Functional expression of the C-X-C chemokine receptor CXCR4 by human bronchial epithelial cells: regulation by proinflammatory mediators

CXCR4 and its ligand stromal cell-derived factor 1alpha (SDF-1alpha) have recently been implicated in the development of airway inflammation in a mouse model of allergic airway disease. Here we report, for the first time, the expression of a functional CXCR4 in primary human normal bronchial epithelial cells and the regulation of CXCR4 gene expression by proinflammatory mediators. Both bradykinin (BK) and IL-1beta induced an accumulation of CXCR4 mRNA in normal bronchial epithelial cells in a time-dependent manner, with peak levels of CXCR4 mRNA reached between 4 and 24 h after stimulation. Ligand activation of CXCR4 in airway epithelial cells resulted in the activation of the extracellular signal-regulated kinase and stress-activated protein kinase/c-Jun amino-terminal kinase signaling pathways and calcium mobilization. Pretreatment of airway epithelial cells with BK or IL-1beta enhanced SDF-1alpha induced phospho-extracellular signal-regulated kinase and calcium mobilization, in addition to increasing the level of CXCR4 protein. Finally, we describe the expression of CXCR4 mRNA and its regulation by BK in vivo in human nasal tissue. CXCR4 mRNA levels are significantly higher in the nasal tissue of symptomatic allergic rhinitis subjects compared with normal subjects. Moreover, BK challenge significantly increased CXCR4 mRNA levels in nasal tissue of mild allergic rhinitis subjects in vivo, but not normal controls. In conclusion, this study demonstrates that human airway epithelial cells respond to proinflammatory mediators by up-regulating the chemokine receptor CXCR4, thus enabling the cells to respond more effectively to constitutively expressed SDF-1alpha. This may lead to enhanced activation of intracellular signaling pathways resulting in the release of mediators involved in inflammatory allergic airway disease.     

Lymphocyte-derived microparticles induce apoptosis of airway epithelial cells through activation of p38 MAPK and production of arachidonic acid

The airway epithelium is critical for the normal integrity and function of the respiratory system. Excessive epithelial cell apoptosis contributes to cell damage and airway inflammation. We previously demonstrated that lymphocyte-derived microparticles (LMPs) induce apoptosis of human bronchial epithelial cells. However, the underlying mechanisms contributing to LMPs-evoked epithelial cell death are largely unknown. Here we used bronchial and lung tissue cultures to confirm the pro-apoptotic effects of LMPs. In cell culture experiments, we found that LMPs induced human airway epithelial cell apoptosis with associated increases in caspase-3 activity. In addition, LMPs treatment triggered oxidative stress in epithelial cells by enhancing production of malondialdehyde, superoxide, and reactive oxygen species (ROS), and by inhibiting production of the antioxidant glutathione. Moreover, decreasing cellular ROS with the antioxidant N-acetylcysteine rescued epithelial cell viability. Together, these results demonstrate an important role for oxidative stress in LMPs-induced cell death. In epithelial cells, LMPs treatment induced phosphorylation of p38 MAPK and arachidonic acid accumulation. Moreover, arachidonic acid was significantly cytotoxic towards LMPs-treated epithelial cells, whereas inhibition of p38 MAPK was protective against these cytotoxic effects. Similarly, inhibition of arachidonic acid production led to decreased caspase-3 activity, thus rescuing airway epithelial cells from LMPs-induced cell death. In conclusion, our results show that LMPs induce airway epithelial cell apoptosis by activating p38 MAPK signaling and stimulating production of arachidonic acid, with consequent increases in oxidative stress and caspase-3 activity. As such, LMPs may be regarded as deleterious markers of epithelial cell damage in respiratory diseases.     

