Yaping Zhang

An assay to evaluate the long-term effects of inflammatory mediators on murine airway smooth muscle: evidence that TNFalpha up-regulates 5-HT(2A)-mediated contraction.

Asthma research is arguably limited by an absence of appropriate animal models to study the pharmacology of inflammatory mediators that affect airway hyperresponsiveness and remodelling. Here we assessed an assay based on mouse tracheal segments cultured for 1–32 days, and investigated contractile responses mediated by muscarinic and 5‐hydroxytryptamine (5‐HT) receptors following long‐term exposure to tumour necrosis factor‐alpha (TNFα). Following culture, in the absence of TNFα, maximum contractile responses to KCl and carbachol were similar, with an increase in response up to day two and a decrease to a stable level after 8 days. Maximal relaxations to isoprenaline were not affected by the culture procedure. The potency of KCl and isoprenaline increased throughout the study. DNA microarray data revealed that global gene expression changes were greater when tissues were introduced to culture than when they were maintained in culture. The morphology of smooth muscle cells was maintained throughout the culture period. 5‐HT induced a weak contraction in both fresh and cultured (up to 8 days) segments. Culture with TNFα produced a time‐ and concentration‐dependent increase in the maximal contraction to 5‐HT, evidently mediated by 5‐HT2A receptors, whereas, the potency for carbachol was reduced. In conclusion, the phenotype of airway smooth muscle remained largely intact during the culture period, even though minor changes were obtained during the first days of culture. The time‐dependent effect of TNFα indicates the importance of studying the long‐term effect of cytokines on the smooth muscle cells in relation to airway hyperresponsiveness and remodelling.

IL-1β induces murine airway 5-HT2A receptor hyperresponsiveness via a non-transcriptional MAPK-dependent mechanism

BackgroundInterleukin 1 beta (IL-1β) is found in bronchoalveolar lavage fluids from asthmatic patients and plays an important role in normal immunoregulatory processes but also in pathophysiological inflammatory responses. The present study was designed to investigate if IL-1β could be involved in the development of airway hyperresponsiveness and if transcriptional mechanisms, epithelium contractile factors and mitogen-activated protein kinase (MAPK) pathways are involved in IL-1β effect.MethodsThe effect of IL-1β on 5-hydroxytryptamine (5-HT) induced bronchoconstriction was evaluated in an in-vitro model for assessment of long-term effects of inflammatory mediators on the airway smooth muscle. Murine tracheal segments were cultured up to 8 days in the absence or presence of IL-1β with subsequent evaluation in a myograph system, along with mRNA quantification, focusing on the role of the epithelium, acetylcholine release, transcriptional mechanisms and MAPK activity.ResultsDuring control conditions, 5-HT induced a relatively weak contraction. Presence of IL-1β increased this response in a time- and concentration-dependent way. The increased concentration-effect curves could be shifted rightwards in a parallel manner by ketanserin, a selective 5-HT2A receptor antagonist, indicating that the responses are mediated by 5-HT2A receptors. The mRNA levels of 5-HT2A receptors were not changed as a consequence of the IL-1β treatment and actinomycin D, a general transcriptional inhibitor, failed to affect the contractile response, suggesting a non-transcriptional mechanism behind this phenomenon. Neither the removal of the epithelium nor the addition of atropine affected the IL-1β induced enhancement of 5-HT2A receptor-mediated contractile response. Application of inhibitors for c-Jun N-terminal kinase (JNK), p38 and extracellular signal-regulated kinase 1 and 2 (ERK1/2) showed that the signaling pathways for JNK and ERK1/2 dominated only in cultured segments (control) whereas JNK and p38 dominated in segments treated with IL-1β.ConclusionIL-1β induces murine airway hyperresponsiveness, via a non-transcriptional up-regulation of 5-HT2A receptor-mediated contractile response. The increase of 5-HT contraction is unrelated to epithelial and cholinergic factors, but is dependent on IL-1β-induced changes of MAPK pathways. The fact that IL-1β can alter airway responses to contractile agents such as 5-HT, via alteration of the intracellular MAPK signal transduction pathways, might provide a new concept for future treatment of asthma.

Interleukin-1beta attenuates endothelin B receptor-mediated airway contractions in a murine in vitro model of asthma: roles of endothelin converting enzyme and mitogen-activated protein kinase pathways.

Background Asthma is a chronic airway disease, known to involve several inflammatory mediators. Little is known about how these mediators interact in order to produce or attenuate even basic features of the disease, like airway hyper‐reactivity and remodelling. Endothelin‐1 (ET‐1) and IL‐1β are two mediators suggested to play important roles in the induction of airway inflammation.

Glucocorticoids suppress transcriptional up-regulation of bradykinin receptors in a murine in vitro model of chronic airway inflammation

Background Glucocorticoids are effective drugs for controlling symptoms and airway inflammation in respiratory diseases such as asthma and chronic obstructive pulmonary disease. However, the mechanisms behind their effects are not fully understood. We have recently demonstrated that prolonged exposure to the pro‐inflammatory mediator tumour necrosis factor‐α (TNF‐α) markedly enhanced contractile responses to des‐Arg9‐bradykinin (selective bradykinin B1 receptor agonist) and bradykinin (selective bradykinin B2 receptor agonist) in murine airways. This increase was paralleled with elevated mRNA levels for bradykinin B1 and B2 receptors, a process involving intracellular mitogen‐activated protein kinase pathways.

