Activation of Transient Receptor Potential Melastatin Family Member 8 (TRPM8) Receptors Induces Proinflammatory Cytokine Expressions in Bronchial Epithelial Cells.

PURPOSE: Cold air is a major environmental factor that exacerbates asthma. Transient receptor potential melastatin family member 8 (TRPM8) is a cold-sensing channel expressed in the airway epithelium. However, its role in airway inflammation remains unknown. We investigated the role of TRPM8 in innate immune responses in bronchial epithelial cells and asthmatic subjects. METHODS: The TRPM8 mRNA and protein expression on BEAS2B human bronchial epithelial cells was examined by real-time polymerase chain reaction (PCR), immunofluorescence staining and western blotting. Additionally, interleukin (IL)-4, IL-6, IL-8, IL-13, IL-25 and thymic stromal lymphopoietin (TSLP) levels before and after menthol, dexamethasone and N-(4-tert-butylphenyl)-4-(3-chloropyridin-2-yl) piperazine-1-carboxamide (BCTC) treatments were measured via real-time PCR. TRPM8 protein levels in the supernatants of induced sputum from asthmatic subjects and normal control subjects were measured using enzyme-linked immunosorbent assay, and mRNA levels in sputum cell lysates were measured using real-time PCR. RESULTS: Treatment with up to 2 mM menthol dose-dependently increased TRPM8 mRNA and protein in BEAS2B cells compared to untreated cells (P < 0.001) and concomitantly increased IL-25 and TSLP mRNA (P < 0.05), but not IL-33 mRNA. BCTC (10 µM) significantly abolished menthol-induced up-regulation of TRPM8 mRNA and protein and IL-25 and TSLP mRNA (P < 0.01). TRPM8 protein levels were higher in the supernatants of induced sputum from asthmatic subjects (n = 107) than in those from healthy controls (n = 19) (P < 0.001), and IL-25, TSLP and IL-33 mRNA levels were concomitantly increased (P < 0.001). Additionally, TRPM8 mRNA levels correlated strongly with those of IL-25 and TSLP (P < 0.001), and TRPM8 protein levels were significantly higher in bronchodilator-responsive asthmatic subjects than in nonresponders. CONCLUSIONS: TRPM8 may be involved in the airway epithelial cell innate immune response and a molecular target for the treatment of asthma.

Toluene diisocyanate exposure induces airway inflammation of bronchial epithelial cells via the activation of transient receptor potential melastatin 8.

Toluene diisocyanate (TDI) is the most important cause of occupational asthma (OA), and various pathogenic mechanisms have been suggested. Of these mechanisms, neurogenic inflammation is an important inducer of airway inflammation. Transient receptor potential melastatin 8 (TRPM8) is a well-established cold-sensing cation channel that is expressed in both neuronal cells and bronchial epithelial cells. A recent genome-wide association study of TDI-exposed workers found a significant association between the phenotype of TDI-induced OA and the single-nucleotide polymorphism rs10803666, which has been mapped to the TRPM8 gene. We hypothesized that TRPM8 located in airway epithelial cells may be involved in the pathogenic mechanisms of TDI-induced OA and investigated its role. Bronchial epithelial cells were treated with TDI in a dose- and time-dependent manner. The expression levels of TRPM8 mRNA and protein were determined by quantitative real-time polymerase chain reaction and western blotting. TDI-induced morphological changes in the cells were evaluated by immunocytochemistry. Alterations in the transcripts of inflammatory cytokines were examined in accordance with TRPM8 activation by TDI. TRPM8 expression at both the mRNA and protein levels was enhanced by TDI in airway epithelial cells. TRPM8 activation by TDI led to significant increases in the mRNA of interleukin (IL)-4, IL-13, IL-25 and IL-33. The increased expression of the cytokine genes by TDI was partly attenuated after treatment with a TRPM8 antagonist. TDI exposure induces increased expression of TRPM8 mRNA in airway epithelial cells coupled with enhanced expression of inflammatory cytokines, suggesting a novel role of TRPM8 in the pathogenesis of TDI-induced OA.

Levodropropizine-Induced Anaphylaxis: Case Series and Literature Review.

