Shiroh Kishioka

Chemokines and cytokines in neuroinflammation leading to neuropathic pain

There has been recent evidence showing the correlation between neuroinflammation owing to the chemokine-cytokine network and neuropathic pain. Chemokines and cytokines are derived from several types of cells in the peripheral and central nervous systems following nerve injury, and are largely involved in the pathogenesis of neuropathic pain. The roles of typical inflammatory cytokines such as interleukin-1β have become the recent center of attention. There is growing evidence that inflammatory chemokines (CCL2, CCL3, and fractalkine) play pivotal roles in neuropathic pain. Further investigations concerning the functions of the chemokine-cytokine network-mediated regulation of neuroinflammation may lead to novel therapeutic strategies against intractable neuropathic pain.

Macrophage inflammatory protein-1α mediates the development of neuropathic pain following peripheral nerve injury through interleukin-1β up-regulation

&NA; In the present study, we investigated the role of the macrophage inflammatory protein‐1&agr; (MIP‐1&agr;) in the pathogenesis of neuropathic pain following partial sciatic nerve ligation (PSL) in mice. MIP‐1&agr; mRNA and its protein were dramatically up‐regulated after PSL, and MIP‐1&agr; was localized on macrophages and Schwann cells in the injured sciatic nerve (SCN). PSL‐induced long‐lasting tactile allodynia and thermal hyperalgesia were prevented by the perineural injection of anti‐MIP‐1&agr; (2 ng). Intraneural (20 ng) and perineural (100 ng) injection of recombinant MIP‐1&agr; elicited tactile allodynia and thermal hyperalgesia in sham‐operated limb. MIP‐1&agr; receptors (CCR1 and CCR5) mRNA and their proteins were also up‐regulated in the SCN after PSL, and were localized on macrophages and Schwann cells. PSL‐induced tactile allodynia was attenuated by perineural injection (0.2 nmol) of siRNA against CCR1 and CCR5. On the other hand, PSL‐induced thermal hyperalgesia was prevented by siRNA against CCR5, but not CCR1. Interleukin‐1&bgr; (IL‐1&bgr;) mRNA and its precursor protein in macrophages and Schwann cells were also up‐regulated in the SCN after PSL, and PSL‐induced neuropathic pain was prevented by the perineural injection of anti‐IL‐1&bgr; (2 ng). PSL‐induced IL‐1&bgr; up‐regulation was suppressed by anti‐MIP‐1&agr; and siRNA against CCR1 and CCR5. Perineural injection of nicotine (20 nmol), a macrophage suppressor, prevented PSL‐induced neuropathic pain and suppressed MIP‐1&agr; and IL‐1&bgr; expressions. In conclusion, we propose a novel critical molecule MIP‐1&agr; derived from macrophages and Schwann cells that appears to play a crucial role in the development of neuropathic pain induced by PSL.

Leptin enhances CC-chemokine ligand expression in cultured murine macrophage.

Despite accumulating evidence, the role of leptin in chemokine expression is poorly understood. In this study, we evaluated the effects of leptin on CC-chemokine ligands (CCLs), CCL3, CCL4, and CCL5 gene expression in cultured murine macrophage, J774A.1 cells. Expression of all these CCLs mRNA was gradually increased and significant up-regulation was observed for 3-12 h exposure to leptin (1 microM). The phosphorylated signal transducer and activator of transcription 3 (pSTAT3) was significantly increased for 5-20 min exposure to leptin, and it was localized in leptin receptor-positive macrophage. Pretreatment with AG490 (100 microM), a janus kinase 2 (JAK2) inhibitor, significantly suppressed leptin-induced pSTAT3 increases and the up-regulation of CCLs mRNA expression. In conclusion, leptin enhances CCLs expression in cultured murine macrophage, through activation of a JAK2-STAT3 pathway. Therefore, a new paradigm of leptin-mediated chemokine expression may lead to the clarification of complex immune systems in future.

Epigenetic Augmentation of the Macrophage Inflammatory Protein 2/C-X-C Chemokine Receptor Type 2 Axis through Histone H3 Acetylation in Injured Peripheral Nerves Elicits Neuropathic Pain

