Human Bronchial Smooth Muscle Cells Research Articles

Pulmonary histone deacetylase is involved in phenotype transformation of airway smooth muscle cells in chronic asthmatic mice

Objective To investigate the changes of histone deacetylase ( HDAC) activity during chronic asthma and its effects on the phenotype and biological activities of airway smooth muscle cells. Methods Chronic asthma model was established with mice using repeated sensitization and challenge with OVA,and the lung was homogenated for total HDAC activity assay. Rat airway smooth muscle cells and human bronchial smooth muscle cells were stimulated with trichostatin A ( TSA) ,an HDAC inhibitor. The effect of HDAC inhibition on phenotype switching was detected by Western Blotting analysis,and its effects on proliferation,migration and contraction of smooth muscle cells were examined by MTT,Transwell and gel contraction,respectively. Results The total HDAC activity was significantly decreased in chronic asthmatic mice compared with saline -treated controls( [0. 371 ± 0. 054]vs[0. 603 ± 0. 034]μmol/( L·μg) ,P 0. 01). Incubation of the rat trachea ring and human bronchial smooth muscle cells with TSA ( 0. 5 μmol/ L) for 12 -24 h increased the expression of α -sm -actin and SM22 -α. TSA significantly decreased cell number after 24 h treatment ( [1. 719 ±0. 044) ×104 vs( [1. 911 ±0. 048]×104,P 0. 05) and 48 h[1. 808 ±0. 009]×104 vs[2. 537 ±0. 01]×104,P 0. 05) compared with DMSO controls. TSA treatment for 24 h also significantly decreased the migrated cells compared with the PDGF -treated cells( 52 ± 7. 5 vs 88 ± 7. 632,P 0. 05). Furthermore,the gel contraction rate was significantly lower in the TSA -treated cells than in the DMSO or PDGF-treated cells( [9. 885 ± 7. 084]% vs [44. 844 ± 3. 808]% and [41. 315 ± 7. 943]% ,P 0. 05). Conclusion HDAC activity is decreased in the lung of chronic asthmatic mice,which may be related to switching of the airway smooth muscle cells to a "contractic" phenotype,but with no increase of their contraction capability.

Cysteinyl‐leukotrienes induce vascular endothelial growth factor production in human monocytes and bronchial smooth muscle cells

Cysteinyl leukotrienes (cysLTs) are suggested to be implicated in the process of airway remodelling in asthma. We investigated the potential for cysLTs to modulate vascular endothelial growth factor (VEGF) expression, a growth factor involved in the angiogenesis of airway remodelling. VEGF mRNA and protein were quantified by real-time PCR and ELISA, respectively. VEGF promoter activation was assessed using luciferase gene-tagged promoter constructs. We found that LTD(4) induction of VEGF in human monocytes and bronchial smooth muscle cells is cysLT1 dependent. Stimulation of HEK293 cells stably expressing cysLT1 or cysLT2 with cysLTs showed a concentration-dependent activation of the VEGF promoter and a time-dependent increase in VEGF mRNA and protein. For the cysLT1-mediated response, mutations of hypoxia-induced factor-1 (HIF-1) sites failed to reduce cysLT-induced VEGF promoter activation and 5' deletions showed that the proximal region containing one AP-1 and four specificity protein 1 (Sp1) sites was necessary. Pretreatment with inhibitors of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK), but not p38, and an overexpression of dominant negative forms of c-Jun, c-Fos or Ras suggested the implication of mitogen-activated protein kinases and AP-1. Mutation of the AP-1-binding element failed to prevent VEGF transactivation suggesting that AP-1 might not act directly on the promoter. Moreover, inhibition of Sp1-dependent transcription by mithramycin completely inhibited VEGF promoter transactivation and VEGF mRNA expression by LTD(4) . Finally, mutations of Sp1 binding elements prevented VEGF promoter transactivation. Our data indicate for the first time that cysLTs can transcriptionally activate VEGF production via cysLT1 receptors, with the involvement of JNK, ERK, the AP-1 complex and Sp1. These findings suggest that cysLTs may be important in the angiogenic process of airway remodelling and potentially provide a previously unknown benefit of using cysLT1 receptor antagonists in the prevention or treatment of airway remodelling in asthma.

