scholarly journals Expression/Activation of PAR-1 in Airway Epithelial Cells of COPD Patients: Ex Vivo/In Vitro Study

2021 ◽  
Vol 22 (19) ◽  
pp. 10703
Author(s):  
Angela Marina Montalbano ◽  
Giuseppina Chiappara ◽  
Giusy Daniela Albano ◽  
Maria Ferraro ◽  
Caterina Di Sano ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Ex Vivo ◽  
In Vitro Study ◽  
Bronchial Epithelial Cell ◽  
Airway Epithelial Cells ◽  
Epithelial Cell Line ◽  
Airway Epithelial ◽  
Bronchial Epithelial ◽  
Copd Patients

The role of PAR-1 expression and activation was described in epithelial cells from the central and distal airways of COPD patients using an ex vivo/in vitro model. PAR-1 immunoreactivity was studied in epithelial cells from surgical specimens of the central and distal airways of COPD patients and healthy control (HC). Furthermore, PAR-1 expression and activation were measured in both the human bronchial epithelial cell line (16HBE) and normal human bronchial epithelial cells (NHBEs) exposed to cigarette smoke extract (CSE) (10%) or thrombin. Finally, cell proliferation, apoptosis, and IL-8 release were detected in stimulated NHBEs. We identified higher levels of PAR-1 expression/activation in epithelial cells from the central airways of COPD patients than in HC. Active PAR-1 increased in epithelial cells from central and distal airways of COPD, with higher levels in COPD smokers (correlated with pack-years) than in COPD ex-smokers. 16HBE and NHBEs exposed to CSE or thrombin showed increased levels of active PAR-1 (localized in the cytoplasm) than baseline conditions, while NHBEs treated with thrombin or CSE showed increased levels of IL-8 proteins, with an additional effect when used in combination. Smoking habits generate the upregulation of PAR-1 expression/activation in airway epithelial cells, and promoting IL-8 release might affect the recruitment of infiltrating cells in the airways of COPD patients.

Glucocorticoid regulation of GM-CSF: evidence for transcriptional mechanisms in airway epithelial cells

1998 ◽  
Vol 275 (2) ◽  
pp. L372-L378 ◽  
Author(s):  
Karissa K. Adkins ◽  
Tricia D. Levan ◽  
Roger L. Miesfeld ◽  
John W. Bloom
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Cell Line ◽  
Bronchial Epithelial Cell ◽  
Airway Epithelial Cells ◽  
Epithelial Cell Line ◽  
Airway Epithelial ◽  
Dexamethasone Treatment ◽  
Bronchial Epithelial ◽  
Gm Csf

Inflammation plays a central role in the pathogenesis of asthma. Glucocorticoids are first-line anti-inflammatory therapy in the treatment of asthma and are effective inhibitors of inflammatory cytokines. Clinical data demonstrate that granulocyte-macrophage colony-stimulating factor (GM-CSF) production by airway epithelial cells may be an important target of inhaled glucocorticoid therapy. We examined the regulatory mechanisms of GM-CSF expression by interleukin-1β (IL-1β) and the synthetic glucocorticoid dexamethasone in the BEAS-2B human bronchial epithelial cell line. IL-1β stimulation resulted in a 15-fold induction of GM-CSF protein, which was associated with a corresponding 47-fold maximal induction of GM-CSF mRNA levels. Treatment with the transcriptional inhibitor actinomycin D before IL-1β stimulation completely abolished induction of GM-CSF mRNA, whereas incubation with cycloheximide had no effect. Taken together, these data demonstrate that IL-1β induction of GM-CSF is mediated through transcriptional mechanisms. Dexamethasone treatment of BEAS-2B cells produced an 80% inhibition of IL-1β-induced GM-CSF protein and a 51% inhibition of GM-CSF mRNA. GM-CSF mRNA was rapidly degraded in these cells, and dexamethasone treatment did not significantly affect this decay rate. We conclude that, in the BEAS-2B bronchial epithelial cell line, IL-1β induction and dexamethasone repression of GM-CSF expression are mediated predominantly through transcriptional mechanisms.

