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Airway epithelial cells, the first barrier of the respiratory tract, play an indispensable role in innate immunity. Integrin β4 (ITGB4) is a structural adhesion molecule that is involved in the pathological progression of acute inflammatory diseases and is downregulated in asthmatic patients. Research has shown that endothelial ITGB4 has proinflammatory properties in acute lung injury (ALI). However, the role of epithelial ITGB4 in a murine ALI model is still unknown. This study investigated the role of ITGB4 in lipopolysaccharide (LPS)-induced ALI. We found that ITGB4 in the airway epithelium had remarkably increased after the introduction of LPS in vivo and in vitro. Then, we constructed airway epithelial cell-specific ITGB4 knockout (ITGB4 -/- ) mice to study its role in ALI. At a time point of 12 h after the tracheal injection of LPS, ITGB4 -/- mice showed increased macrophages (mainly M1-type macrophages) and neutrophil infiltration into the lungs; inflammation-related proteins including interleukin (IL)-6, tumor necrosis factor, and IL-17A were significantly elevated compared to their levels in ITGB4 +/+ mice. Furthermore, we investigated the role of ITGB4 in the anti-inflammatory response. Intriguingly, in the ITGB4 -/-  + LPS group, we found significantly reduced expression of anti-inflammatory factors, including IL-10 messenger RNA (mRNA) and ARG-1 mRNA. We also observed that monocyte chemotactic protein (MCP-1) increased significantly both in vivo and in vitro. Airway epithelium activates macrophages, most likely driven by MCP-1, which we confirmed in the coculture of epithelia and macrophages. These phenomena indicate that ITGB4 in airway epithelial cells plays an important role in the process of inflammation and activation of macrophages in ALI. Overall, these data demonstrated a novel link between airway epithelial ITGB4 and the inflammatory response in LPS-induced ALI., (© 2021 Wiley Periodicals LLC.)

In this study we tried to explore whether calcitonin gene-related peptide (CGRP) regulates the potential antigen uptaking ability of human bronchial epithelial cells (HBECs) and promoting the differentiation of Th1/Th2. We found that CGRP increased the uptake of fluorescein isothiocyanate labeled ovalbumin (FITC-OVA) by HBECs using fluorescence microscopy and flow cytometry analysis. MTT assay showed that T cells proliferated in a dose-dependent manner in the presence of OVA-pretreated HBECs and CGRP inhibited the proliferation of T cells. CGRP decreased secretion of IFN-γ, while it had no influence on secretion of IL-4 by ELISA. Our data suggest that CGRP enhanced HBECs antigen uptake ability and inhibits HBECs induced T cells proliferation.

Oxidative stress induced by hypoxia/ischemia resulted in the excessive reactive oxygen species (ROS) and the relative inadequate antioxidants. As the initial barrier to environmental pollutants and allergic stimuli, airway epithelial cell is vulnerable to oxidative stress. In recent years, the antioxidant effect of hydrogen sulfide (H 2 S) has attracted much attention. Therefore, in this study, we explored the impact of H 2 S on CoCl 2 -induced cell injury in 16HBE14o- cells. The effect of CoCl 2 on the cell viability was detected by Cell Counting Kit (CCK-8) and the level of ROS in 16HBE14o- cells in response to varying doses (100-1000 μ mol/L) of CoCl 2 (a common chemical mimic of hypoxia) was measured by using fluorescent probe DCFH-DA. It was shown that, in 16HBE14o- cells, CoCl 2 acutely increased the ROS content in a dose-dependent manner, and the increased ROS was inhibited by the NaHS (as a donor of H 2 S). Moreover, the calcium ion fluorescence probe Fura-2/AM and fluorescence dye Rh123 were used to investigate the intracellular calcium concentration ([Ca 2+ ] i ) and mitochondria membrane potential (MMP) in 16HBE14o- cells, respectively. In addition, we examined apoptosis of 16HBE14o- cells with Hoechst 33342. The results showed that the CoCl 2 effectively elevated the Ca 2+ influx, declined the MMP, and aggravated apoptosis, which were abrogated by NaHS. These results demonstrate that H 2 S could attenuate CoCl 2 -induced hypoxia injury via reducing ROS to perform an agonistic role for the Ca 2+ influx and MMP dissipation.