House dust mite extract downregulates C/EBP in asthmatic bronchial smooth muscle cells

Reduced translation of CEBPA mRNA has been associated with increased proliferation of bronchial smooth muscle (BSM) cells of asthma patients. Here, we assessed the effect of house dust mite (HDM) extracts on the cell proliferation ([(3)H]-thymidine incorporation), inflammation (interleukin (IL)-6 release) and upstream translation regulatory proteins of CCAAT/enhancer-binding protein (C/EBP)α in human BSM cells of healthy controls and asthmatic patients. HDM extract significantly increased IL-6 protein and proliferation of BSM cells of asthma patients only. HDM extract reduced the C/EBPα expression in BSM cells of asthma patients, which coincided with significantly increased levels of calreticulin (CRT) protein, an inhibitor of CEBPA mRNA translation. HDM extract elicited both protease-dependent and -independent responses, which were mediated via protease-activated receptor (PAR)2 and CRT, respectively. In conclusion, HDM extract reduced CEBPA mRNA translation, specifically in asthmatic BSM cells, and 1) upregulated CRT, 2) activated PAR2, and increased 3) IL-6 expression and 4) the proliferation of asthmatic BSM cells. Hence, HDM exposure contributes to inflammation and remodelling by a nonimmune cell-mediated mechanism via a direct interaction with BSM cells. These findings may potentially explain several pathological features of this disease, in particular BSM cell hyperplasia.

Signalling pathway of isophorone diisocyanate-responsive interleukin-8 in airway smooth muscle cells

This study is the first to analyse the soluble factors secreted by the bronchial epithelium after exposure to isophorone diisocyanate (IPDI) that are responsible for increasing migration and proliferation of primary normal human bronchial smooth muscle cells (BSMCs). We treated immortalised, nontumorigenic human bronchial epithelial cells (cell line BEAS-2B) and primary normal human bronchial epithelial cells (HBEC) with IPDI, and then collected the conditioned culture media (IPDI-BEAS-2B-CM and IPDI-HBEC-CM, respectively), which was added to BSMCs. Exposure of BEAS-2B cells and HBECs to IPDI increased interleukin (IL)-8 production. Culture of BSMCs with IPDI-BEAS-2B-CM and IPDI-HBEC-CM increased BSMC proliferation and migration, which are major features in asthma-related airway remodelling. Induction of BSMC proliferation and migration by IPDI-BEAS-2B-CM and IPDI-HBEC-CM was associated with increased focal adhesion kinase (FAK), Src, extracellular signal-regulated kinase (ERK)1/2 and AKT activation. Blocking FAK with a specific inhibitor significantly decreased BSMC migration and proliferation by inhibiting ERK1/2 activation. FAK and ERK1/2 inhibitor also decreased IPDI-BEAS-2B-CM-, IPDI-HBEC-CM- and recombinant human IL-8-mediated BSMC proliferation and migration, whereas blocking Rnd3 using small interfering RNA failed to affect BSMC proliferation, suggesting that Rnd3 was only involved in the regulation of BSMC migration. Our study suggests that inhibition of IL-8 or IL-8-mediated FAK/ERK/Rnd3 signalling is an attractive therapeutic target for IPDI-mediated asthma.

