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, 29 (13), 4096-108

JNK-induced MCP-1 Production in Spinal Cord Astrocytes Contributes to Central Sensitization and Neuropathic Pain

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JNK-induced MCP-1 Production in Spinal Cord Astrocytes Contributes to Central Sensitization and Neuropathic Pain

Yong-Jing Gao et al. J Neurosci.

Abstract

Our previous study showed that activation of c-jun-N-terminal kinase (JNK) in spinal astrocytes plays an important role in neuropathic pain sensitization. We further investigated how JNK regulates neuropathic pain. In cultured astrocytes, tumor necrosis factor alpha (TNF-alpha) transiently activated JNK via TNF receptor-1. Cytokine array indicated that the chemokine CCL2/MCP-1 (monocyte chemoattractant protein-1) was strongly induced by the TNF-alpha/JNK pathway. MCP-1 upregulation by TNF-alpha was dose dependently inhibited by the JNK inhibitors SP600125 (anthra[1,9-cd]pyrazol-6(2H)-one) and D-JNKI-1. Spinal injection of TNF-alpha produced JNK-dependent pain hypersensitivity and MCP-1 upregulation in the spinal cord. Furthermore, spinal nerve ligation (SNL) induced persistent neuropathic pain and MCP-1 upregulation in the spinal cord, and both were suppressed by D-JNKI-1. Remarkably, MCP-1 was primarily induced in spinal cord astrocytes after SNL. Spinal administration of MCP-1 neutralizing antibody attenuated neuropathic pain. Conversely, spinal application of MCP-1 induced heat hyperalgesia and phosphorylation of extracellular signal-regulated kinase in superficial spinal cord dorsal horn neurons, indicative of central sensitization (hyperactivity of dorsal horn neurons). Patch-clamp recordings in lamina II neurons of isolated spinal cord slices showed that MCP-1 not only enhanced spontaneous EPSCs but also potentiated NMDA- and AMPA-induced currents. Finally, the MCP-1 receptor CCR2 was expressed in neurons and some non-neuronal cells in the spinal cord. Together, we have revealed a previously unknown mechanism of MCP-1 induction and action. MCP-1 induction in astrocytes after JNK activation contributes to central sensitization and neuropathic pain facilitation by enhancing excitatory synaptic transmission. Inhibition of the JNK/MCP-1 pathway may provide a new therapy for neuropathic pain management.