Levodropropizine is commonly used as an antitussive drug for acute and chronic cough. It is a non-opioid agent with peripheral antitussive action via the modulation of sensory neuropeptide levels in the airways. Thus, levodropropizine has a more tolerable profile than opioid antitussives. However, we experienced 3 cases of levodropropizine-induced anaphylaxis. Three patients commonly presented with generalized urticaria, dyspnea, and collapse after taking cold medication including levodropropizine. To find out the culprit drug, we performed skin tests, oral provocation tests (OPTs), and basophil activation tests (BATs). Two patients were confirmed as having levodropropizine-induced anaphylaxis by OPTs, and one of them showed positive to skin prick tests (SPTs). The other patient was confirmed by skin tests and BATs. When we analyzed pharmacovigilance data related to levodropropizine collected for 5 years, most cases (78.9%) had allergic reactions, such as rash, urticaria, angioedema, and anaphylaxis. Therefore, physicians should consider that levodropropizine can be a culprit drug, when anaphylaxis occurs after taking anti-cough or common cold medication.

The Basophil Activation Test Is Safe and Useful for Confirming Drug-Induced Anaphylaxis.

The basophil activation test (BAT) has been suggested as a complementary method for diagnosing drug allergies. The aim of this study was to evaluate the clinical utility of this test in patients with drug-induced anaphylaxis. In total, 19 patients, all of whom had a history of moderate to severe anaphylaxis, were enrolled. None of the causative drugs had available in vitro tests or reliable skin tests; these drugs included, among others, first and second-generation cephalosporins, H2 blockers, and muscle relaxants. The BAT yielded positive results in 57.9% of the cases, which was similar those results of skin prick and intradermal tests (42.1% and 57.9%, respectively). When basophils were double labelled with CD63 and CD203c, both of which are basophil activation markers, the positive rate was increased from 57.9% to 73.7%. Therefore, the results of this study confirm that the BAT is a quick, reliable, and safe diagnostic tool for patients with drug-induced anaphylaxis.

Bee Pollen-Induced Anaphylaxis: A Case Report and Literature Review.

Bee pollen is pollen granules packed by honey bees and is widely consumed as natural healthy supplements. Bee pollen-induced anaphylaxis has rarely been reported, and its allergenic components have never been studied. A 40-year-old male came to the emergency room with generalized urticaria, facial edema, dyspnea, nausea, vomiting, abdominal pain, and diarrhea 1 hour after ingesting one tablespoon of bee pollen. Oxygen saturation was 91%. His symptoms resolved after injection of epinephrine, chlorpheniramine, and dexamethasone. He had seasonal allergic rhinitis in autumn. Microscopic examination of the bee pollen revealed Japanese hop, chrysanthemum, ragweed, and dandelion pollens. Skin-prick with bee pollen extracts showed positive reactions at 0.1 mg/mL (A/H ratio > 3+). Serum specific IgE to ragweed was 25.2, chrysanthemum 20.6, and dandelion 11.4 kU/L; however, Japanese hop, honey-bee venom and yellow-jacket venom were negative (UniCAP®, Thermo Fisher Scientific, Uppsala, Sweden). Enzyme-linked immunosorbent assay (ELISA) confirmed serum specific IgE to bee-pollen extracts, and an ELISA inhibition assay for evaluation of cross-allergenicity of bee pollen and other weed pollens showed more than 90% of inhibition with chrysanthemum and dandelion and ~40% inhibition with ragweed at a concentration of 1 µg/mL. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and IgE-immunoblot analysis revealed 9 protein bands (11, 14, 17, 28, 34, 45, 52, 72, and 90 kDa) and strong IgE binding at 28-34 kDa, 45 and 52 kDa. In conclusion, healthcare providers should be aware of the potential risk of severe allergic reactions upon ingestion of bee pollen, especially in patients with pollen allergy.

Schedule-Dependent Effect of Epigallocatechin-3-Gallate (EGCG) with Paclitaxel on H460 Cells.

BACKGROUND: Epigallocatechin-3-gallate (EGCG), a major biologically active component of green tea, has anti-cancer activity in human and animal models. We investigated the schedule-dependent effect of EGCG and paclitaxel on growth of NCI-H460 non-small cell lung cancer cells. METHODS: To investigate the combined effect of EGCG (E) and paclitaxel (P), combination indices (CIs) were calculated, and cell cycle analysis was performed. For the effect on cell apoptosis, western blot analysis was also performed. RESULTS: CI analysis demonstrated that both concurrent and sequential E → P treatments had antagonistic effects (CIs >1.0), but sequential P → E had synergistic effects (CIs <1.0), on the growth inhibition of NCI-H460 cells. In the cell cycle analysis, although paclitaxel induced G2/M cell cycle arrest and increased the sub-G1 fraction, concurrent EGCG and paclitaxel treatments did not have any additive or synergistic effects compared with the paclitaxel treatment alone. However, western blot analysis demonstrated that sequential P → E treatment decreased the expression of Bcl-2 and procaspase-3 and increased poly(ADP-ribose) polymerase (PARP) cleavage; while minimal effects were seen with concurrent or sequential E → P treatments. CONCLUSION: Concurrent or sequential E → P treatment had opposite effects to P → E treatment, where P → E treatment showed a synergistic effect on growth inhibition of NCI-H460 cells by inducing apoptosis. Thus, the efficacy of EGCG and paclitaxel combination treatment seems to be schedule-dependent.