Although there is growing evidence showing that the involvement of chemokines in the pathogenesis of neuropathic pain is associated with neuroinflammation, the details are unclear. We investigated the C-X-C chemokine ligand type 2 [macrophage inflammatory protein 2 (MIP-2)]/C-X-C chemokine receptor type 2 (CXCR2) axis and epigenetic regulation of these molecules in neuropathic pain after peripheral nerve injury. Expression of MIP-2 and CXCR2 were up-regulated and localized on accumulated neutrophils and macrophages in the injured sciatic nerve (SCN) after partial sciatic nerve ligation (PSL). Perineural injection of MIP-2-neutralizing antibody (anti-MIP-2) or the CXCR2 antagonist N-(2-bromophenyl)-N′-(2-hydroxy-4-nitrophenyl)urea (SB225002) prevented PSL-induced tactile allodynia and thermal hyperalgesia. Perineural injection of recombinant MIP-2 elicited neuropathic pain-like behaviors. Anti-MIP-2 suppressed neutrophil accumulation in the SCN after PSL. Neutrophil depletion by intraperitoneal injection of Ly6G antibody attenuated PSL-induced neuropathic pain. Both anti-MIP-2 and SB225002 suppressed up-regulation of inflammatory cytokines and chemokines in the injured SCN. In addition, acetylation of histone H3 [lysine (Lys9)-acetylated histone H3 (AcK9-H3)] on the promoter region of MIP-2 and CXCR2 was increased in the injured SCN after PSL. Expression of AcK9-H3 was observed in the nuclei of neutrophils and macrophages surrounding the epineurium. Administration of the histone acetyltransferase inhibitor anacardic acid suppressed the up-regulation of MIP-2 and CXCR2 in the SCN after PSL and resulted in the prevention of PSL-induced neuropathic pain. Taken together, these results show that augmentation of the MIP-2/CXCR2 axis by hyperacetylation of histone H3 on the promoter region of MIP-2 and CXCR2 located in the injured peripheral nerve elicits chronic neuroinflammation through neutrophil accumulation, leading to neuropathic pain.

Leptin derived from adipocytes in injured peripheral nerves facilitates development of neuropathic pain via macrophage stimulation

Nerve injury may result in neuropathic pain, characterized by allodynia and hyperalgesia. Accumulating evidence suggests the existence of a molecular substrate for neuropathic pain produced by neurons, glia, and immune cells. Here, we show that leptin, an adipokine exclusively produced by adipocytes, is critical for the development of tactile allodynia through macrophage activation in mice with partial sciatic nerve ligation (PSL). PSL increased leptin expression in adipocytes distributed at the epineurium of the injured sciatic nerve (SCN). Leptin-deficient animals, ob/ob mice, showed an absence of PSL-induced tactile allodynia, which was reversed by the administration of leptin to the injured SCN. Perineural injection of a neutralizing antibody against leptin reproduced this attenuation. Macrophages recruited to the perineurium of the SCN expressed the leptin receptor and phosphorylated signal transducer and activator of transcription 3 (pSTAT3), a transcription factor downstream of leptin. PSL also up-regulated the accepted mediators of neuropathic pain—namely, cyclooxygenase-2, inducible nitric oxide synthase, and matrix metalloprotease-9—in the injured SCN, with transcriptional activation of their gene promoters by pSTAT3. This up-regulation was partly reproduced in a macrophage cell line treated with leptin. Administration of peritoneal macrophages treated with leptin to the injured SCN reversed the failure of ob/ob mice to develop PSL-induced tactile allodynia. We suggest that leptin induces recruited macrophages to produce pronociceptive mediators for the development of tactile allodynia. This report shows that adipocytes associated with primary afferent neurons may be involved in the development of neuropathic pain through adipokine secretion.

Modulation of Opioid Actions by Nitric Oxide Signaling

Nitric oxide (NO) plays pivotal roles in controlling physiological functions, participates in pathophysiological intervention, and is involved in mechanisms underlying beneficial or untoward actions of therapeutic agents. Endogenous nitric oxide is formed by three isoforms of nitric oxide synthase: endothelial, neurogenic and inducible. The former two are constitutively present mainly in the endothelium and nervous system, respectively, and the latter one is induced by lipopolysaccharides or cytokines mainly in mitochondria and glial cells. Constitutively formed nitric oxide modulates the actions of morphine and related analgesics by either enhancing or reducing antinociception. Tolerance to and dependence on morphine or its withdrawal syndrome are likely prevented by nitric oxide synthase inhibition. Information concerning modulation of morphine actions by nitric oxide is undoubtedly useful in establishing new strategies for efficient antinociceptive treatment and for minimizing noxious and unintended reactions.

Up-regulation of tumor necrosis factor-alpha in spinal cord contributes to vincristine-induced mechanical allodynia in mice.