Figures

Figure 1.
Figure 1.
TNF-α induces JNK1 activation via TNF receptor type 1 in astrocytes. A, Double staining of GFAP (astrocyte marker, green) and DAPI (nuclear marker, blue) shows that all DAPI+ cells also express GFAP-IR. Scale bar, 50 μm. B, C, Western blot shows that TNF-α induces transient activation of JNK1 in astrocytes from wild-type (WT) mice (B) but not in astrocytes from TNFR1 null mice (C). D, Density of pJNK1 bands, which is normalized to GAPDH loading control and expressed as ratio of nontreated control.
Figure 2.
Figure 2.
Cytokine array reveals a JNK-dependent upregulation of chemokines in astrocytes after TNF-α stimulation. A, Illustration of the cytokine array that contains 40 different antibodies with duplicates. The array also contains three positive control (PC) proteins. For more details, see the instructions of the manufacturer (R & D Systems). B, TNF-α treatment (10 ng/ml) for 1 h markedly increases the expression of the chemokines IP-10 (IFN-γ-inducible protein 10/CXCL10), KC (keratinocyte-derived chemokine/CXCL1), and MCP-1 in astrocytes. Note that the PC protein levels in the white boxes of the two arrays do not change. C, Pretreatment of the JNK inhibitor D-JNKI-1 (50 μm) reduces TNF-α (10 ng/ml, 1 h)-induced upregulation of the chemokines. Note that the PC protein levels in the white boxes of the two arrays are comparable. The two arrays (blots) in B and C were processed under the same conditions after stimulation. This experiment was repeated two to three times.
Figure 3.
Figure 3.
ELISA demonstrates a JNK-dependent MCP-1 upregulation in astrocytes after TNF-α stimulation. A, TNF-α (10 ng/ml) induces a time-dependent upregulation of MCP-1 in astrocytes. B, TNF-α fails to increase MCP-1 expression in astrocytes prepared from TNFR1 null mice. C, JNK inhibitors SP600125 and D-JNKI-1 dose dependently inhibit TNF-α (10 ng/ml, 1 h)-induced MCP-1 upregulation. D, MEK inhibitor U0126, but not p38 inhibitor SB203580, reduces TNF-α-induced MCP-1 upregulation. E, F, TNF-α (10 ng/ml, 1 h) also induces MCP-1 release in the astrocyte culture medium (E), which is inhibited by the JNK inhibitors SP600125 and D-JNKI-1 (F). *p < 0.05, **p < 0.01, ***p < 0.001 versus TNF-α treatment (positive control); n = 3.
Figure 4.
Figure 4.
Spinal administration of TNF-α induces JNK-dependent pain hypersensitivity and MCP-1 upregulation in the spinal cord. A–C, Spinal injection of TNF-α (20 ng) induces mechanical allodynia (A), heat hyperalgesia (B), and MCP-1 increase in spinal cord (C). D–F, Pretreatment of the JNK inhibitor D-JNKI-1 (50 nmol, i.p.), starting 1 h before TNF-α injection, reduces TNF-α-induced mechanical allodynia (D), heat hyperalgesia (E), and MCP-1 upregulation (F). *p < 0.05, **p < 0.01, ***p < 0.001 versus corresponding saline control, n = 5.
Figure 5.
Figure 5.
SNL induces neuropathic pain and MCP-1 upregulation in the spinal cord, in a JNK-dependent manner. A, SNL induces persistent MCP-1 increase in the L5 spinal cord. *p < 0.05, **p < 0.01 versus naive control; ++p < 0.01 versus 3 d sham control; n = 3. B–D, Systemic application of D-JNKI-1 (50 nmol, i.p., once a day for 3 d, indicated by small arrows) attenuates SNL-induced MCP-1 increase (B), mechanical allodynia (C), and heat hyperalgesia (D). *p < 0.05 versus corresponding saline control; n = 5. E, F, Intrathecal application of MCP-1 neutralizing antibody, 3 d after SNL, partially reverses SNL-induced mechanical allodynia (E) and heat hyperalgesia (F). *p < 0.05, ***p < 0.001 versus control serum; n = 5. BL, Baseline.
Figure 6.
Figure 6.
SNL induces MCP-1 upregulation in spinal cord astrocytes. A–C, MCP-1 expression in the spinal cord of naive animals (A) and SNL animals at 3 d (B) and 10 d (C). Scale bar, 200 μm. D–F, High-magnification images of A–C, indicated in the white boxes of A–C, show the dorsal horn of the ipsilateral spinal cord. Scale bar, 100 μm. G–I, Double staining shows that MCP-1 is colocalized with GFAP, a marker for astrocytes (G), but not with NeuN, a marker for neurons (H) or OX-42, a marker for microglia (I). Scale bar, 50 μm.
Figure 7.
Figure 7.
Spinal injection of MCP-1 induces heat hyperalgesia and ERK activation in superficial dorsal horn neurons. A, Spinal injection of MCP-1 (10 or 100 ng) induces a dose-dependent heat hyperalgesia. *p < 0.05, **p < 0.01, ***p < 0.001 versus saline (A); n = 5. BL, baseline. B, C, Spinal injection of MCP-1 (100 ng) induces pERK at 30 min after the injection in superficial dorsal horn. D, Majority of pERK-immunoreactive cells in intact spinal cord (in vivo) express the neuronal marker NeuN. E, F, Incubation of isolated spinal cord slices with MCP-1 (100 ng/ml, 5 min) induces substantial ERK activation in the superficial dorsal horn. G, Majority of pERK-immunoreactive cells in spinal cord slices (ex vivo) express NeuN. Scale bars, 100 μm. BL, Baseline.
Figure 8.
Figure 8.
Bath application of MCP-1 increases excitatory synaptic transmission and AMPA- and NMDA-induced currents. A, Patch-clamp recording of sEPSCs shows increase in the frequency and amplitude of sEPSC after perfusion of MCP-1 (100 ng/ml, 2 min). Aa and Ab are enlarged recording before and after MCP-1 treatment, respectively. B, C, Ratio of the frequency and amplitude of sEPSCs after MCP-1 treatment. **p < 0.01 versus pretreatment baseline. D–G, Patch-clamp recording demonstrates increase in AMPA-induced currents (D, E) and NMDA-induced currents (F, G) after MCP-1 treatment (100 ng/ml, 2 min). The amplitude of AMPA- and NMDA-induced currents is shown in E and G, respectively. *p < 0.05 versus pretreatment baseline. Inside each column, the number of total recorded neurons and number of responding neurons are indicated (B, C, E, G).
Figure 9.
Figure 9.
Expression of CCR2 in the spinal cord. A–C, Spinal cord from CCR2–GFP-report mice shows CCR2 expression in NeuN-positive neurons in the deep (A, B, B′) and superficial (C, C′) dorsal horn. B is a double staining of CCR2–GFP and NeuN. B′ shows high-magnification images of A and B (inset squares). Arrows indicate double-labeled cells. Scale bars: B, C, 100 μm; B′, C′, 25 μm. D, RT-PCR shows CCR2 mRNA expression in the spleen, DRG, and spinal cord (SC) of naive mice after 20–35 cycles of amplification. E–G, In situ hybridization shows CCR2 mRNA expression in spinal cord in normal and nerve injury conditions using a CCR2 antisense probe. CCR2 mRNA-positive cells are not detectable in naive animals (E). Three days after SNL, CCR2 mRNA-positive neurons are seen in deep dorsal horn and ventral horn (F). E′ and F′ are high-magnification images of E and F (inset squares). Hybridization using a control sense probe shows no signal in the spinal cord of SNL animals (G). Scale bars: E–G, 200 μm; E′, F′, 100 μm.

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