Epithelial to mesenchymal transition of mesothelial cells in tuberculous pleurisy.

PURPOSE: Tuberculous pleurisy is the most frequent extrapulmonary manifestation of tuberculosis. In spite of adequate treatment, pleural fibrosis is a common complication, but the mechanism has not been elucidated. This study is to determine whether epithelial to mesenchymal transition (EMT) of mesothelial cells occurs in tuberculous pleurisy. MATERIALS AND METHODS: Normal pleural mesothelial cells, isolated from irrigation fluids during operations for primary spontaneous pneumothorax, were characterized by immunofluorescence and reverse transcription polymerase chain reaction (RT-PCR). These cells were treated in vitro with various cytokines, which were produced in the effluents of tuberculous pleurisy. The isolated cells from the effluents of tuberculous pleurisy were analyzed by immunofluorescence and RT-PCR analysis. RESULTS: The isolated cells from the irrigation fluid of primary spontaneous pneumothorax had epithelial characteristics. These cells, with transforming growth factor-ß1 and/or interleukin-1ß treatment, underwent phenotypic transition from epithelial to mesenchymal cells, with the loss of epithelial morphology and reduction in cytokeratin and E-cadherin expression. Effluent analysis from tuberculous pleurisy using immunofluorescence and RT-PCR demonstrated two phenotypes that showed mesenchymal characteristics and both epithelial & mesencymal characteristics. CONCLUSION: Our results suggest that pleural mesothelial cells in tuberculous pleurisy have been implicated in pleural fibrosis through EMT.

Transforming growth factor beta1 induces epithelial-to-mesenchymal transition of A549 cells.

Idiopathic pulmonary fibrosis (IPF) comprises an aggregate of mesenchymal cells. However, the cellular origin of these mesenchymal phenotypes remains unclear. Transforming growth factor beta1 (TGF-beta1) has been known as the main cytokine involved in the pathogenesis of IPF. We examined whether the potent fibrogenic cytokine TGF-beta1 could induce the epithelial-to-mesenchymal transition (EMT) in the human alveolar epithelial cell line, A549, and determined whether snail expression is associated with the phenotypic changes observed in the A549 cells. EMT was investigated with cells morphology changes under phase-contrast microscopy, western blotting, and indirect immunofluorescence stains. E-cadherin and transcription factor, snail, were also evaluated by measuring mRNA levels using reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. The data showed that TGF-beta1 induced A549 cells with epithelial cell characteristics to undergo EMT in a concentration-dependent manner. Following TGF-beta1 treatment, A549 cells induced EMT characterized by cells morphological changes, loss of epithelial markers Ecaherin and cytokeratin, increased stress fiber reorganization by F-actin, and cytokeratin replacement by vimentin. Although IL-1beta failed to induce A549 cells to undergo EMT, the combination of TGF-beta1 and IL-1beta showed synergy effects in cells morphology changes and the expression of mesenchymal markers. The snail expression study using RT-PCR analysis provided that loss of E-cadherin expression was associated with snail expression. Stimulation of A54 cells with TGF-beta1 plus IL-1beta revealed a higher level of snail expression. Our data showed that EMT of A549 cells might be closely associated with snail expression.

Nordihydroguaiaretic acid, an antioxidant, inhibits transforming growth factor-beta activity through the inhibition of Smad signaling pathway.

Transforming growth factor-beta (TGF-beta) and its family are potent and multi-functional cytokines that affect various fundamental biological events. TGF-beta has a unique signaling pathway that is carried by Smad family, and many recent studies showed the extensive crosstalk between Smad pathway and other signaling pathway. There were also clear evidences for the involvement of oxidative events in TGF-beta signaling pathway. To elucidate the role of oxidative events in carrying TGF-beta signals, we examined the effect of various antioxidants on TGF-beta activities in osteoblastic cell line. Among the examined compounds, we found nordihydroguaiaretic acid (NDGA) has a unique and strong inhibitory effect on various TGF-beta activities. Since the majority of TGF-beta activities are mediated by Smad, we questioned whether NDGA blocks the Smad signaling pathway. The result showed that NDGA inhibits the translocation of Smad2 to the nucleus. Further study revealed the strong inhibitory effect of NDGA on the phosphorylation of Smad2. This result may be important for designing chemical modulators of TGF-beta and its family related events and may provide new insights into the action mechanism of antioxidant.