Efficacy is a contributing factor to the clinical onset of bronchodilation of inhaled β2-adrenoceptor agonists

Inhaled beta(2) adrenoceptor (beta(2) AR) agonists are widely used as bronchodilator therapies for asthma and COPD. Different agonists have varying rates of onset of action, e.g. indacaterol and salbutamol are effective bronchodilators within 5 min whereas salmeterol takes 15 min to achieve significant bronchodilation over baseline (Brookman et al., Curr Med Res Opin 23:3113-3122, 2007). This has been attributed to differences in the lipophilicity of the agonists such that hydrophobic ligands take longer to diffuse into tissue and may even access the receptor via the membrane compartment (Anderson et al., Eur Respir J 7:569-578, 1994). While this holds true for salmeterol and salbutamol, the relatively high lipophilicity of indacaterol should result in a slower onset of action. Here we have explored the possibility that the efficacy of these ligands may also contribute to their onset of action. We have characterised efficacy and rate of cyclic adenosine monophosphate (cAMP) accumulation in primary human bronchial smooth muscle cells using a competition assay (AlphaScreen, Perkin Elmer) and in HEK 293-GloSensor cells endogenously expressing the beta(2) AR using a luminescence readout. For all agonists tested, cAMP was generated in a concentration-dependent manner. For both assay formats, the relative efficacies were unchanged, with isoprenaline > formoterol > indacaterol > salbutamol > salmeterol. The rate of cAMP generation varied for each agonist and correlated well with intrinsic efficacy in that the high-efficacy agonists promoted the most rapid rise in cAMP levels. We have demonstrated that the rate of cAMP accumulation is influenced by agonist efficacy and that this, in combination with lipophilicity, may explain why beta(2) AR agonists demonstrate differences in their onset of action.

Pharmacology and immunological actions of a herbal medicine ASHMITM on allergic asthma

AbstractAllergic asthma is a chronic and progressive inflammatory disease for which there is no satisfactory treatment. Studies reported tolerability and efficacy of an anti‐asthma herbal medicine intervention (ASHMI) for asthma patients, developed from traditional Chinese medicine. To investigate the pharmacological actions of ASHMI on early‐ and late‐phase airway responses (EAR and LAR), Ovalbumin (OVA)‐sensitized mice received 6 weeks of ASHMI treatment beginning 24 h following the first intratracheal OVA challenge. EAR were determined 30 min following the fourth challenge and LAR 48 h following the last challenge. ASHMI effects on cytokine secretion, murine tracheal ring contraction and human bronchial smooth muscle cell prostaglandin (PG) production were also determined.ASHMI abolished EAR, which was associated with significantly reduced histamine, leukotriene C4, and OVA‐specific IgE levels, as well as LAR, which was associated with significantly reduced bronchoalveolar lavage fluid (BALF) eosinophils, decreased airway remodeling, and lower Th2 cytokine levels in BALF and splenocyte cultures. Furthermore, ASHMI inhibited contraction of murine tracheal rings and increased production of the potent smooth muscle relaxer PGI2. ASHMI abrogation of allergic airway responses is associated with broad effects on asthma pathological mechanisms. Copyright © 2009 John Wiley & Sons, Ltd.

Azithromycin reduces the viability of human bronchial smooth muscle cells

The macrolide antibiotic azithromycin has an antiproliferative and autophagic effect on rabbit tracheal smooth muscle cells (SMCs). The purpose of this study is to investigate the effect of azithromycin on human bronchial SMCs. Human bronchial SMCs were treated with azithromycin (10(-5) M) in the presence or absence of 10% fetal bovine serum (FBS). Cell number was estimated using the Cell Titer 96 AQ(ueous) One Solution Assay. Induction of autophagy was studied by observation of cell morphology in cells treated or not with the autophagy inhibitor, 3-methyladenine (3-MA), as well as by Lysotracker Red staining of lysosomes. Activation of apoptosis was assessed with flow cytometry after annexin staining. Incubation with azithromycin for 24, 48 or 72 h reduced viability in FBS-deprived cells, as well as cells cultured in FBS-containing medium. Azithromycin treatment resulted in the formation of cytoplasmic vacuoles that could not be prevented by 3-MA. Furthermore, 3-MA did not reverse the effect of azithromycin on the viability of SMCs. There was an increase in the number of lysosomes in cells treated with azithromycin. Finally, azithromycin increased the percentage of early apoptotic cells. In conclusion, azithromycin reduces the viability of human bronchial SMCs possibly by leading to apoptotic cell death.