Our previous studies provided evidence that bombesin receptor-activated protein (BRAP), encoded by C6ORF89, is widely expressed in human airway epithelial cells and may play a role in the stress response of lung epithelia. In this study, we demonstrated that BRAP has a regulatory effect on NF-κB transcriptional activity in cultured human bronchial epithelial cells (HBECs). BRAP overexpression by gene transfer inhibited both basal and inducible NF-κB transcriptional activity in HBECs, whereas BRAP knockdown had the opposite effect. BRAP was shown to regulate NF-κB activity by enhancing histone deacetylase (HDAC) activity. In addition, BRAP might increase HDAC activity that leads to NF-κB activation via its putative C-terminal domain. Our study suggests that the BRAP protein is an important regulator of immune and inflammatory responses in the human airway epithelium., (© 2015 Wiley Periodicals, Inc.)

Bombesin receptor activated protein (BRAP) was identified in a bacterial two-hybrid screen for proteins interacting with bombesin receptor subtype-3 (BRS-3). We found that BRAP is widely expressed in the airway epithelium of human lungs and may play a role during the stress response of lung epithelium. In this work, we explored the potential roles of BRAP in the antigen presenting function of human bronchial epithelial cells (HBECs). Overexpression of a BRAP recombinant protein in a human bronchial epithelial cell line resulted in a reduction of FITC-OVA uptake by HBECs, which indicated that the antigen uptake ability is inhibited. The analysis of the protein expression of surface molecules including B7 homologs and the major histocompactability complex (MHC) class II molecules showed that the expression levels of HLA-DR and B7DC increased while the levels of B7-H1 and B7.2 decreased. Since those surface molecules are all related to antigen presenting process, the altered expression pattern of those molecules provides further evidence showing that BRAP overexpression leads to a change in antigen presenting function of HBECs. Moreover, overexpression of BRAP in HBECs caused a decrease of co-cultured lymphocytes proliferation and a changed pattern of cytokines produced by lymphocytes in the presence of FITC-OVA, which indicated that changes in the maturation pattern and functions of co-cultured lymphocytes were induced by BRAP overexpression. Overall, our results suggested that overexpression of BRAP may play a role during the antigen presenting process of bronchial epithelium by inhibiting the antigen uptake ability of bronchial epithelial cells., (Copyright © 2012 Wiley Periodicals, Inc.)

Adhesion molecules play important roles in airway hyperresponsiveness or airway inflammation. Our previous study indicated catenin alpha-like 1 (CTNNAL1), an alpha-catenin-related protein, was downregulated in asthma patients and animal model. In this study, we observed that the expression of CTNNAL1 was increased in lung tissue of the ozone-stressed Balb/c mice model and in acute ozone stressed human bronchial epithelial cells (HBEC). In order to identify the possible DNA-binding proteins regulating the transcription of CTNNAL1 gene in HBEC, we designed 8 oligo- nucleotide probes corresponding to various regions of the CTNNAL1 promoter in electrophoretic mobility shift assays (EMSA). We detected 5 putative transcription factors binding sites within CTNNAL1 promoter region that can recruit LEF-1, AP-2α and CREB respectively by EMSA and antibody supershift assay. Chromatin immunoprecipitation (ChIP) assay verified that AP-2 α and LEF-1 could be recruited to the CTNNAL1 promoter. Therefore we further analyzed the functions of putative AP-2 and LEF-1 sites within CTNNAL1 promoter by site-directed mutagenesis of those sites within pGL3/FR/luc. We observed a reduction in human CTNNAL1 promoter activity of mutants of both AP-2α and LEF-1 sites. Pre-treatment with ASOs targeting LEF-1and AP-2α yielded significant reduction of ozone-stress-induced CTNNAL1 expression. The activation of AP-2α and LEF-1, followed by CTNNAL1 expression, showed a correlation during a 16-hour time course. Our data suggest that a robust transcriptional CTNNAL1 up-regulation occurs during acute ozone-induced stress and is mediated at least in part by ozone-induced recruitments of LEF-1 and AP-2α to the human CTNNAL1 promoter.

