Kyungpyo Park

A Critical Role of Toll-like Receptor 2 in Nerve Injury-induced Spinal Cord Glial Cell Activation and Pain Hypersensitivity

The activation of spinal cord glial cells has been implicated in the development of neuropathic pain upon peripheral nerve injury. The molecular mechanisms underlying glial cell activation, however, have not been clearly elucidated. In this study, we found that damaged sensory neurons induce the expression of tumor necrosis factor-α, interleukin-1β, interleukin-6, and inducible nitric-oxide synthase genes in spinal cord glial cells, which is implicated in the development of neuropathic pain. Studies using primary glial cells isolated from toll-like receptor 2 knock-out mice indicate that damaged sensory neurons activate glial cells via toll-like receptor 2. In addition, behavioral studies using toll-like receptor 2 knock-out mice demonstrate that the expression of toll-like receptor 2 is required for the induction of mechanical allodynia and thermal hyperalgesia due to spinal nerve axotomy. The nerve injury-induced spinal cord microglia and astrocyte activation is reduced in the toll-like receptor 2 knock-out mice. Similarly, the nerve injury-induced pro-inflammatory gene expression in the spinal cord is also reduced in the toll-like receptor 2 knock-out mice. These data demonstrate that toll-like receptor 2 contributes to the nerve injury-induced spinal cord glial cell activation and subsequent pain hypersensitivity.

Activation of glia and microglial p38 MAPK in medullary dorsal horn contributes to tactile hypersensitivity following trigeminal sensory nerve injury

Abstract Glial activation is known to contribute to pain hypersensitivity following spinal sensory nerve injury. In this study, we investigated mechanisms by which glial cell activation in medullary dorsal horn (MDH) would contribute to tactile hypersensitivity following inferior alveolar nerve and mental nerve transection (IAMNT). Activation of microglia and astrocytes was monitored at 2 h, 1, 3, 7, 14, 28, and 60 days using immunohistochemical analysis with OX‐42 and GFAP antibodies, respectively. Tactile hypersensitivity was significantly increased at 1 day, and this lasted for 28 days after IAMNT. Microglial activation, primarily observed in the superficial laminae of MDH, was initiated at 1 day, maximal at 3 days, and maintained until 14 days after IAMNT. Astrocytic activation was delayed compared to that of microglia, being more profound at 7 and 14 days than at 3 days after IAMNT. Both tactile hypersensitivity and glial activation appeared to gradually reduce and then return to the basal level by 60 days after IAMNT. There was no significant loss of trigeminal ganglion neurons by 28 days following IAMNT, suggesting that degenerative changes in central terminals of primary afferents might not contribute to glial activation. Minocycline, an inhibitor of microglial activation, reduced microglial activation, inhibited p38 mitogen‐activated protein kinase (MAPK) activation in microglia, and significantly attenuated the development of pain hypersensitivity in this model. These results suggest that glial activation in MDH plays an important role in the development of neuropathic pain and activation of p38 MAPK in hyperactive microglia contributes to pain hypersensitivity in IAMNT model.

TLR3-mediated signal induces proinflammatory cytokine and chemokine gene expression in astrocytes: Differential signaling mechanisms of TLR3-induced IP-10 and IL-8 gene expression

Viral infection is one of the leading causes of brain encephalitis and meningitis. Recently, it was reported that Toll‐like receptor‐3 (TLR3) induces a double‐stranded RNA (dsRNA)‐mediated inflammatory signal in the cells of the innate immune system, and studies suggested that dsRNA may induce inflammation in the central nervous system (CNS) by activating the CNS‐resident glial cells. To explore further the connection between dsRNA and inflammation in the CNS, we have studied the effects of dsRNA stimulation in astrocytes. Our results show that the injection of polyinosinic‐polycytidylic acid (poly(I:C)), a synthetic dsRNA, into the striatum of the mouse brain induces the activation of astrocytes and the expression of TNF‐α, IFN‐β, and IP‐10. Stimulation with poly(I:C) also induces the expression of these proinflammatory genes in primary astrocytes and in CRT‐MG, a human astrocyte cell line. Furthermore, our studies on the intracellular signaling pathways reveal that poly(I:C) stimulation activates IκB kinase (IKK), extracellular signal‐regulated kinase (ERK), and c‐Jun N‐terminal kinase (JNK) in CRT‐MG. Pharmacological inhibitors of nuclear factor‐κB (NF‐κB), JNK, ERK, glycogen synthase kinase‐3β (GSK‐3β), and dsRNA‐activated protein kinase (PKR) inhibit the expression of IL‐8 and IP‐10 in astrocytes, indicating that the activation of these signaling molecules is required for the TLR3‐mediated chemokine gene induction. Interestingly, the inhibition of PI3 kinase suppressed the expression of IP‐10, but upregulated the expression of IL‐8, suggesting differential roles for PI3 kinase, depending on the target genes. These data suggest that the TLR3 expressed on astrocytes may initiate an inflammatory response upon viral infection in the CNS.