Synthesis and in-vitro antitumour activity of new naphthyridine derivatives on human pancreatic cancer cells

Abstract Objectives The aim of the study was to evaluate the antitumour effect in vitro of newly synthesized 7-substituted 2,3-dihydro-1,8-naphthyridines. Methods Characterization tools included cell viability assay, caspase 3/7 induction, DNA fragmentation, fibroblast growth factor type 1 receptor kinase inhibition, and in-vitro antiangiogenic analysis. Key findings Treatment of MIA PaCa-2 human pancreatic cancer cells with test compounds showed time- and concentration-dependent cytotoxicity with IC50 values in the micromolar range. Compounds with an aminoalkyl or a diaminoalkyl side chain at the 7-position exhibited remarkable cytotoxicity, whereas the presence of a methyl group or a cyclic amine in the same position led to a significant decrease in their biological activity. Cytotoxicity screening demonstrated that the most active was compound 11 (mean 50% inhibition of cell proliferation (IC50) 11 μM). This compound had an in-vitro antitumour efficacy superior to 5-fluorouracil (the lowest cell viability value after treatment (Emax) 0.2% and 19%, respectively) and proved to be less toxic than 5-fluorouracil against non-cancerous human oral epithelial cells. In addition, compound 11 induced apoptosis in MIA PaCa-2 cells and it was able to promote antiangiogenic effects in vitro. Finally, its cytotoxicity was enhanced in pancreatic cancer cells stimulated with fibroblast growth factor, while no substantial effect was observed on human bronchial smooth muscle cells stimulated with the same growth factor. Conclusions These findings suggest that 1,8-naphthyridine derivatives are a promising class of compounds in cancer research. In particular, the antitumour activity of compound 11 is worth further investigation.

Mechanism of Inhibitory Effect of Prednisolone on RhoA Upregulation in Human Bronchial Smooth Muscle Cells

RhoA plays an important role in Ca(2+) sensitization of bronchial smooth muscle in antigen-induced airway hyperresponsiveness (AHR). Glucocorticoids are now the most effective anti-inflammatory treatment for asthma, and inhaled corticosteroids are the most effective long-term control therapy for persistent asthma. To determine the mechanism of the inhibitory action of glucocorticoids on AHR in allergic bronchial asthma, that of prednisolone on RhoA upregulation was investigated using cultured human bronchial smooth muscle cells (hBSMCs). The upregulation of RhoA induced by interleukin (IL)-13 and tumor necrosis factor (TNF)-alpha, major mediators for development of AHR, was observed in hBSMCs. Prednisolone partly inhibited the IL-13-induced RhoA upregulation and RhoA promoter activity, although prednisolone had no effects on the activations of signal transducers and activators of transcription (STAT)6 and nuclear factor (NF)-kappaB. Increased expression and promoter activity of RhoA induced by TNF-alpha was completely inhibited by prednisolone, although the activation of NF-kappaB failed to be inhibited by prednisolone in hBSMCs. These findings suggest that prednisolone might inhibit NF-kappaB-induced transcription via interaction between glucocorticoid receptor (GR), resulting in an inhibition of RhoA upregulation induced by IL-13 and TNF-alpha.

Induction of RhoA gene expression by interleukin-4 in cultured human bronchial smooth muscle cells

RhoA, a small GTPase, is one of the key proteins of smooth muscle contraction. In allergic asthma, an upregulation of RhoA in bronchial smooth muscle has been suggested. However, the mechanism of its upregulation has not yet been clarified. In the present study, the effects of interleukin-4 (IL-4), one of the T-helper 2 cytokines, on RhoA mRNA expression and promoter activity of RhoA gene were examined in cultured human bronchial smooth muscle cells (hBSMCs). The quantitative real-time RT-PCR analyses revealed that incubation of hBSMCs with IL-4 (10, 30 and 100 ng/mL, for 24 hr) caused an increase in RhoA mRNA in a concentration-dependent manner. In luciferase reporter gene assay using hBSMCs that were transfected with luciferase constructs and were then stimulated with IL-4 (100 ng/mL), an importance of the most proximal STAT6 binding region (78-70 bp upstream of the transcription initiation site) was suggested. It is thus possible that IL-4 is capable of upregulating RhoA by promoting its transcription in hBSMCs. The proximal STAT6 binding region is required for the IL-4-induced increase in promoter activity of the human RhoA gene.