Chronic treatment with vincristine (VCR) causes mechanical allodynia as an adverse effect. We previously reported that peripheral macrophage-derived interleukin-6 played a critical role in VCR-induced allodynia. However, the involvement of glial cell activation and central sensitization in VCR-induced allodynia is still unclear. In this study, we focused on tumor necrosis factor-alpha (TNF-alpha) in spinal cord, and investigated the role of TNF-alpha in VCR-induced allodynia in mice. VCR (0.1mg/kg, i.p.) was administered to mice once per day for 7 days. The expression of TNF-alpha mRNA and the protein in spinal cord was evaluated by quantitative real-time PCR and immunohistochemistry, respectively. In VCR-treated mice, TNF-alpha mRNA gradually increased and was significantly up-regulated on day 7. As measured by immunohistochemistry, microglia and astrocytes were activated in the spinal dorsal horn on day 7 of VCR administration. The immunoreactivity of TNF-alpha was co-localized in some of the activated microglia and astrocytes. In behavioral analysis, a neutralizing antibody of TNF-alpha, which was injected intrathecally on days 0, 3, and 6, significantly attenuated VCR-induced mechanical allodynia on days 4 and 7. These results suggest that VCR treatments elicited the activation of glial cells in spinal cord, and up-regulated TNF-alpha in these cells may play an important role in VCR-induced mechanical allodynia.

CC-chemokine ligand 4/macrophage inflammatory protein-1β participates in the induction of neuropathic pain after peripheral nerve injury.

Neuropathic pain is caused by neural damage or dysfunction and neuropathic pain‐related symptoms are resistant to conventional analgesics. Neuroinflammation due to the cytokine‐chemokine network may play a pivotal role in neuropathic pain. We demonstrate that macrophage inflammatory protein‐1β (MIP‐1β) participates in neuropathic pain.

The critical role of invading peripheral macrophage-derived interleukin-6 in vincristine-induced mechanical allodynia in mice

Although the clinical use of vincristine is limited by its adverse effect, neuropathic pain, the mechanism of this effect is poorly understood. Recently, reports demonstrated that inflammatory and immune responses play an important role in the neuropathic pain that follows peripheral nerve injury. In this study, we examined the role of macrophage-derived interleukin (IL)-6 in vincristine-induced mechanical allodynia. Vincristine sulfate (0.01-0.1 mg/kg, i.p.) was administered to male ICR mice and BALB/c mice once per day for 7 or 14 days. Mechanical allodynia was evaluated by withdrawal responses, using von Frey filaments from day 0 to day 28. In both ICR mice and BALB/c mice, significant dose-dependent increases in the percentage of withdrawal responses were observed from day 3 to day 28 following repeated administration of vincristine (0.1 mg/kg). As determined by immunohistochemistry, the number of macrophages in the region of the sciatic nerve and lumbar dorsal root ganglion was significantly increased on day 7 of vincristine administration. The expression of IL-6 was increased by vincristine administration and was co-localized in the invading macrophage. Moreover, a neutralizing antibody of IL-6, which was injected into areas surrounding the sciatic nerve on day 0, 3, and 6, significantly attenuated vincristine-induced mechanical allodynia from day 7 to day 28. In addition, the incidence of vincristine-induced mechanical allodynia in IL-6 knockout mice was lower than that in wild type mice from day 3 to day 28. These results suggest that the invading peripheral macrophage-derived IL-6 plays a critical role in vincristine-induced mechanical allodynia.

Peroxisome Proliferator-Activated Receptor Gamma Activation Relieves Expression of Behavioral Sensitization to Methamphetamine in Mice

Peroxisome proliferator-activated receptor (PPAR) is a ligand-activated transcriptional factor that regulates lipid metabolism and inflammation. Behavioral sensitization is an experimental model of psychostimulant psychosis; it is elicited by repeated administration of psychostimulants and has recently been implicated in brain inflammation. We examined the involvement of PPARγ, one of the isotypes of PPAR, in development of behavioral sensitization to the stimulant effect of methamphetamine (METH) (1 mg/kg, subcutaneously) in mice. Repeated administration of METH (once daily for 5 days) enhanced the locomotor-activating effect of METH, which was reproduced by METH challenge on withdrawal day 7 (test day 12). The protein level and the activity of PPARγ were significantly increased in the nuclear fraction of whole brain after 5 days of METH administration (test day 5) and on withdrawal day 7 (test day 12). Both pioglitazone and ciglitazone (PPARγ agonists; 0.5–5.0 μg, intracerebroventricularly (i.c.v.), once daily) prevented the expression of behavioral sensitization to METH challenge on withdrawal day 7, but not the sensitization that occurred during repeated administration of METH. In addition, the magnitude of expression of behavioral sensitization was augmented by treatments with GW9662 (a PPARγ antagonist; 0.5–5.0 μg i.c.v., once daily) during the withdrawal period. The pioglitazone-induced alleviation of behavioral sensitization was synergistically facilitated by simultaneous i.c.v. injection of 9-cis-retinoic acid (1.0 μg), an agonist for the retinoid X receptor which is a ligand-activated nuclear receptor that forms heterodimers with PPAR. These results suggest that PPARγ has a significant role in the expression of behavioral sensitization to METH in mice.