Functional Expression of Thermo-transient Receptor Potential Channels in Dental Primary Afferent Neurons IMPLICATION FOR TOOTH PAIN

Temperature signaling can be initiated by members of transient receptor potential family (thermo-TRP) channels. Hot and cold substances applied to teeth usually elicit pain sensation. This study investigated the expression of thermo-TRP channels in dental primary afferent neurons of the rat identified by retrograde labeling with a fluorescent dye in maxillary molars. Single cell reverse transcription-PCR and immunohistochemistry revealed expression of TRPV1, TRPM8, and TRPA1 in subsets of such neurons. Capsaicin (a TRPV1 agonist), menthol (a TRPM8 agonist), and icilin (a TRPM8 and TRPA1 agonist) increased intracellular calcium and evoked cationic currents in subsets of neurons, as did the appropriate temperature changes (>43 °, <25 °, and <17 °C, respectively). Some neurons expressed more than one TRP channel and responded to two or three corresponding stimuli (ligands or thermal stimuli). Immunohistochemistry and single cell reverse transcription-PCR following whole cell recordings provided direct evidence for the association between the responsiveness to thermo-TRP ligands and expression of thermo-TRP channels. The results suggest that activation of thermo-TRP channels expressed by dental afferent neurons contributes to tooth pain evoked by temperature stimuli. Accordingly, blockade of thermo-TRP channels will provide a novel therapeutic intervention for the treatment of tooth pain.

Inhibitory effects of autoantibodies on the muscarinic receptors in Sjögren's syndrome

Sjögrens syndrome (SS) is a systemic autoimmune disease that involves reduced salivary secretions. Recently, circulating autoantibodies from SS patients against the type 3 muscarinic cholinergic receptor (M3R) has been reported in the sera of SS patients. However, the role of these autoantibodies in the development of SS has not been elucidated. In this study, purified IgG was obtained from the sera of 11 SS patients, and its inhibitory effect on the M3R of the salivary glands was evaluated using RT-PCR, microspectrofluorimetry, immunohistochemistry, and Western blot analysis. Stimulation with carbachol (CCh) evoked a [Ca2+]i transient in the fura-2 loaded HSG cells. However, pretreatment of the cells with SS IgG (0.5 mg/ml) for 12 or 24 h significantly reduced the magnitude of the CCh-induced [Ca2+]i transient (CICT). We found that the magnitude of CICT was decreased by 62–45% when cells were pretreated with the SS IgG. However, the [Ca2+]i response to ATP was not altered by the pretreatment of SS IgG. The effect of SS IgG on CICT was abrogated by the inclusion of excessive competitive peptides that encode the amino-acid sequence of M3R, which was not recapitulated by nonspecific peptides. The inhibitory effect of SS IgG on the aquaporin (AQP)-5 expression was also examined. After confirming the apical localization of AQP-5 along with its increase by pilocarpine (10−5 M), we examined whether SS IgG had an effect on pilocarpine-induced AQP-5 trafficking to the apical membrane (APM) using rat parotid acinar cells. After incubating the cells with SS IgG for 12 h, the amount of pilocarpine-induced AQP-5 significantly decreased compared to the control groups. In conclusion, autoantibodies from the SS patients inhibit the function of the human M3R that is mediated by Ca2+ mobilization and AQP-5 trafficking. Our results could partly explain the underlying mechanisms of glandular dysfunction and associated features of impaired autonomic function in SS patients.

