Yohji Fukazawa

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.

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.

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.

Activation of nicotinic acetylcholine receptors on bone marrow-derived cells relieves neuropathic pain accompanied by peripheral neuroinflammation

Emerging evidence indicates that chronic neuroinflammation plays a pivotal role in neuropathic pain. We explored whether activation of the nicotinic acetylcholine receptor (nAChRs) pathway on peripheral immune cells improves neuropathic pain. Mice were subjected to partial sciatic nerve ligation (PSL). Enhanced green fluorescent protein (EGFP)-chimeric mice were generated by transplantation of EGFP(+) bone marrow (BM) cells from EGFP-transgenic mice into wild-type mice. EGFP(+) BM-derived cells infiltrated the injured sciatic nerve (SCN) of EGFP-chimeric mice, and these cells were found to be F4/80(+) macrophages and Ly6G(+) neutrophils. The protein expression of nAChR subunit α4 and α7 were up-regulated in the injured SCN. Increased α4 and α7 subunits were localized on both BM-derived macrophages and neutrophils. When nicotine (20nmol) was perineurally administered once a day for 4days (days 0-3), PSL-induced tactile allodynia and thermal hyperalgesia were significantly prevented. Relieving effects of nicotine on neuropathic pain were reversed by co-administration of mecamylamine (20nmol), a non-selective antagonist for nAChRs. PSL-induced up-regulation of inflammatory cytokines and chemokines was suppressed by perineural administration of nicotine. Taken together, the expression of α4β2 and α7 subtypes of nAChRs may be increased on circulating macrophages and neutrophils in injured peripheral nerves. Activation of nAChRs on immune cells may relieve neuropathic pain accompanied by the suppression of neuroinflammation.

Vascular endothelial growth factor signaling in injured nerves underlies peripheral sensitization in neuropathic pain

Chronic neuroinflammation may be a critical component of intractable inflammatory diseases, including neuropathic pain. Because angiogenesis as a result of vascular endothelial growth factor (VEGF) signaling plays a pivotal role in inflammation, we focused on the mechanisms of VEGF‐regulated neuropathic pain in mice. The mRNA and protein expression of VEGFA were up‐regulated in the injured sciatic nerve after partial sciatic nerve ligation (PSL). VEGFA was localized to accumulated macrophages and neutrophils derived from bone marrow. Up‐regulation of VEGFA was mediated by histone H3 acetylation and trimethylation in its promoter region. VEGF receptors (VEGFR1 and VEGFR2) were localized to vascular endothelial cells or macrophages. By ex vivo fluorescence imaging and immunohistochemistry using DiI fluorescence, progression of angiogenesis was observed in the injured sciatic nerve after PSL. Perineural administration of pharmacological inhibitors of VEGFA and VEGFR tyrosine kinases prevented tactile allodynia and thermal hyperalgesia caused by PSL. Moreover, we determined the contribution of VEGF‐ and CXC‐chemokine receptor 4‐expressing angiogenic macrophages to neuropathic pain. Taken together, VEGFA is up‐regulated in injured peripheral nerves and participates in angiogenesis and prolonged pain behaviors through its receptors. We propose that VEGFA‐related components may underlie peripheral sensitization leading to neuropathic pain.

Macrophage-T Cell Interactions Mediate Neuropathic Pain through the Glucocorticoid-induced Tumor Necrosis Factor Ligand System

Background: Peripheral neuroinflammation is associated with neuropathic pain. Results: Inhibiting the glucocorticoid-induced tumor neurosis factor receptor ligand (GITRL) system in infiltrating macrophages and T cells in the injured nerve attenuated neuropathic pain. Conclusion: GITRL expressed by macrophages drives cytokine release and T cell activation, resulting in neuropathic pain via GITR-dependent actions. Significance: Macrophage-T cell interactions via the GITRL system provides insights into controlling neuropathic pain. Peripheral neuroinflammation caused by activated immune cells can provoke neuropathic pain. Herein, we investigate the actions of macrophages and T cells through glucocorticoid-induced tumor neurosis factor receptor ligand (GITRL) and its receptor (GITR) in neuropathic pain. After partial sciatic nerve ligation (PSL) in enhanced green fluorescent protein (eGFP) chimeric mice generated by the transplantation of eGFP+ bone marrow cells, eGFP+ macrophages, and T cells markedly migrated to the injured site after PSL. Administration of agents to deplete macrophages (liposome-clodronate and Clophosome-ATM) or T cells (anti-CD4 antibody and FTY720) could suppress PSL-induced thermal hyperalgesia and tactile allodynia. The expression levels of co-stimulatory molecules GITRL and GITR were increased on infiltrating macrophages and T cells, respectively. The perineural injection of a GITRL neutralizing antibody that could inhibit the function of the GITRL-GITR pathway attenuated PSL-induced neuropathic pain. Additionally, the induction of inflammatory cytokines and the accumulation of GITR+ T cells in the injured SCN were abrogated after macrophage depletion by Clophosome-ATM. In conclusion, GITRL expressed on macrophages drives cytokine release and T cell activation, resulting in neuropathic pain via GITR-dependent actions. The GITRL-GITR pathway might represent a novel target for the treatment of neuropathic pain.