MicroRNAs and Their Therapeutic Potential for Human Diseases: MiR-133a and Bronchial Smooth Muscle Hyperresponsiveness in Asthma

MicroRNAs (miRNAs) play important roles in normal and diseased cell functions. The small-GTPase RhoA is one of the key proteins of bronchial smooth muscle (BSM) contraction, and an upregulation of RhoA has been demonstrated in BSMs of experimental asthma. Although the mechanism of RhoA upregulation in the diseased BSMs is not fully understood, recent observations suggest that RhoA translation is controlled by a miRNA, miR-133a, in cardiomyocytes. Similarly, in human BSM cells (hBSMCs), our recent studies revealed that an upregulation of RhoA was induced when the function of endogenous miR-133a was inhibited by its antagomir. Treatment of hBSMCs with interleukin-13 (IL-13) caused an upregulation of RhoA and a downregulation of miR-133a. In a mouse model of allergic bronchial asthma, increased expression of IL-13 and RhoA and the BSM hyperresponsiveness were observed. The level of miR-133a was significantly decreased in BSMs of the diseased animals. These findings suggest that RhoA expression is negatively regulated by miR-133a in BSMs and that the miR-133a downregulation causes an upregulation of RhoA, resulting in an augmentation of the contraction. MiR-133a might be a key regulator of BSM hyperresponsiveness and provide us with new insight into the treatment of airway hyperresponsiveness in asthmatics.

The proximal STAT6 and NF-κB sites are responsible for IL-13- and TNF-α-induced RhoA transcriptions in human bronchial smooth muscle cells

RhoA protein is involved in the Ca(2+) sensitization of bronchial smooth muscle (BSM) contraction, and an upregulation of RhoA in BSMs has been suggested in allergic bronchial asthma. However, the mechanism of upregulation of RhoA remains poorly understood. In the present study, the transcriptional regulation of human RhoA gene was investigated in cultured human BSM cells stimulated with IL-13 and TNF-alpha, both of which have an ability to upregulate RhoA protein. Luciferase-based assay showed that the RhoA promoter activity was augmented by both IL-13 and TNF-alpha. The deletion studies revealed a significant level of promoter activity between the 112 bp upstream and the transcription start site, which contains the STAT6 (78-70 bp upstream) and NF-kappaB (84-74 bp upstream) binding regions. The promoter activity was also decreased significantly by the mutations of these regions. Thus, the current study for the first time characterized the transcriptional regulation of the human RhoA gene. The findings also suggest that STAT6 and NF-kappaB are important for the upregulation of RhoA in human BSM induced by IL-13 and TNF-alpha, both of which are major cytokines in the pathogenesis of allergic bronchial asthma.

Interleukin-4 upregulates RhoA protein via an activation of STAT6 in cultured human bronchial smooth muscle cells