Signaling and proapoptotic functions of transformed cell-derived reactive oxygen species

Transformed fibroblasts generate extracellular superoxide anions through the recently identified membrane-associated NADPH oxidase. These cell-derived superoxide anions exhibit signaling functions such as regulation of proliferation and maintenance of the transformed state. Their dismutation product hydrogen peroxide regulates the intracellular level of catalase, whose activity has been observed to be upregulated in certain transformed cells. After glutathione depletion, transformed cell-derived reactive oxygen species (ROS) exhibit apoptosis-inducing potential through the metal-catalyzed Haber-Weiss reaction. Moreover, transformed cell-derived ROS represent key elements for selective and efficient apoptosis induction by natural antitumor systems (such as fibroblasts, granulocytes and macrophages). These effector cells release peroxidase, which utilizes target cell-derived hydrogen peroxide for HOCl synthesis. In a second step, HOCl interacts with target cell-derived superoxide anions and forms apoptosis-inducing hydroxyl radicals. In a parallel signaling pathway, effector cell-derived NO interacts with target cell-derived superoxide anions and generates the apoptosis inducer peroxynitrite. Therefore, transformed cell-derived ROS determine transformed cells as selective targets for induction of apoptosis by these effector systems. It is therefore proposed that transformed cell derived ROS interact with associated cells to exhibit directed and specific signaling functions, some of which are beneficial and some of which can become detrimental to transformed cells.     

Reduced Superoxide Dismutase in Lung Cells of Patients with Asthma

Lung cells recovered from symptomatic patients with asthma generate increased amounts of reactive oxygen species (ROS). Animal and in vitro studies indicate that ROS can reproduce many of the features of asthma. The ability of ROS to produce the clinical features of asthma may depend on an individual's lung antioxidant defenses. Patients with asthma are reported to have reduced antioxidant defenses in peripheral blood, but little is known about the antioxidant defenses of their lung cells. To define lung cell antioxidant defenses in asthma, the glutathione concentration and the glutathione reductase, glutathione peroxidase, catalase, and superoxide dismutase (SOD) activities were measured in cells recovered by bronchoalveolar lavage (BAL cells) and by bronchial brushing (bronchial epithelial cells, HBEC) from normal subjects and patients with asthma. Superoxide dismutase activity was reduced 25% in BAL cells (p < .05) and nearly 50% in HBEC (p < .02) from patients with asthma. Alterations in the other antioxidants were not identified. A direct relationship was found between airway reactivity to methacholine, measured as PC₂₀FEV₁, and HBEC SOD activity (r² = 89; p < .005), but not between airway reactivity and the other antioxidants. The finding of reduced SOD activity in lung cells of patients with asthma suggests that diminished SOD activity serves as a marker of the inflammation characterizing asthma. Alternatively, it may play a role in the development or severity of the disease. © 1997 Elsevier Science Inc.     

Kaempferol Inhibits Endoplasmic Reticulum Stress-Associated Mucus Hypersecretion in Airway Epithelial Cells And Ovalbumin-Sensitized Mice

Mucus hypersecretion is an important pathological feature of chronic airway diseases, such as asthma and pulmonary diseases. MUC5AC is a major component of the mucus matrix forming family of mucins in the airways. The initiation of endoplasmic reticulum (ER)-mediated stress responses contributes to the pathogenesis of airway diseases. The present study investigated that ER stress was responsible for airway mucus production and this effect was blocked by the flavonoid kaempferol. Oral administration of ≥10 mg/kg kaempferol suppressed mucus secretion and goblet cell hyperplasia observed in the bronchial airway and lung of BALB/c mice sensitized with ovalbumin (OVA). TGF-β and tunicamycin promoted MUC5AC induction after 72 h in human bronchial airway epithelial BEAS-2B cells, which was dampened by 20 μM kaempferol. Kaempferol inhibited tunicamycin-induced ER stress of airway epithelial cells through disturbing the activation of the ER transmembrane sensor ATF6 and IRE1α. Additionally, this compound demoted the induction of ER chaperones such as GRP78 and HSP70 and the splicing of XBP-1 mRNA by tunicamycin. The in vivo study further revealed that kaempferol attenuated the induction of XBP-1 and IRE1α in epithelial tissues of OVA-challenged mice. TGF-β and tunicamycin induced TRAF2 with JNK activation and such induction was deterred by kaempferol. The inhibition of JNK activation encumbered the XBP-1 mRNA splicing and MUC5AC induction by tunicamycin and TGF-β. These results demonstrate that kaempferol alleviated asthmatic mucus hypersecretion through blocking bronchial epithelial ER stress via the inhibition of IRE1α-TRAF2-JNK activation. Therefore, kaempferol may be a potential therapeutic agent targeting mucus hypersecretion-associated pulmonary diseases.     