Morphine has an antinociceptive effect through activation of the okadaic-acid-sensitive Ser/Thr protein phosphatases PP2A and PP5 estimated by tail-pinch test in mice

Although the serine/threonine protein kinases involved in the pharmacological action of morphine are well recognized, the critical contribution of serine/threonine protein phosphatase (PP) has been appreciated on to a slight degree. We examined the involvement of subtypes of serine/threonine protein phosphatase (PP) in the antinociceptive effect of morphine in mice. The antinociceptive effect of subcutaneously administered morphine was attenuated by simultaneously intracerebroventricular (i.c.v.) or intrathecal (i.t.) injection of okadaic acid (OA), a PP inhibitor. To reveal which subtypes of PPs participated in the antinociceptive effect of morphine, mice received i.c.v. or i.t. injections of antisense oligodeoxynucleotide (AS-ODN) directed against either the PP 2 A or PP5 subtypes of PPs before assessment of morphine-induced antinociception. Pretreatment with AS-ODN against PP 2 A or PP5 via each route weakened the antinociceptive effect of morphine, accompanied by reduction of expression levels of PP in the periaqueductal gray (PAG) and the spinal cord. Subcutaneously administered morphine increased activity of OA-sensitive PPs in the PAG and the spinal cord in a dose-dependent manner; this was prevented by concurrent administration of naloxone. These results suggest that PP 2 A and PP5 are involved in the antinociceptive effect of morphine in mice.

Activation of Extracellular Signal-Regulated Kinase in Sciatic Nerve Contributes to Neuropathic Pain After Partial Sciatic Nerve Ligation in Mice

BACKGROUND: The mitogen-activated protein kinase family plays an important role in several types of pain. However, the detailed role of phosphorylated extracellular signal-regulated kinase (pERK) in the region of injured peripheral nerve is poorly understood. In this study, we investigated whether pERK in injured sciatic nerve contributes to neuropathic pain induced by partial sciatic nerve ligation (PSL) in mice. METHODS: Mice received PSL; pERK1/2 (p44/42) in sciatic nerve was measured by both Western blotting and immunohistochemistry. U0126 (an ERK kinase inhibitor) was injected twice, an intraneural injection (20 nmol/2 &mgr;L) 30 min before PSL, and a perineural injection (20 nmol/10 &mgr;L) on Day 1 after PSL. Thermal hyperalgesia and tactile allodynia induced by PSL were evaluated by the thermal paw withdrawal test and the von Frey test, respectively. RESULTS: As measured by Western blotting, in sham-operated mice, the levels of pERK1/2 in sciatic nerve were constant and the same as those in naive mice across Days 1-14. In PSL-operated mice, a significant increase in pERK1/2 was observed on Day 1 after PSL and persisted until Day 3. As measured by immunohistochemistry, immunoreactivity of pERK1/2 in PSL-operated sciatic nerve was markedly increased in comparison with that in sham-operated sciatic nerve on Day 1 after PSL. In the sciatic nerve on Day 1 after PSL, as indicated by double immunostaining, the increased immunoreactivity of pERK1/2 was colocalized with glial fibrillary acidic protein (GFAP), a marker of Schwann cells, but not F4/80, a marker of macrophages. PSL-induced thermal hyperalgesia was significantly attenuated by treatment with U0126 on Days 3, 7, and 14 after PSL. The PSL-induced tactile allodynia was also significantly attenuated by treatment with U0126 on Days 7 and 14 after PSL. CONCLUSION: Activation of ERK in Schwann cells of the injured peripheral nervous system may play an important role in the development of neuropathic pain. Our results suggest that pERK itself and ERK-related mediators are potential therapeutic targets for the treatment of neuropathic pain.