Interleukin-4 (IL-4) is believed to play a role in allergic bronchial asthma, and has been suggested to cause hyperresponsiveness of airway smooth muscle. In the present study, the effects of IL-4 on the expression of RhoA protein, a monomeric GTP-binding protein that contributes to the contraction of smooth muscle, were determined in cultured human bronchial smooth muscle cells (hBSMCs). Incubation of hBSMCs with IL-4 (100 ng/mL) caused a distinct phosphorylation of signal transducer and activator of transcription 6 (STAT6), a major signal transducer activated by IL-4, indicating that IL-4 is capable of activating signal transduction in the hBSMCs directly. IL-4 also caused a significant increase in the expression level of RhoA protein: the peak of the upregulation of RhoA protein was observed at 12–24 h after the IL-4 treatment. Both the phosphorylation of STAT6 and the upregulation of RhoA protein induced by IL-4 were inhibited by the co-incubation with AS1517499, a selective inhibitor of STAT6, in a concentration-dependent fashion. These findings suggest that IL-4 is capable of inducing an upregulation of RhoA via an activation of STAT6 in cultured hBSMCs. The RhoA upregulation induced by IL-4, one of the Th2 cytokines upregulated in the airways of allergic bronchial asthmatics, might result in an augmentation of bronchial smooth muscle contractility, that is one of the causes of airway hyperresponsiveness.

Characterization of Primary Cilia in Human Airway Smooth Muscle Cells

Considerable evidence indicates a key role for primary cilia of mammalian cells in mechanochemical sensing. Dysfunctions of primary cilia have been linked to the pathogenesis of several human diseases. However, cilia-related research has been limited to a few cell and tissue types; to our knowledge, no literature exists on primary cilia in airway smooth muscle (ASM). The aim of this study was to characterize primary cilia in human ASM. Primary cilia of human bronchial smooth muscle cells (HBSMCs) were examined using immunofluorescence confocal microscopy, and scanning and transmission electron microscopy. HBSMC migration and injury repair were examined by scratch-wound and epidermal growth factor (EGF)-induced migration assays. Cross-sectional images of normal human bronchi revealed that primary cilia of HBSMCs within each ASM bundle aggregated at the same horizontal level, forming a "cilium layer." Individual cilia of HBSMCs projected into extracellular matrix and exhibited varying degrees of deflection. Mechanochemical sensing molecules, polycystins, and alpha2-, alpha5-, and beta1-integrins were enriched in cilia, as was EGF receptor, known to activate jointly with integrins during cell migration. Migration assays demonstrated a ciliary contribution to HBSMC migration and wound repair. The primary cilia of ASM cells exert a role in sensing and transducing extracellular mechanochemical signals and in ASM injury repair. Defects in ASM ciliary function could potentially affect airway wall maintenance and/or remodeling, possibly relating to the genesis of bronchiectasis in autosomal dominant polycystic kidney disease, a disease of ciliopathy.

Synthesis and in-vitro antitumour activity of new naphthyridine derivatives on human pancreatic cancer cells

The aim of the study was to evaluate the antitumour effect in vitro of newly synthesized 7-substituted 2,3-dihydro-1,8-naphthyridines. Characterization tools included cell viability assay, caspase 3/7 induction, DNA fragmentation, fibroblast growth factor type 1 receptor kinase inhibition, and in-vitro antiangiogenic analysis. Treatment of MIA PaCa-2 human pancreatic cancer cells with test compounds showed time- and concentration-dependent cytotoxicity with IC50 values in the micromolar range. Compounds with an aminoalkyl or a diaminoalkyl side chain at the 7-position exhibited remarkable cytotoxicity, whereas the presence of a methyl group or a cyclic amine in the same position led to a significant decrease in their biological activity. Cytotoxicity screening demonstrated that the most active was compound 11 (mean 50% inhibition of cell proliferation (IC50) 11 mum). This compound had an in-vitro antitumour efficacy superior to 5-fluorouracil (the lowest cell viability value after treatment (E(max)) 0.2% and 19%, respectively) and proved to be less toxic than 5-fluorouracil against non-cancerous human oral epithelial cells. In addition, compound 11 induced apoptosis in MIA PaCa-2 cells and it was able to promote antiangiogenic effects in vitro. Finally, its cytotoxicity was enhanced in pancreatic cancer cells stimulated with fibroblast growth factor, while no substantial effect was observed on human bronchial smooth muscle cells stimulated with the same growth factor. These findings suggest that 1,8-naphthyridine derivatives are a promising class of compounds in cancer research. In particular, the antitumour activity of compound 11 is worth further investigation.