Systemic administration of minocycline inhibits formalin-induced inflammatory pain in rat

It has been demonstrated that spinal microglial activation is involved in formalin-induced pain and that minocycline, an inhibitor of microglial activation, attenuate behavioral hypersensitivity in neuropathic pain models. We investigated whether minocycline could have any anti-nociceptive effect on inflammatory pain, after intraperitonial administration of minocycline, 1 h before formalin (5%, 50 microl) injection into the plantar surface of rat hindpaw. Minocycline (15, 30, and 45 mg/kg) significantly decreased formalin-induced nociceptive behavior during phase II, but not during phase I. The enhancement in the number of c-Fos-positive cells in the L4-5 spinal dorsal horn (DH) and the magnitude of paw edema induced by formalin injection during phase II were significantly reduced by minocycline. Minocycline inhibited synaptic currents of substantia gelatinosa (SG) neurons in the spinal DH, whereas membrane electrical properties of dorsal root ganglion neurons were not affected by minocycline. Analysis with OX-42 antibody revealed the inhibitory effect of minocycline on microglial activation 3 days after formalin injection. These results demonstrate the anti-nociceptive effect of minocycline on formalin-induced inflammatory pain. In addition to the well-known inhibitory action of minocycline on microglial activation, the anti-edematous action in peripheral tissue, as well as the inhibition of synaptic transmission in SG neurons, is likely to be associated with the anti-nociceptive effect of minocycline.

The association of cholesteryl ester transfer protein polymorphism with high-density lipoprotein cholesterol and coronary artery disease in Koreans

Cholesteryl ester transfer protein (CETP) is a key protein involved in high‐density lipoprotein cholesterol (HDL‐C) metabolism. It is known to affect plasma HDL‐C levels, and its genetic regulation may be involved in the development of coronary artery disease (CAD). The aim of this study was to determine the frequency of the CETP Taq1B polymorphism in Koreans, and to investigate its relationship with plasma HDL‐C levels and CAD. One‐hundred and nineteen patients with significant CAD and 106 controls were examined with respect to their genotypes, lipid profiles and other risk factors of CAD. The genotype frequencies of B1B1:B1B2:B2B2 in males and females were 35.5%:50%:14.5% and 34.7%:42.6%:22.7%, respectively, which is comparable to previous reports in other ethnic groups. The B1B1 homozygote was associated with significantly lower HDL‐C levels in females (p = 0.049) and non‐smoking males (p = 0.037). After controlling for gender, body mass index (BMI) and smoking, the TaqIB polymorphism was still significantly associated with HDL‐C levels (p = 0.046) and explained 5.4% of the HDL‐C variation in this study. By univariate analysis, the B1B1 homozygote was a significant predictor of CAD (p = 0.043), and this was confirmed by multivariate analysis with traditional risk factors, i.e. the B1B1 homozygote was an independent predictor of CAD (p = 0.026, odds ratio = 1.97, 95% confidence interval: 1.08–3.57). In conclusion, the B1B1 homozygote of the CETP Taq1B polymorphism is associated with low HDL‐C levels in females and non‐smoking males, and may be an independent genetic risk factor of CAD in the Korean population.

Antibodies interfering with the type 3 muscarinic receptor pathway inhibit gastrointestinal motility and cholinergic neurotransmission in Sjögren's syndrome.

OBJECTIVE In primary Sjögrens syndrome (SS), impairment of the gastrointestinal (GI) tract is common, and includes reduced esophageal motor function, delayed gastric emptying, and abnormalities in colonic motility; the pathogenesis is as yet unknown. We undertook this study to investigate the role of functional antibodies to the type 3 muscarinic receptor (M3R) in GI dysfunction associated with primary SS. METHODS Muscle strip and whole-organ functional assays were used to determine whether IgG with anti-M3R activity from patients with primary SS disrupted neurotransmission in tissue from throughout the mouse GI tract. Specificity of the autoantibody for the M3R was determined using knockout mice that were deficient in the expression of muscarinic receptor subtypes. RESULTS Functional antibodies to the M3R inhibited neuronally mediated contraction of smooth muscle from throughout the GI tract and disrupted complex contractile motility patterns in the colon. The autoantibodies were not active on tissue from mice that lacked the M3R, providing compelling evidence of the direct interaction of patient autoantibodies with the M3R. CONCLUSION Our results indicate that anti-M3R autoantibodies have the potential to mediate multiple dysfunctions of the GI tract in primary SS, ranging from reduced esophageal motor activity to altered colonic motility. We hypothesize that altered GI motility forms part of a broader autonomic dysfunction mediated by pathogenic anti-M3R autoantibodies in primary SS.