Ecto 5'-nucleotidase and nonspecific alkaline phosphatase. Two AMP-hydrolyzing ectoenzymes with distinct roles in human airways

In human airways, extracellular adenosine regulates epithelial functions supporting mucociliary clearance, an important airway defense mechanism against bacterial infection. Thus, defining the mechanisms of adenosine generation is critical for elucidating the role of this nucleoside in airway homeostasis. In this study, we identified the source of adenosine on the mucosal surface of human airway epithelia. Polarized primary cultures of human nasal or bronchial epithelial cells were assayed for transepithelial transport, cytosolic and cell surface adenosine production. Ussing chamber experiments indicated that serosal 1 microM [(3)H]adenosine was not transported to the mucosal compartment. Messenger RNA for the cytosolic AMP-specific 5'-nucleotidase (CN-I) was not detected in human bronchial epithelial cells, suggesting that mucosal adenosine did not originate from intracellular pools. In contrast, extracellular 0.1 mm ATP was rapidly dephosphorylated into adenosine on the mucosal epithelial surface. We identified two ectonucleotidases that mediated the conversion of AMP to adenosine: ecto 5'-nucleotidase (ecto 5'-NT, CD73) and alkaline phosphatase (AP). Both mucosal and serosal epithelial surfaces displayed ecto 5'-NT activity (K(m) = 14 microM, V(max) = 0.5 nmol x min(-1) x cm(-2)), whereas AP activity was restricted to the mucosal surface (K(m,)(high) = 36 microM, V(max) = 1.2 nmol x min(-1) x cm(-2); K(m,)(low) = 717 microM, V(max) = 2.8 nmol x min(-1) x cm(-2)). In bronchial cultures and tissues, ecto 5'-NT accounted for >80% of total activity toward 0.01 mm AMP, compared with <15% for 5 mm AMP. The proximal airway AP isoform was identified as nonspecific AP (NS AP) by levamisole sensitivity and mRNA expression. The two ectoenzymes presented opposite airway distributions, ecto 5'-NT and NS AP mRNA dominating in higher and lower airways, respectively. Collectively, these experiments support a major role for extracellular nucleotide catalysis and for ecto 5'-NT and NS AP in the regulation of adenosine concentrations on airway surfaces.     

Down-regulation of secreted lymphocyte antigen-6/urokinase-type plasminogen activator receptor-related peptide-1 (SLURP-1), an endogenous allosteric α7 nicotinic acetylcholine receptor modulator, in murine and human asthmatic conditions

Whereas acetylcholine (ACh) acts as a bronchoconstrictor and stimulator of mucus secretion from bronchial epithelium, it acts via α7 nicotinic Ach receptors (nAChRs) on macrophages in the airways to exert anti-inflammatory effects by reducing synthesis of pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α). Moreover, the effects of ACh are modified by secreted ly-6/urokinase-type plasminogen activator receptor-related peptide-1 (SLURP-1), a positive allosteric modulator of α7 nAChR signaling. Our aim was to explore the roles played by SLURP-1 in the pathophysiology of asthma by assessing SLURP-1 expression in the OVA-sensitized murine asthma model and in cultured human bronchial epithelial cells. Using real-time PCR we found that expression of SLURP-1 mRNA is down-regulated in the lungs of asthmatic model mice, as compared to healthy mice. In addition, immunohistochemical studies confirmed the diminished expression of SLURP-1 in the bronchioles of asthmatic mice, and showed it was due to extensive metaplasia of mucus-secreting cells and the concomitant loss of ciliated epithelial cells. Expression of SLURP-1 mRNA and protein was also significantly down-regulated in human epithelial cells stimulated with the pro-inflammatory cytokine interleukin-13 (IL-13), which is related to asthmatic condition. Thus SLURP-1 appears to be down-regulated in both an animal model of asthma and human epithelial cells treated with an inflammatory cytokine related to asthma. Those findings suggest that diminished expression of SLURP-1 in asthma attenuates its negative regulation of airway inflammation, and that perhaps changes in SLURP-1 expression could serve as a marker of airway damage in asthma.     