Cystic fibrosis transmembrane conductance regulator (CFTR) is an apical membrane chloride channel critical to the regulation of fluid, chloride, and bicarbonate transport in epithelia and other cell types. The most common cause of cystic fibrosis (CF) is the abnormal trafficking of CFTR mutants. Therefore, understanding the cellular machineries that transit CFTR from the endoplasmic reticulum to the cell surface is important. Vasoactive intestinal polypeptide (VIP) plays an important role in CFTR-dependent chloride transport. The present study was designed to observe the affection of VIP on the trafficking of CFTR, and channel gating in human bronchial epithelium cells (HBEC). Confocal microscopy revealed CFTR immunofluorescence extending from the apical membrane deeply into the cell cytoplasm. After VIP treatment, apical extension of CFTR immunofluorescence into the cell was reduced and the peak intensity of CFTR fluorescence shifted towards the apical membrane. Western blot showed VIP increased cell surface and total CFTR. Compared with the augmented level of total CFTR, the surface CFTR increased more markedly. Immunoprecipitation founded that the mature form of CFTR had a marked increase in HBEC treated with VIP. VIP led to a threefold increase in Cl(-) efflux in HBEC. Glibenclamide-sensitive and DIDS-insensitive CFTR Cl(-) currents were consistently observed after stimulation with VIP (10(-8) mol/L). The augmentation of CFTR Cl(-) currents enhanced by VIP (10(-8) mol/L) was reversed, at least in part, by the protein kinase A (PKA) inhibitor, H-89 and the protein kinase C (PKC) inhibitor, H-7, suggesting PKA and PKC participate in the VIP-promoted CFTR Cl(-) currents., (Copyright © 2010 Wiley-Liss, Inc.)

Bombesin receptor subtype 3 (BRS-3), the orphan bombesin receptor, may play a role in the regulation of stress responses in lung and airway epithelia. Bombesin receptor activated protein (BRAP )is a novel protein we found in our previous study which interacts with BRS-3. This study was designed to observe the subcellular location and wound repair function of BRAP in human bronchial epithelial cells (HBECs). BRAP ORF was amplified by RT-PCR and ligated to pEGFP-C1 vector, and then the recombinant plasmid pEGFP-C1-BRAP was transfected into Hela cells. The location of BRAP protein was observed by laser confocal microscope, and the expression of it was analyzed by Western-blot. At the same time,we built the recombinant plasmid pcDNA3.1(+)-BRAP, transfected it into HBECs and observed its impact on cell cycle and wound repair of HBECs. The results showed that BRAP locates in membrane and cytoplasm and increases significantly in transfected cells. Flow cytometry results demonstrated that the recombinant plasmid increases S phase plus G2 phase of cell cycle by 25%. Microscopic video analysis system showed that the repair index of wounded HBECs increases by 20% through stable expression of BRAP. The present study demonstrated that BRAP locates in the membrane and cytoplasm, suggesting that this protein is a cytoplasm protein, which promotes cell cycle and wound repair of HBECs.

Integrin beta 4 (ITGB4) is a structural adhesion molecule which engages in maintaining the integrity of airway epithelial cells. Its specific cytomembrane structural feature strongly indicates that ITGB4 may engage in many signaling pathways and physiologic processes. However, in addition to adhesion, the specific biologic significance of ITGB4 in airway epithelial cells is almost unknown. In this article, we investigated the expression and functional properties of ITGB4 in airway epithelial cells in vivo and in vitro. Human bronchial epithelial cell line (16HBE14O-cells) and primary rat tracheal epithelial cells (RTE cells) were used to determine ITGB4 expression under ozone tress or mechanical damage, respectively. An ovalbumin (OVA)-challenged asthma model was used to investigate ITGB4 expression after antigen exposure in vivo. In addition, an ITGB4 overexpression vector and ITGB4 silence virus vector were constructed and transfected into RTE cells. Then, wound repair ability and anti-oxidation capacity was evaluated. Our results demonstrated that, on the edge of mechanically wounded cell areas, ITGB4 expression was increased after mechanical injury. After ozone stress, upregulation expression of ITGB4 was also detected. In the OVA-challenged asthma model, ITGB4 expression was decreased on airway epithelial cells accompanying with structural disruption and damage of anti-oxidation capacity. Besides, our study revealed that upregulation of ITGB4 promotes wound repair ability and anti-oxidative ability, while such abilities were blocked when ITGB4 was silenced. Taken together, these results showed that ITGB4 was a new interesting molecule involved in the regulation of wound repair and anti-oxidation processes for airway epithelial cells.