Down-Regulation of miR-133a Contributes to Up-Regulation of RhoA in Bronchial Smooth Muscle Cells

Augmented bronchial smooth muscle (BSM) contraction is one of the causes of bronchial hyperresponsiveness. The protein RhoA and its downstream pathways have now been proposed as a new target for asthma therapy. MicroRNAs (miRNAs) play important roles in normal and diseased cell functions, and a contribution of miR-133 to RhoA expression has been suggested in cardiomyocytes. To make clear the mechanism(s) of up-regulation of RhoA observed in the BSMs of experimental asthma, the role of miR-133a in RhoA expression was tested. Total proteins and RNAs (containing miRNAs) were extracted from cultured human BSM cells (hBSMCs) that were treated with antagomirs and/or IL-13, and bronchial tissues of BALB/c mice that were sensitized and repeatedly challenged with ovalbumin. RhoA protein and miR-133a were detected by immunoblotting and quantified real-time reverse transcriptase-polymerase chain reaction, respectively. In hBSMCs, an up-regulation of RhoA was observed when the function of endogenous miR-133a was inhibited by its antagomir. Treatment of hBSMCs with IL-13 caused an up-regulation of RhoA and a down-regulation of miR-133a. In bronchial tissues of the repeatedly ovalbumin-challenged mice, a significant increase in RhoA was observed. Interestingly, the level of miR-133a was significantly decreased in BSMs of the challenged mice. These findings suggest that RhoA expression is negatively regulated by miR-133a in BSMs. IL-13 might, at least in part, contribute to the reduction of miR-133a.

Proinflammatory and Th2 Cytokines Regulate the High Affinity IgE Receptor (FcεRI) and IgE-Dependant Activation of Human Airway Smooth Muscle Cells

BackgroundThe high affinity IgE receptor (FcεRI) is a crucial structure for IgE-mediated allergic reactions. We have previously demonstrated that human airway smooth muscle (ASM) cells express the tetrameric (αβγ2) FcεRI, and its activation leads to marked transient increases in intracellular Ca2+ concentration, release of Th-2 cytokines and eotaxin-1/CCL11. Therefore, it was of utmost importance to delineate the factors regulating the expression of FcεRI in human (ASM) cells.Methodology/Principal FindingsIncubation of human bronchial and tracheal smooth muscle (B/TSM) cells with TNF-α, IL-1β or IL-4 resulted in a significant increase in FcεRI-α chain mRNA expression (p<0.05); and TNF-α, IL-4 enhanced the FcεRI-α protein expression compared to the unstimulated control at 24, 72 hrs after stimulation. Interestingly, among all other cytokines, only TNF-α upregulated the FcεRI-γ mRNA expression. FcεRI-γ protein expression remained unchanged despite the nature of stimulation. Of note, as a functional consequence of FcεRI upregulation, TNF-α pre-sensitization of B/TSM potentially augmented the CC (eotaxin-1/CCL11 and RANTES/CCL5, but not TARC/CCL17) and CXC (IL-8/CXCL8, IP-10/CXCL10) chemokines release following IgE stimulation (p<0.05, n = 3). Furthermore, IgE sensitization of B/TSM cells significantly enhanced the transcription of selective CC and CXC chemokines at promoter level compared to control, which was abolished by Lentivirus-mediated silencing of Syk expression.Conclusions/SignificanceOur data depict a critical role of B/TSM in allergic airway inflammation via potentially novel mechanisms involving proinflammatory, Th2 cytokines and IgE/FcεRI complex.