Signaling intermediates required for NF-κB activation and IL-8 expression in CF bronchial epithelial cells

2003 ◽  
Vol 284 (2) ◽  
pp. L307-L315 ◽  
Author(s):  
Jing Li ◽  
Xa Dwight Johnson ◽  
Svetlana Iazvovskaia ◽  
Alan Tan ◽  
Anning Lin ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Cell Line ◽  
Bronchial Epithelial Cell ◽  
Airway Epithelial Cells ◽  
Epithelial Cell Line ◽  
Airway Epithelial ◽  
Independent Manner ◽  
Bronchial Epithelial ◽  
Erk Activation ◽  
Iκb Kinase

Ligation of the asialoGM1 Pseudomonas aeruginosa pilin receptor has been demonstrated to induce IL-8 expression in airway epithelial cells via an NF-κB-dependent pathway. We examined the signaling pathways required for asialoGM1-mediated NF-κB activation in IB3 cells, a human bronchial epithelial cell line derived from a cystic fibrosis (CF) patient, and C-38 cells, the rescued cell line that expresses a functional CF transmembrane regulator. Ligation of the asialoGM1 receptor with specific antibody induced greater IL-8 expression in IB3 cells than C-38 cells, consistent with the greater density of asialoGM1 receptors in CF phenotype cells. AsialoGM1-mediated activation of NF-κB, IκB kinase (IKK), and ERK was also greater in IB3 cells. With the use of genetic inhibitors, we found that IKK-β and NF-κB-inducing kinase are required for maximal NF-κB transactivation and transcription from the IL-8 promoter. Finally, although ERK activation was required for maximal asialoGM1-mediated IL-8 expression, inhibition of ERK signaling had no effect on IKK or NF-κB activation, suggesting that ERK regulates IL-8 expression in an NF-κB-independent manner.

Hog barn dust extract stimulates IL-8 and IL-6 release in human bronchial epithelial cells via PKC activation

2002 ◽  
Vol 93 (1) ◽  
pp. 289-296 ◽  
Author(s):  
D. J. Romberger ◽  
V. Bodlak ◽  
S. G. Von Essen ◽  
T. Mathisen ◽  
T. A. Wyatt
Keyword(s):  
Epithelial Cells ◽  
Polymyxin B ◽  
Bronchial Epithelial Cell ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Initial Exposure ◽  
Bronchial Epithelial ◽  
Pkc Activation ◽  
Dust Extract

Hog barn workers have an increased incidence of respiratory tract symptoms and demonstrate an increase in lung inflammatory mediators, including interleukin (IL)-8 and IL-6. Utilizing direct kinase assays for protein kinase C (PKC) activation, we demonstrated that dust from hog confinement facilities, or hog dust extract (HDE), augments PKC activity of human airway epithelial cells in vitro. A 5% dilution of HDE typically stimulates an approximately twofold increase in human bronchial epithelial cell (HBEC) PKC activity compared with control medium-treated cells. This increase in PKC is observed with 15 min of HDE treatment, and kinase activity reaches peak activity by 1–2 h of HDE treatment before returning to baseline PKC levels between 6 and 24 h. The classic PKC inhibitor, calphostin C, blocks HDE-stimulated PKC activity and associated IL-8 and IL-6 release. Desensitization to HDE stimulation of PKC activation does not appear to occur because subsequent exposures to HDE after an initial exposure result in further augmentation of PKC. Detoxification of HDE with polymyxin B to remove endotoxin did not change PKC activation or IL-8 release, suggesting that endotoxin is not solely responsible for HDE augmentation of PKC. These data support the hypothesis that HDE exposure augments HBEC IL-8 and IL-6 release via a PKC-dependent pathway.

Resting and Cytokine-Stimulated Human Small Airway Epithelial Cells Recognize and Engulf Apoptotic Eosinophils

Blood ◽  
1999 ◽  
Vol 94 (8) ◽  
pp. 2827-2835 ◽  
Author(s):  
Garry M. Walsh ◽  
Darren W. Sexton ◽  
Morgan G. Blaylock ◽  
Catherine M. Convery
Keyword(s):  
Epithelial Cells ◽  
Interleukin 1 ◽  
Airway Epithelial Cells ◽  
Small Airway ◽  
Airway Epithelial ◽  
Human Bronchial Epithelial Cells ◽  
Bronchial Epithelial ◽  
Small Airway Epithelial Cells ◽  
Necrosis Factor