To investigate abnormalities of cystic fibrosis transmembrane conductance regulator (CFTR) expression in chronic inflammatory airway diseases and its regulation mechanisms, the present study was designed to observe the expression of CFTR, CFTR chloride current and the possible relevant signal pathways in in vitro and in vivo bronchial epithelium by using real-time PCR, immunofluorescence, Western blot and whole cell patch-clamp. The results demonstrated that CFTR staining was decreased in rat airway epithelium under ozone stress. Ozone stress also down-regulated CFTR protein and mRNA expression and CFTR chloride current in cultured human bronchial epithelial cells (HBEC). STAT1 signal pathway was checked to investigate the signal mechanism. It was found that pretreatment with STAT1 inhibitor attenuated the down-regulated CFTR expression induced by ozone stress. We also observed that ozone stress accelerated the phosphorylation of STAT1 in HBEC, which could be influenced by some signaling molecules related to the early transduction of cellular stress. Furthermore, reactive oxygen species inhibitors N-acetylcysteine and nitric oxide synthase inhibitor aminoguanidine increased the expression of CFTR. Ozone stress could down-regulate the expression of CFTR and decrease CFTR chloride current in HBEC. The signal mechanism which referred to cascade events in cells included early oxidative stress signal transmission molecules, and subsequently transcription modulator STAT1.

Antigen presenting is the initial step of the immune responses. In order to verify that human bronchial epithelial cells (HBECs) can express antigen presentation molecules, which can be modulated by intrapulmonary regulatory peptides, the present study was designed to examine the expressions of human leukocyte antigen DR (HLA-DR), CD80 and CD86 in resting or ozone-stressed HBECs by using immunocytochemistry and flow cytometry analysis. The results showed that HBECs expressed HLA-DR, CD80 and the expressions of HLA-DR and CD80 molecules were down-regulated under ozone stress. While VIP, P3513 and CGRP upregulated the expression of HLA-DR in resting or ozone-stressed HBECs, they had different effects on CD80 expression. VIP did not influence the expression of CD80 under resting state, but increased the expression of CD80 under ozone stress. CGRP decreased CD80 expression in resting HBECs, but increased CD80 expression in ozone-stressed HBECs. P3513 increased CD80 expression in resting HBECs, but decreased CD80 expression in ozone-stressed HBECs. The expression of CD86 was absent in resting or ozone-stressed HBECs. The results obtained demonstrate that HBECs have the capability to act as antigen presenting cells and the expression of HLA-DR and costimulatory molecules can be modulated by intrapulmonary regulatory peptides.

Adhesion molecules play vital roles in airway hyperresponsiveness (AHR) or airway inflammation. Our previous study indicated that adhesion molecule catenin alpha-like 1 (CTNNAL1) is relevant closely to asthma susceptibility, but its biological function or significance is still unclear. In the present study, we observed the temporal and spatial distribution of CTNNAL1 expression in mouse lung tissue with the OVA-sensitized asthma model and found that the level of CTNNAL1 mRNA showed a prominent negative correlation with pulmonary resistance (R(L)). To study the function of CTNNAL1 in airway, effects of CTNNAL1 on proliferation and wound repair activity of human bronchial epithelial cells (HBEC) was investigated with antisense oligonucleotide (ASO) technique. The results showed that: (1) CTNNAL1 ASO could decelerate the repairing velocity and proliferation of HBEC; (2) CTNNAL1 expression was increased on the edge cells of mechanic wounded area in culture; (3) extracellular matrix component fibronectin (Fn) obviously promoted wound repair activity and proliferation of HBEC, which could be blocked by CTNNAL1 ASO; (4) Western blot showed that Fn could promote FAK phosphorylation, which also be inhibited by CTNNAL1 ASO. In conclusion, the level of CTNNAL1 mRNA expression is highly correlated to airway resistance; CTNNAL1 may contribute to the wound repair and proliferation of HBEC. Furthermore, it may serve to Fn mediated cell-extracellular adhesion and its signal transduction., (Copyright 2007 Wiley-Liss, Inc.)

It is commonly accepted that airway hyperresponsiveness (AHR) is a chronic airway inflammation although the exact mechanism of its pathogenesis is still unclear. In the past ten years, an epithelial defect hypothesis has gradually gained supports from the main stream. Airway epithelium is no longer considered only as a simple mechanic barrier but an active interface between the inner and outer environment. Bronchial epithelial cells play a critical role in maintenance of homeostasis in the airway local microenvironment through a wide range of physiologic functions including anti-oxidation, exocrine/endocrine secretions, mucus production and antigen presentation under health and stressed/inflamed/injured conditions. It is reasonably hypothesized that disruption of these functional processes or defects in airway epithelium integrity may be the initial steps leading to airway hyperresponsiveness such as in asthma and chronic obstructive pulmonary disease.

