, contralateral paw of CB₂ ^−/− mice.

Figure 4.

Development of neuropathic pain in male IFN-γ^−/− and IFN-γ^−/−/CB₂ ^−/− knock-out animals. A single tight ligature around one-third or one-half of sciatic nerve was made to induce the neuropathic pain. Mice were tested in the ipsilateral and contralateral paw for evaluating mechanical allodynia (von Frey model) and thermal hyperalgesia (plantar model) on days 3, 6, 8, 10, and 15 after surgery. Mechanical allodynia data are expressed as mean ± SEM percentage values of basal responses of sham-operated mice. Thermal hyperalgesia data are expressed as mean ± SEM values of withdrawal latencies. The black stars represent comparisons between time points in IFN-γ^−/− animals (n = 8 for nerve injury, n = 6 for sham). The white stars represent comparison between time points in IFN-γ^−/−/CB₂ ^−/− mice (n = 7 for nerve injury; n = 3 for sham). One star, p < 0.05; two stars, p < 0.01; three stars, p < 0.001.

Figure 5.

IFN-γ expression by astrocytes and neuronal cells in the spinal cord of CB₂ ^+/+ and CB₂ ^−/− mice after sciatic nerve injury. A, Representative low-magnification images of double immunofluorescence staining with GFAP (astrocyte: red, CY3-conjugated secondary anti-rabbit Ab) and IFN-γ (green, streptavidin Alexa Fluor 488) recorded with 10× objective in the lumbar dorsal horn of sciatic nerve injury CB₂ ^+/+ and CB₂ ^−/− mice. B, Confocal microscopy of representative spinal cord sections after double immunofluorescence staining for microglial marker iba-1 or astrocyte marker GFAP (red, CY3-conjugated secondary anti rabbit Ab) and for IFN-γ (green, streptavidin Alexa Fluor 488). IFN-γ expression in GFAP⁺ astrocytes and not iba-1 microglial cells in the lumbar dorsal horn of CB₂ ^+/+ and CB₂ ^−/− mice. C, IFN-γ immunoreactivity in neuron-like cells as evidenced by neuronal morphology in the ventral horn of spinal cords of neuropathic WT mice. IFN-γ expression was visualized by staining of cryostat sections with rat anti-IFN-γ antibody and a biotinylated anti-rat secondary antibody followed by streptavidin Alexa Fluor 488 (green). D, Colocalization of GFAP with IFN-γ in the ipsilateral dorsal horn of neuropathic CB₂ ^−/− mice. GFAP was visualized by staining of cryostat sections with rabbit anti-GFAP antibody followed by CY3-conjugated secondary anti-rabbit antibody (red).

Figure 6.

Intensity correlation analysis for the colocalization of GFAP with IFN-γ in CB₂ ^+/+ and CB₂ ^−/− mice after sciatic nerve injury. A, Representative intensity correlation plots of GFAP (astrocyte) (red channel) and IFN-γ (green channel) in the ipsilateral dorsal horn of a CB₂ ^−/− mice after nerve injury. GFAP was visualized by staining of cryostat sections with rabbit anti-GFAP antibody followed by CY3-conjugated secondary anti-rabbit antibody (red). IFN-γ expression was visualized by staining of cryostat sections with rat anti-IFN-γ antibody and a biotinylated anti-rat secondary antibody followed by streptavidin Alexa Fluor 488 (green). B, Scatter plot of pixel staining intensities of the same image. C, Image of the product of the differences of the mean values (PDM) of the same image. D, Intensity correlation quotients for the different experimental groups (n = 5). The white bars represent the contralateral dorsal horn, and the black bars represent the ipsilateral dorsal horn. Error bars indicate SEM. The black stars represent comparisons between sham-operated and nerve injury, or between ipsilateral and contralateral paw. The white stars represent comparisons between genotypes. Two stars, p < 0.01; three stars, p < 0.001.

Figure 7.

The CB₂ agonist JWH-133 reduces IFN-γ-inducible iNOS and CCR2 mRNA expression in microglial cells. Relative expression levels of CB₂, iNOS, and CCR2 mRNA were determined by quantitative real-time PCR. BV-2 microglial cells were stimulated either with 50 U/ml IFN-γ for 15 h alone, or in the presence of JWH-133 (5 μm) with or without CB₂ receptor antagonist SR144528 (1 μm). cDNA was generated from BV-2 cells and expression of CB₂, iNOS, and CCR2 mRNA was evaluated by real-time RT-PCR. Quantitative RT-PCR results are expressed as a ratio of average copies per copy of GAPDH for mean values ± SEM of n = 6 for all conditions. *p < 0,05; **p < 0,005 (BV-2 cells stimulated with IFN-γ vs with IFN-γ in the presence of 5 μm JWH-133).

Figure 8.

Hypothetical mechanism to explain the modulation of neuropathic pain through CB₂ receptor activation. The release of IFN-γ by activated astrocytes and neurons plays an important role in the neuroinflammatory process leading to the development of neuropathic pain. IFN-γ promotes microglia activation by the induction of several inflammatory pathways, including an enhancement in iNOS and CCR2 activity. The activated microglia promote consolidation and progression of the neuropathic pain state. CB₂ receptors on microglial cells would 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. CB₂ receptors located in neurons could also participate in the neuropathic pain response by decreasing the production of IFN-γ. These inhibitory effects would restrict the activation of microglial cells and attenuate the development of neuropathic pain. In the absence of CB₂ receptors, IFN-γ 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.