Eosinophils, which are prominent cells in asthmatic inflammation, undergo apoptosis and are recognized and engulfed by phagocytic macrophages in vitro. We have examined the ability of human small airway epithelial cells (SAEC) to recognize and ingest apoptotic human eosinophils. Cultured SAEC ingested apoptotic eosinophils but not freshly isolated eosinophils or opsonized erythrocytes. The ability of SAEC to ingest apoptotic eosinophils was enhanced by interleukin-1 (IL-1) or tumor necrosis factor  (TNF) in a time- and concentration-dependent fashion. IL-1 was found to be more potent than TNF and each was optimal at 10−10 mol/L, with a significant (P < .05) effect observed at 1 hour postcytokine incubation that was maximal at 5 hours. IL-1 stimulation not only increased the number of SAEC engulfing apoptotic eosinophils, but also enhanced their capacity for ingestion. The amino sugars glucosamine, n-acetyl glucosamine, and galactosamine significantly inhibited uptake of apoptotic eosinophils by both resting and IL-1–stimulated SAEC, in contrast to the parent sugars glucose, galactose, mannose, and fucose. Incubation of apoptotic eosinophils with the tetrapeptide RGDS, but not RGES, significantly inhibited their uptake by both resting and IL-1–stimulated SAEC, as did monoclonal antibody against vβ3 and CD36. Thus, SAEC recognize apoptotic eosinophils via lectin- and integrin-dependent mechanisms. These data demonstrate a novel function for human bronchial epithelial cells that might represent an important mechanism in the resolution of eosinophil-induced asthmatic inflammation.

scholarly journals Development and validation of an open-source, disposable, 3D-printed in vitro environmental exposure system for Transwell® culture inserts

2020 ◽  
Author(s):  
Abiram Chandiramohan ◽  
Mohammedhossein Dabaghi ◽  
Jennifer A. Aguiar ◽  
Nicholas Tiessen ◽  
Mary Stewart ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Exposure System ◽  
Bronchial Epithelial ◽  
Human Airway ◽  
3D Printed ◽  
Air Liquid Interface ◽  
Whole Smoke

AbstractAccessible in vitro models recapitulating the human airway that are amenable to study whole cannabis smoke exposure are needed for immunological and toxicological studies that inform public health policy and recreational cannabis use. In the present study, we developed and validated a novel 3D printed In Vitro Exposure System (IVES) that can be directly applied to study the effect of cannabis smoke exposure on primary human bronchial epithelial cells.Using commercially available design software and a 3D printer, we designed a four-chamber Transwell® insert holder for exposures to whole smoke. Software was used to model gas distribution, concentration gradients, velocity profile and shear stress within IVES. Following simulations, primary human bronchial epithelial cells cultured at air-liquid interface on Transwell® inserts were exposed to whole cannabis smoke. Following 24 hours, outcome measurements included cell morphology, epithelial barrier function, lactate dehydrogenase (LDH) levels, cytokine and gene expression.Whole smoke delivered through IVES possesses velocity profiles consistent with uniform gas distribution across the four chambers and complete mixing. Airflow velocity ranged between 1.0-1.5 μm s−1 and generated low shear stresses (<< 1 Pa). Human airway epithelial cells exposed to cannabis smoke using IVES showed changes in cell morphology and disruption of barrier function without significant cytotoxicity. Cannabis smoke elevated IL-1 family cytokines and elevated CYP1A1 and CYP1B1 expression relative to control.IVES represents an accessible, open-source, exposure system that can be used to model varying types of cannabis smoke exposures with human airway epithelial cells grown under air-liquid interface culture conditions.

scholarly journals MiR-223 is increased in lungs of patients with COPD and modulates cigarette smoke-induced pulmonary inflammation

2021 ◽  
Author(s):  
Mirjam P. Roffel ◽  
Tania Maes ◽  
Corry-Anke Brandsma ◽  
Maarten van den Berge ◽  
Bart M. Vanaudenaerde ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Cigarette Smoke ◽  
Pulmonary Inflammation ◽  
Airway Epithelial Cells ◽  
Negative Regulator ◽  
Stage Ii ◽  
Airway Epithelial ◽  
Copd Patients ◽  
Deficient Mice

Since microRNA (miR)-223-3p modulates inflammatory responses and COPD is associated with amplified pulmonary inflammation, we hypothesized that miR-223-3p plays a role in COPD pathogenesis. Expression of miR-223-3p was measured in lung tissue of 2 independent cohorts with COPD GOLD stage II-IV patients, never smokers and smokers without COPD. The functional role of miR-223-3p was studied in deficient mice and upon overexpression in airway epithelial cells from COPD and controls. We observed higher miR-223-3p levels in patients with COPD stage II-IV compared to (non)-smoking controls, and levels were associated with higher neutrophil numbers in bronchial biopsies of COPD patients. MiR-223-3p expression was also increased in lungs and bronchoalveolar lavage of cigarette smoke (CS)-exposed mice. CS-induced neutrophil and monocyte lung infiltration was stronger in miR-223 deficient mice upon acute (5 days) exposure, but attenuated upon sub-chronic (4 weeks) exposure. Additionally, miR-223 deficiency attenuated acute and sub-chronic CS-induced lung infiltration of dendritic cells and T lymphocytes. Finally, in vitro overexpression of miR-223-3p in non-COPD airway epithelial cells suppressed CXCL8 and GM-CSF secretion and gene expression of the pro-inflammatory transcription factor TRAF6. Importantly, this suppressive effect of miR-223-3p was compromised in COPD-derived cultures. In conclusion, we demonstrate that miR-223-3p is increased in lungs of COPD patients and CS-exposed mice, and is associated with neutrophilic inflammation. In vivo data indicate that miR-223 acts as negative regulator of acute CS-induced neutrophilic and monocytic inflammation. In vitro data suggests that miR-223-3p does so by suppressing pro-inflammatory airway epithelial responses, which is less effective in COPD epithelium.