Evaluation of Cysteinyl Leukotriene-Induced Contraction of Human Cultured Bronchial Smooth Muscle Cells

Background: Cysteinyl leukotrienes (cysLTs) are major mediators involved in bronchial asthma, particularly bronchial constriction. However, a contractile response of human bronchial smooth muscle cells (hBSMCs) to cysLTs has not been well characterized at cellular level. Methods: A contraction assay using collagen gel embedded with cultured hBSMCs was established to analyze contractile responses at cellular level. Contractile responses to several constrictors including cysLTs were evaluated in 6-day cultured gels with varying fetal bovine serum (FBS) concentrations. Results: Removal of FBS from the culture for the designated time periods resulted in increased contractile responses. CysLT-induced contraction of the gel became more pronounced and reproducible. CysLT₁R expression on the hBSMCs was significantly increased by the removal of FBS. Conclusion: Contractile responses of hBSMCs to cysLTs can be evaluated at cellular level for the first time. This experimental system may be useful for the in vitro evaluation and future development of cysLTs antagonists.

T cells and eosinophils in bronchial smooth muscle cell death in asthma

Bronchial smooth muscle cells (SMC) proliferate, express adhesion molecules, secrete cytokines and thus efficiently contribute to the pathogenesis of asthma. The aim of the study was to investigate whether, and by which mechanism, T cells and eosinophils can cause death of airway SMC. The T cell- and eosinophil-induced cell death was analysed in primary human bronchial SMC cultures as well as in bronchial biopsy specimens from non-asthmatic and asthmatic individuals. Bronchial SMC death showed characteristic morphological features of apoptosis in 3-6 days cultures with inflammatory cytokines (IFN-gamma, TNF-alpha), soluble death ligands [sFasL, TNF-related apoptosis-inducing ligand (TRAIL)] and activated T-helper type 1 (Th1) and Th2 cell supernatants. The recombinant eosinophil cationic protein induced SMC necrosis within 1 h. Resting SMC expressed the death receptors TNFR1, TNFR2, Fas, TRAILR1, TRAILR2 and membrane FasL as a death-inducing ligand. IFN-gamma and TNF-alpha up-regulated TNFR1, TNFR2, Fas and membrane FasL on SMC. TNF-alpha up-regulated TRAILR1 and TRAILR2; sFasL up-regulated TNFR2. The intracellular caspase-3 activation in SMC was significantly increased by IFN-gamma, sFasL, TRAIL, Th1 and Th2 cell supernatants. Increased expression of TRAIL in asthmatics, but not in non-asthmatic individuals was demonstrated in situ. The apoptosis receptors TRAILR1 and TRAILR2 were expressed in SMC and epithelial cells both in healthy and asthmatic biopsies. Prominent apoptosis of SMC was observed in fatal asthma, but not intermittent asthma biopsies. The demonstration of bronchial SMC death both by apoptosis and necrosis indicates the essential role of T cells and eosinophils in the bronchial tissue injury particularly in the severe asthma.

IL-13 induces translocation of NF-κB in cultured human bronchial smooth muscle cells

Background and purpose: Interleukin-13 (IL-13), a major Th2 cytokine, plays an important role in bronchial asthma, including mucus production, inflammation and airway hyperresponsiveness. Although IL-13 through its binding to IL-4 receptor α (IL-4Rα/IL-13Rα1 uses the canonical signal transducer and activator of transcription 6 (STAT6)-signaling pathway to mediate these tissue responses, recent studies have demonstrated that other signaling pathways may also be involved in. In the present study, whether IL-13 induces an activation of nuclear factor (NF)-κB, inflammatory transcription factor, was investigated in human bronchial smooth muscle cells (hBSMCs). Methods: Nuclear proteins were extracted from cultured hBSMCs treated with tumor necrosis factor (TNF)-α (10 ng/mL) or IL-13 (100 ng/mL), and assayed for activated NF-κB and STAT6 by Western blotting. Result: Treatments with TNF-α and IL-13 induced a translocation of NF-κB to nuclei in hBSMCs. In addition, coincubation with BMS-345541 (0.3 μM), an inhibitor of NF-κB (IκB) kinase (IKK) inhibitor, markedly inhibited the translocation of NF-κB. Conclusion: Our results suggest for the first time that IL-13 activates NF-κB in hBSMCs.