TGFβ1 induces IL‐6 and inhibits IL‐8 release in human bronchial epithelial cells: The role of Smad2/3

Human bronchial epithelial (HBE) cells contribute to asthmatic airway inflammation by secreting cytokines, chemokines, and growth factors, including interleukin (IL)‐6, IL‐8 and transforming growth factor (TGF) β1, all of which are elevated in asthmatic airways. This study examines the signaling pathways leading to TGFβ1 induced IL‐6 and IL‐8 in primary HBE cells from asthmatic and non‐asthmatic volunteers. HBE cells were stimulated with TGFβ1 in the presence or absence of signaling inhibitors. IL‐6 and IL‐8 protein and mRNA were measured by ELISA and real‐time PCR respectively, and cell signaling kinases by Western blot. TGFβ1 increased IL‐6, but inhibited IL‐8 production in both asthmatic and non‐asthmatic cells; however, TGF induced significantly more IL‐6 in asthmatic cells. Inhibition of JNK MAP kinase partially reduced TGFβ1 induced IL‐6 in both cell groups. TGFβ1 induced Smad2 phosphorylation, and blockade of Smad2/3 prevented both the TGFβ1 modulated IL‐6 increase and the decrease in IL‐8 production in asthmatic and non‐asthmatic cells. Inhibition of Smad2/3 also increased basal IL‐8 release in asthmatic cells but not in non‐asthmatic cells. Using CHIP assays we demonstrated that activated Smad2 bound to the IL‐6, but not the IL‐8 promoter region. We conclude that the Smad2/3 pathway is the predominant TGFβ1 signaling pathway in HBE cells, and this is altered in asthmatic bronchial epithelial cells. Understanding the mechanism of aberrant pro‐inflammatory cytokine production in asthmatic airways will allow the development of alternative ways to control airway inflammation. J. Cell. Physiol. 225: 846–854, 2010. © 2010 Wiley‐Liss, Inc.     

TLR3- and Th2 cytokine-dependent production of thymic stromal lymphopoietin in human airway epithelial cells

Thymic stromal lymphopoietin (TSLP) is elevated in asthma and triggers dendritic cell-mediated activation of Th2 inflammatory responses. Although TSLP has been shown to be produced mainly by airway epithelial cells, the regulation of epithelial TSLP expression has not been extensively studied. We investigated the expression of TSLP in cytokine- or TLR ligand-treated normal human bronchial epithelial cells (NHBE). The mRNA for TSLP was significantly up-regulated by stimulation with IL-4 (5.5-fold) and IL-13 (5.3-fold), weakly up-regulated by TNF-alpha, TGF-beta, and IFN-beta, and not affected by IFN-gamma in NHBE. TSLP mRNA was only significantly up-regulated by the TLR3 ligand (dsRNA) among the TLR ligands tested (66.8-fold). TSLP was also induced by in vitro infection with rhinovirus. TSLP protein was detected after stimulation with dsRNA (120 +/- 23 pg/ml). The combination of TNF-alpha and IL-4 produced detectable levels of TSLP protein (40 +/- 13 pg/ml). In addition, TSLP was synergistically enhanced by a combination of IL-4 and dsRNA (mRNA; 207-fold, protein; 325 +/- 75 pg/ml). The induction of TSLP by dsRNA was dependent upon NF-kappaB and IFN regulatory factor 3 (IRF-3) signaling via TLR3 as indicated by a study with small interfering RNA. The potent topical glucocorticoid fluticasone propionate significantly suppressed dsRNA-dependent TSLP production in NHBE. These results suggest that the expression of TSLP is induced in airway epithelial cells by stimulation with the TLR3 ligand and Th2 cytokines and that this response is suppressed by glucocorticoid treatment. This implies that respiratory viral infection and the recruitment of Th2 cytokine producing cells may amplify Th2 inflammation via the induction of TSLP in the asthmatic airway.     