Association of endothelial constitutive nitric oxide synthase gene polymorphism with acute coronary syndrome in Koreans

Objective: To examine the effects of two polymorphisms of the endothelial constitutive nitric oxide synthase (ecNOS) gene, 4a/4b(A:B) located in intron 4 and Glu298Asp(G:T) located in exon 7, on the development of acute coronary syndromes (ACS). Methods: 164 patients with ACS and 142 control participants were investigated for genotype and conventional risk factors. Genotype was determined by polymerase chain reaction and restriction fragment length polymorphism analysis. Results: Genotype and allele frequencies of the A:B polymorphism in the ACS group (0.15:0.85 for AA+AB:BB, 0.09:0.91 for A:B) differed from those in the control group (0.26:0.74 for AA+AB:BB, 0.15:0.85 for A:B). However, genotype and allele frequencies of the G:T polymorphism in the ACS group (0.22:0.78 for TT+TG:GG, 0.11:0.89 for T:G) were similar to those in the control group (0.17:0.83 for TT+TG:GG, 0.09:0.91 for T:G). Multiple logistic regression analysis showed that the non-BB (AA+AB) and the non-BB+GG genotypes were significant protective factors against ACS (odds ratios 0.49 and 0.34, 95% confidence intervals 0.26 to 0.93 and 0.14 to 0.83, respectively). In addition, linear association analysis showed that the percentage of ACS patients was significantly lower in the genotype group non-BB+GG than in the genotype group BB+non-GG (39.6% v 62.7%, p  =  0.01). Conclusions: The non-BB genotype of the ecNOS 4a/4b gene polymorphism is a protective factor against the development of ACS. The GG genotype of the ecNOS Glu298Asp polymorphism exerts a benefit in addition to the non-BB genotype in the Korean population.

Histamine H1 Receptor Induces Cytosolic Calcium Increase and Aquaporin Translocation in Human Salivary Gland Cells

One of the common side effects of antihistamine medicines is xerostomia (dry mouth). The current consensus is that antihistamine-induced xerostomia comes from an antimuscarinic effect. Although the effect of antihistamines on salivary secretion is both obvious and significant, the cellular mechanism whereby this happens is still unclear because of the lack of knowledge of histamine signaling in human salivary glands. Here, we have studied histamine receptors and the effect of antihistamines on human submandibular acinar cells. In primary cultured human submandibular gland and a HSG cell line, histamine increased the intracellular Ca2+ concentration. The histamine-induced cytosolic free Ca2+ concentration ([Ca2+]i) increase was inhibited by histamine H1 receptor-specific antagonists, and the expression of the functional histamine H1 receptor was confirmed by reverse transcription–polymerase chain reaction. Interestingly, histamine pretreatment did not inhibit a subsequent carbachol-induced [Ca2+]i rise without “heterologous desensitization.” Chlorpheniramine inhibited a carbachol-induced [Ca2+]i increase at a 100-fold greater concentration than histamine receptor antagonism, whereas astemizole and cetrizine showed more than 1000-fold difference, which in part explains the xerostomia-inducing potency among the antihistamines. Notably, histamine resulted in translocation of aquaporin-5 to the plasma membrane in human submandibular gland cells and green fluorescent protein-tagged aquaporin-5 expressing HSG cells. We found that histidine decarboxylase and the histamine H1 receptor are broadly distributed in submandibular gland cells, whereas choline acetyltransferase is localized only at the parasympathetic terminals. Our results suggest that human salivary gland cells express histamine H1 receptors and histamine-synthesizing enzymes, revealing the cellular mechanism of antihistamine-induced xerostomia.