Diesel exhaust particles upregulate eotaxin gene expression in human bronchial epithelial cells via nuclear factor-κB-dependent pathway

2003 ◽  
Vol 284 (6) ◽  
pp. L1055-L1062 ◽  
Author(s):  
Hajime Takizawa ◽  
Shinji Abe ◽  
Hitoshi Okazaki ◽  
Tadashi Kohyama ◽  
Isamu Sugawara ◽  
...  
Keyword(s):  
Gene Expression ◽  
Epithelial Cells ◽  
Diesel Exhaust ◽  
Airway Epithelial Cells ◽  
Diesel Exhaust Particles ◽  
Pyrrolidine Dithiocarbamate ◽  
Airway Epithelial ◽  
Bronchial Epithelial ◽  
Exhaust Particles

Fine particles derived from diesel engines, diesel exhaust particles (DEP), have been shown to augment gene expression of several inflammatory cytokines in human airway epithelial cells in vitro. However, it remains unclear whether or not DEP have any effect on the expression and production of eotaxin, an important chemokine involved in eosinophil recruitment into the airways. We studied the effects of DEP by using a conventional suspended DEP and by a recently established in vitro cell exposure system to diesel exhaust (Abe S, Takizawa H, Sugawara I, and Kudoh S, Am J Respir Cell Mol Biol 22: 296–303, 2000). DEP showed a dose-dependent stimulatory effect on eotaxin production by normal human peripheral airway epithelial cells as well as by bronchial epithelial cell line BET-1A as assessed by specific ELISA. mRNA levels increased by DEP were shown by RT-PCR. DEP showed an additive effect on IL-13-stimulated eotaxin expression. DEP induced NF-κB activation by EMSA as previously reported but did not induce signal transducer and activator of transcription (STAT) 6 activation according to Western blot analysis. Finally, antioxidant agents ( N-acetyl cysteine and pyrrolidine dithiocarbamate), which inhibited NF-κB activation but failed to affect STAT6 activation, almost completely attenuated DEP-induced eotaxin production, whereas these agents failed to attenuate IL-13-induced eotaxin production. These findings suggested that DEP stimulated eotaxin gene expression via NF-κB-dependent, but STAT6-independent, pathways.

scholarly journals Autocrine Acetylcholine, Induced by IL-17A via NFκB and ERK1/2 Pathway Activation, Promotes MUC5AC and IL-8 Synthesis in Bronchial Epithelial Cells

2016 ◽  
Vol 2016 ◽  
pp. 1-16 ◽  
Author(s):  
Angela Marina Montalbano ◽  
Giusy Daniela Albano ◽  
Anna Bonanno ◽  
Loredana Riccobono ◽  
Caterina Di Sano ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Anticholinergic Drug ◽  
Airway Epithelial Cells ◽  
Bronchial Epithelial Cells ◽  
Choline Uptake ◽  
Airway Epithelial ◽  
Related Activity ◽  
Bronchial Epithelial ◽  
Pathway Activation

IL-17A is overexpressed in the lung during acute neutrophilic inflammation. Acetylcholine (ACh) increases IL-8 and Muc5AC production in airway epithelial cells. We aimed to characterize the involvement of nonneuronal components of cholinergic system on IL-8 and Muc5AC production in bronchial epithelial cells stimulated with IL-17A. Bronchial epithelial cells were stimulated with recombinant human IL-17A (rhIL-17A) to evaluate the ChAT expression, the ACh binding and production, the IL-8 release, and the Muc5AC production. Furthermore, the effectiveness of PD098,059 (inhibitor of MAPKK activation), Bay11-7082 (inhibitor of IkBαphosphorylation), Hemicholinium-3 (HCh-3) (choline uptake blocker), and Tiotropium bromide (Spiriva®) (anticholinergic drug) was tested in ourin vitromodel. We showed that rhIL-17A increased the expression of ChAT, the levels of ACh binding and production, and the IL-8 and Muc5AC production in stimulated bronchial epithelial cells compared with untreated cells. The pretreatment of the cells with PD098,059 and Bay11-7082 decreased the ChAT expression and the ACh production/binding, while HCh-3 and Tiotropium decreased the IL-8 and Muc5AC synthesis in bronchial epithelial cells stimulated with rhIL-17A. IL-17A is involved in the IL-8 and Muc5AC production promoting, via NFκB and ERK1/2 pathway activation, the synthesis of ChAT, and the related activity of autocrine ACh in bronchial epithelial cells.