Nicotine enhances murine airway contractile responses to kinin receptor agonists via activation of JNK- and PDE4-related intracellular pathways.

BackgroundNicotine plays an important role in cigarette-smoke-associated airway disease. The present study was designed to examine if nicotine could induce airway hyperresponsiveness through kinin receptors, and if so, explore the underlying mechanisms involved.MethodsMurine tracheal segments were cultured for 1, 2 or 4 days in serum-free DMEM medium in presence of nicotine (1 and 10 μM) or vehicle (DMSO). Contractile responses induced by kinin B1 receptor agonist, des-Arg9-bradykinin, and B2 receptor agonist, bradykinin, were monitored with myographs. The B1 and B2 receptor mRNA expressions were semi-quantified using real-time PCR and their corresponding protein expressions assessed with confocal-microscopy-based immunohistochemistry. Various pharmacological inhibitors were used for studying intracellular signaling pathways.ResultsFour days of organ culture with nicotine concentration-dependently increased kinin B1 and B2 receptor-mediated airway contractions, without altering the kinin receptor-mediated relaxations. No such increase was seen at day 1 or day 2. The airway contractile responses to 5-HT, acetylcholine and endothelin receptor agonists remained unaffected by nicotine. Two different neuronal nicotinic receptor antagonists MG624 and hexamethonium blocked the nicotine-induced effects. The enhanced contractile responses were accompanied by increased mRNA and protein expression for both kinin receptors, suggesting the involvement of transcriptional mechanisms. Confocal-microscopy-based immunohistochemistry showed that 4 days of nicotine treatment induced activation (phosphorylation) of c-Jun N-terminal kinase (JNK), but not extracellular signal-regulated kinase 1 and 2 (ERK1/2) and p38. Inhibition of JNK with its specific inhibitor SP600125 abolished the nicotine-induced effects on kinin receptor-mediated contractions and reverted the enhanced receptor mRNA expression. Administration of phosphodiesterase inhibitors (YM976 and theophylline), glucocorticoid (dexamethasone) or adenylcyclase activator (forskolin) suppressed the nicotine-enhanced airway contractile response to des-Arg9-bradykinin and bradykinin.ConclusionsNicotine induces airway hyperresponsiveness via transcriptional up-regulation of airway kinin B1 and B2 receptors, an effect mediated via neuronal nicotinic receptors. The underlying molecular mechanisms involve activation of JNK- and PDE4-mediated intracellular inflammatory signal pathways. Our results might be relevant to active and passive smokers suffering from airway hyperresponsiveness, and suggest new therapeutic targets for the treatment of smoke-associated airway disease.

Interleukin-1beta up-regulates tumor necrosis factor receptors in the mouse airways.

Cytokines like interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha), released during the inflammatory process, play important roles in the development of airway hyperresponsiveness. The effects of these cytokines are mediated by cell surface receptors, specific for each cytokine. The expression of cytokine receptors is a dynamic process, where receptors can be up- or down-regulated in response to changes in the environment. One such environmental factor is the presence of cytokines per se. The present study was designed to evaluate the effects of IL-1beta on the expression of its corresponding receptor IL-1 RI, as well as on the closely related TNFalpha receptors TNF RI and TNF RII in airways using a mouse organ culture assay and intranasal inoculation model. Immunohistochemical staining was used to quantify expressional differences between fresh and cultured tracheal segments. In the fresh, uncultured, segments, IL-1 RI and TNF RI were seen in the epithelial layer and TNF RI in the smooth muscle layer. After 4 days of culture, the expression of TNF RI decreased in the epithelial layer, whereas the corresponding expression of IL-1 RI and TNF RI in the smooth muscle remained unchanged. When culture was performed in the presence of IL-1beta, the expression of IL-1 RI and TNF RI in the epithelial cells and TNF RI in the smooth muscle cells increased. TNF RII was not detected in either fresh or cultured trachea, but after treatment with IL-1beta an expression was found in both the epithelial layer and in the smooth muscle cells. The IL-1beta-induced increased expression, on TNF RI and TNF RII in the smooth muscle ex vivo and in the lung parenchyma after intranasal challenge in vivo, was verified at the mRNA level using real-time RT PCR. To summarize, presence of IL-1beta increases the expression of IL-1 R1 and TNF RI and induces expression of TNF RII in the airway wall. It is not inconceivable that these alterations of the IL-1 and TNF receptors may have important functional implications for the development of hyperresponsiveness in inflammatory airway diseases like asthma.