The absence of supplemental induction of neurotrophin receptors on the bronchial epithelia during bronchial asthma may lead to the epithelial denudation

Bronchial asthma is characterized by airway inflammation, bronchial hyperresponsiveness, reversible bronchial obstruction, eosinophilia, and specific IgE response. Involved in this pathogenesis are neurotrophins such as nerve growth factor or brain-derived neurotrophic factor, which mediate their inflammatory effects through their receptors like tropomyosin-receptor kinases and pan-neurotrophin receptor p75. In addition, the induction of the above-mentioned mediators promotes increased surviving capacities for various cells of bronchial mucosa, which are in contact with noxious stimuli. In contrast to a wide variety of cellular populations, the denudation of local epithelia during bronchial inflammation, associated with potentiation of bronchial hyperresponsiveness mediated by subepithelial neuronal fibers is observed. In the context of these processes, bronchial epithelial cells show an increased neurotrophin synthesis, associated with decreased expression levels of relative receptors. This pattern contrasts again to other resident or emigrated cellular populations within bronchial tissue, which show both neurotrophin and neurotrophin receptor induction. It seems that apart from destructive abilities of specific or nonspecific noxious stimuli on the bronchial epithelia, the presence of depleted survival stimuli due to lack of neurotrophin receptors expression on the mentioned cellular population play a strategical role in epithelial destruction. This effect might be initiated by the asthmatic patient in order to provide airway hyperresponsiveness and thereby reduce the contact with harmful stimuli due to bronchial obstruction.     

Glucocorticoid and estrogen receptors are reduced in mitochondria of lung epithelial cells in asthma

Mitochondrial glucocorticoid (mtGR) and estrogen (mtER) receptors participate in the coordination of the cell's energy requirement and in the mitochondrial oxidative phosphorylation enzyme (OXPHOS) biosynthesis, affecting reactive oxygen species (ROS) generation and induction of apoptosis. Although activation of mtGR and mtER is known to trigger anti-inflammatory signals, little information exists on the presence of these receptors in lung tissue and their role in respiratory physiology and disease. Using a mouse model of allergic airway inflammation disease and applying confocal microscopy, subcellular fractionation, and Western blot analysis we showed mitochondrial localization of GRα and ERβ in lung tissue. Allergic airway inflammation caused reduction in mtGRα, mtERβ, and OXPHOS enzyme biosynthesis in lung cells mitochondria and particularly in bronchial epithelial cells mitochondria, which was accompanied by decrease in lung mitochondrial mass and induction of apoptosis. Confirmation and validation of the reduction of the mitochondrial receptors in lung epithelial cells in human asthma was achieved by analyzing autopsies from fatal asthma cases. The presence of the mitochondrial GRα and ERβ in lung tissue cells and especially their reduction in bronchial epithelial cells during allergic airway inflammation suggests a crucial role of these receptors in the regulation of mitochondrial function in asthma, implicating their involvement in the pathophysiology of the disease.     

A novel inhibitory role of microRNA-224 in particulate matter 2.5-induced asthmatic mice by inhibiting TLR2

Epidemiological studies have shown that elevated concentrations of particulate matter 2.5 (PM2.5) correlate with increased incidence of asthma. Studies have highlighted the implication of microRNAs (miRNAs) in asthmatic response. Here, the objective of this study is to explore the effect of miR-224 on PM2.5-induced asthmatic mice. Ovalbumin (OVA) was utilized to establish asthmatic mouse models, which were then exposed to PM2.5, followed by miR-224 expression detection. Next, lesions and collagen deposition area in lung tissue, ratio Treg/Th17, the expression of TLR4 and MYD88, inflammation, eosinophils (EOS) and airway remodelling were evaluated in OVA mice after injection with miR-224 agomir. Following isolation of mouse primary bronchial epithelial cells, miR-224 mimic and TLR2/TLR4 inhibitor were introduced to assess inflammation and the expression of TGF-β, MMP9, TIMP-1, Foxp3, RORγt, TLR2, TLR4 and MYD88. After exposure to PM2.5, lesions and collagen deposition were promoted in lung tissues, inflammation and EOS were increased in bronchoalveolar lavage fluid (BALF), and airway remodelling was enhanced in OVA mice. miR-224 was down-regulated, whereas TLR2/TLR4/MYD88 was up-regulated in OVA mice after treatment with PM2.5, accompanied by Treg/Th17 immune imbalance. Of note, bioinformatic prediction and dual luciferase reporter gene assay confirmed that TLR2 was a target gene of miR-224. Overexpressed miR-224 reduced expression of TGF-β, MMP9, TIMP-1 and RORγt and inflammation but increased Foxp3 expression in bronchial epithelial cells through down-regulating TLR2. In summary, overexpressed miR-224 suppressed airway epithelial cell inflammation and airway remodelling in PM2.5-induced asthmatic mice through decreasing TLR2 expression.