scholarly journals Hypoxia and the hypoxia-regulated transcription factor HIF-1α suppress the host defence of airway epithelial cells

2017 ◽  
Vol 23 (4) ◽  
pp. 373-380 ◽  
Author(s):  
Markus Polke ◽  
Frederik Seiler ◽  
Philipp M Lepper ◽  
Andreas Kamyschnikow ◽  
Frank Langer ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Epithelial Cells ◽  
Inflammatory Mediators ◽  
Airway Epithelial Cells ◽  
Innate Immune ◽  
Epithelial Cell Line ◽  
Airway Epithelial ◽  
Immune Functions ◽  
Low Oxygen ◽  
Bronchial Epithelial

Chronic diseases of the respiratory tract, such as cystic fibrosis, are associated with mucosal and systemic hypoxia. Innate immune functions of airway epithelial cells are required to prevent and control infections of the lung parenchyma. The transcription factor hypoxia-inducible factor 1α (HIF-1α) regulates cellular adaptation to low oxygen conditions. Here, we show that hypoxia and HIF-1α regulate innate immune mechanisms of cultured human bronchial epithelial cells (HBECs). Exposure of primary HBECs to hypoxia or the prolyl hydroxylase inhibitor dimethyloxaloylglycine (DMOG) resulted in a significantly decreased expression of inflammatory mediators (IL-6, IFN-γ-induced protein 10) in response to ligands for TLRs (flagellin, polyI:C) and Pseudomonas aeruginosa, whereas the expression of inflammatory mediators was not affected by hypoxia or DMOG in the absence of microbial factors. Small interfering RNA-mediated knockdown of HIF-1α in HBECs and in the bronchial epithelial cell line Calu-3 resulted in increased expression of inflammatory mediators. The inflammatory response was decreased in lungs of mice stimulated with inactivated P. aeruginosa under hypoxia. These data suggest that hypoxia suppresses the innate immune response of airway epithelial cells via HIF-1α.

β-Adrenergic agonist modulation of monocyte adhesion to airway epithelial cells in vitro

2000 ◽  
Vol 278 (1) ◽  
pp. L139-L147 ◽  
Author(s):  
Debra J. Romberger ◽  
Peggy Heires ◽  
Stephen I. Rennard ◽  
Todd A. Wyatt
Keyword(s):  
Cell Adhesion ◽  
Epithelial Cells ◽  
Airway Epithelial Cells ◽  
Bronchial Epithelial Cells ◽  
Airway Epithelial ◽  
Human Bronchial Epithelial Cells ◽  
Adrenergic Agonist ◽  
Bronchial Epithelial

β-Adrenergic agonists are commonly used in the treatment of obstructive airway diseases and are known to modulate cAMP-dependent processes of airway epithelial cells. However, little is known regarding the ability of cAMP-dependent mechanisms to influence cell-cell interactions within the airway. Thus we investigated the role of the β-adrenergic agonist isoproterenol in modulating the ability of human bronchial epithelial cells to support the adhesion of THP-1 cells, a monocyte/macrophage cell line, in vitro. We demonstrated that pretreatment of human bronchial epithelial cells (HBECs) with 10 μM isoproterenol or 100 μM salbutamol augments the adhesion of fluorescently labeled THP-1 cells to HBEC monolayers by ∼40–60%. The increase in THP-1 cell adhesion occurred with 10 min of isoproterenol pretreatment of HBECs and gradually declined but persisted with up to 24 h of isoproterenol exposure. Exposure of THP-1 cells to isoproterenol or salbutamol before the adhesion assays did not result in an increase in adhesion to HBECs, suggesting that the isoproterenol modulation was primarily via changes in epithelial cells. A specific protein kinase A inhibitor, KT-5720, inhibited subsequent isoproterenol augmentation of THP-1 cell adhesion, further supporting the role of cAMP-dependent mechanisms in modulating THP-1 cell adhesion to HBECs.

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