MAPK/NF-κB-dependent upregulation of kinin receptors mediates airway hyperreactivity: A new perspective for the treatment

Airway hyperreactivity (AHR) is a major feature of asthmatic and inflammatory airways. Cigarette smoke exposure, and bacterial and viral infections are well-known environmental risk factors for AHR, but knowledge about the underlying molecular mechanisms on how these risk factors lead to the development of AHR is limited. Activation of intracellular mitogen-activated protein kinase (MAPK)/nuclear factor-kappa B (NF-κB) and their related signal pathways including protein kinase C (PKC), phosphoinositide 3-kinase (PI3K) and protein kinase A (PKA) signaling pathways may result in airway kinin receptor upregulation, which is suggested to play an important role in the development of AHR. Environmental risk factors trigger the production of pro-inflammatory mediators such as tumor necrosis factor-α (TNF-α) and interleukins (ILs) that activate intracellular MAPK- and NF-κB-dependent inflammatory pathways, which subsequently lead to AHR via kinin receptor upregulation. Blockage of intracellular MAPK/NF-κB signaling prevents kinin B₁ and B₂ receptor expression in the airways, resulting in a decrease in the response to bradykinin (kinin B₂ receptor agonist) and des-Arg⁹-bradykinin (kinin B₁ receptor agonist). This suggests that MAPK- and NF-κB-dependent kinin receptor upregulation can provide a novel option for treatment of AHR in asthmatic as well as in other inflammatory airway diseases.

Long-term exposure to IL-1beta enhances Toll-IL-1 receptor-mediated inflammatory signaling in murine airway hyperresponsiveness.

Toll-interleukin-1 (Toll-IL-1) receptor signaling may play a key role in the development of airway hyperreactivity (AHR) and chronic airway inflammatory diseases such as asthma. Previously, we have demonstrated that pro-inflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta), induce AHR. However, the underlying intracellular signaling mechanisms that lead to AHR remain elusive. In order to see if the Toll-IL-1 receptor-mediated inflammatory signal pathways are involved in the development of AHR, the present study was designed to use a real-time PCR array, a sensitive and powerful tool, consisting of 84 genes related to Toll-IL-1 receptor signal pathways. Murine tracheal segments were organ cultured for four days in the presence and absence of IL-1beta. The Toll-IL-1 receptor-mediated inflammatory signal gene profile was studied using the real-time PCR-based cDNA array. The key gene expressions that were altered were verified by immunohistochemistry using confocal microscopy. Tracheal ring segment contractile responsiveness to the inflammatory mediator bradykinin was monitored using a sensitive myograph system. The results showed that after exposed to IL-1beta for four days, the tracheal segments exhibited increased mRNA expression of 67 genes (out of the 84 genes in the array), although expression reached statistical significance for only 16 of these genes. There were 14 genes that showed only a tendency towards a decrease in mRNA expression following IL-1beta treatment. Immunohistochemistry confirmed that protein expression for CD14, RP105, MCP-1 and phosphorylated IkappaB-alpha were increased in both the airway epithelial and smooth muscle cells. In order to link the activation of Toll-IL-1 receptor-mediated inflammatory signal mechanisms to the AHR, the anti-inflammatory drug dexamethasone, was used. Dexamethasone not only completely abolished the IL-1beta-induced AHR to bradykinin, but also abrogated the increased mRNA expression for inflammatory mediators, IL-6, IFN-gamma and Cox-2. In conclusion, long-term exposure of murine airway to IL-1beta induces up- and down-regulation of mRNA expression for Toll-IL-1 receptor signal molecules, with a significant increase in the expression of 16 genes that contribute to the development of airway inflammation and AHR. Understanding cytokine-induced activation of the Toll-IL-1 receptor-mediated inflammatory signaling mechanisms may provide new options for the treatment of airway inflammation and AHR.

Nicotine Exaggerates LPS-Induced Airway Hyperreactivity via JNK-Mediated Up-regulation of Toll-Like Receptor 4

Tobacco smokers often display increased airway hyperreactivity (AHR) when faced with bacterial infections. The present study uses a murine organ-culture model to dissect the mechanisms involved in this exaggerated smooth muscle response. Nicotine simulates the effects of smoking, and LPS represents bacterial infection. Contractile responses of isolated murine tracheal segments were analyzed in myographs after organ culture with increasing concentrations of LPS and/or nicotine for 4 days with or without specific MAPK inhibitors. Nicotines effect on the expression of cell surface Toll-like receptors (TLRs), MCP-1, COX-2, and TNF-α were examined by real-time PCR. Increased protein expression was verified by immunohistochemistry. LPS concentration-dependently increased contractile responses to bradykinin and des-Arg(9)-bradykinin. A combination of nicotine and low-dose LPS caused powerful synergistic contractions along with increased kinin receptor expression. Specific kinin B1 and B2 receptor inhibitors blocked this reaction. Nicotine increased mRNA and protein expression of TLR4 and -6 in the epithelium and smooth muscle layer, with MCP-1 and COX-2 mRNA increasing in parallel. Specific inhibition of JNK attenuated nicotines effects. In conclusion, long-term exposure to nicotine up-regulated the expression of TLR4 and -6 via a JNK-related pathway, causing an exaggeration of the LPS-induced local airway inflammation and increased AHR. This might offer a mechanistic explanation to the increased AHR seen in tobacco smokers confronted with bacterial infections.