Previously, we found that bombesin receptor subtype 3 (BRS-3) significantly increased in an ozone-stressed airway hyperresponsiveness animal model and resulted in induced wound repair and protection from acute lung injury. In the present study, we determined molecular mechanisms of BRS-3 regulation in human BECs (bronchial epithelial cells) in response to ozone stress. Ten oligonucleotide probes corresponding to various regions of the BRS-3 promoter were used in EMSA (electrophoretic mobilityshift assays). Four were found to have an enhanced mobility shift with extracts from ozone-stressed cells. On the basis of the assay of mutated probes binding with extracts and antibody supershift, they were verified as MTF-1 (metal-regulatory-element-binding transcription factor-1), PPARalpha (peroxisome-proliferator-activated receptor alpha), AP-2alpha (activator protein 2alpha) and HSF-1 (heat-shock factor 1). Next, ChIP (chromatin immunoprecipitation) assay, site-directed mutagenesis technology and antisense oligonucleotide technology were used to observe these transcription factors associated with the BRS-3 promoter. Only AP-2alpha and PPARalpha increased ozone-inducible DNA binding on the BRS-3 promoter and BRS-3 expression. The time courses of AP-2alpha and PPARalpha activation, followed by BRS-3 expression, were also examined. It was shown that ozone-inducible BRS-3 expression and AP-2alpha- and PPARalpha-binding activity correlated over a 48 h period. The translocation of PPARalpha was observed by immunofluorescence assay, which showed that PPARalpha nuclear translocation increased after ozone exposure. Our data suggest that AP-2alpha and PPARalpha may be especially involved in this ozone-inducible up-regulation mechanism of BRS-3 expression.

The present study was designed to investigate the role of bombesin receptor subtype 3 (BRS-3) in airway wound repair. The results showed that: (1) There was few expression of BRS-3 mRNA in the control group. In contrast, the expression of BRS-3 mRNA was gradually increased in the early 2 days, and peaked on the fourth day, and then decreased in the ozone-stressed AHR animal. BRS-3 mRNA was distributed in the ciliated columnar epithelium, monolayer columnar epithelium cells, scattered mesenchymal cells and Type II alveolar cells; (2) The wound repair and proliferation of bronchial epithelial cells (BECs) were accelerated in a concentration-dependent manner by BRS-3 activation with P3513, which could be inhibited by PKA inhibitor H89. The study demostrated that activation of BRS-3 may play an important role in wound repair of AHR.

Objective: To investigate the role and mechanism of bombesin receptor subtype 3 (BRS-3) in the proliferation and protection against injury of human brochial epithelial cells (HBECs)., Methods: Effect of P3513 (a specific agonist of BRS-3) on the proliferation of HBECs was observed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method; the release rate of 3H-Udr, and LDH activity, catalase activity, and the expression of cadherin and integrin beta1 were also analyzed under O3 stress with or without P3513 treatment., Results: The proliferation of HBECs was accelerated by P3513 in a concentration-dependent manner (10(-9) approximately 10(-7) mol/L). Ozone stress could promote the release rate of 3H-Udr, and LDH activity, which could be inhibited by P3513. P3513 could promote the activity of catalase. The effect of proliferation and protection against injury caused by P3513 could be inhibited by W7 (calmodulin inhibitor), PD98059 (tyrosin kinase inhibitor) and H89 (PKA inhibitor). P3513 could stimulate the expression of caderin and integrinbeta1 of ozone-stressed HBECs., Conclusion: Activation of BRS-3 caused by P3513 may play an important role in protecting HBECs from oxidant injury, and the signal pathway is possibly relevant to Ca2+, MEK and PKA.

A complex network of regulatory neuropeptides controls airway inflammation reaction, in which airway epithelial cells adhering to and activating leukocytes is a critical step. To study the effect of intrapulmonary regulatory peptides on adhesion of polymorphonuclear leukocytes (PMNs) to bronchial epithelial cells (BECs) and its mechanism, several regulatory peptides including vasoactive intestinal peptide (VIP), epidermal growth factor (EGF), endothelin-1 (ET-1) and calcitonin gene-related peptide (CGRP), were investigated. The results demonstrated that VIP and EGF showed inhibitory effects both on the secretion of IL-1, IL-8 and the adhesion of PMNs to BECs, whereas ET-1 and CGRP had the opposite effect. Anti-intercellular adhesion molecule-1 (ICAM-1) antibody could block the adhesion of PMNs to ozone-stressed BECs. Using immunocytochemistry and reverse transcription-polymerase chain reaction (RT-PCR), it was shown that VIP and EGF down-regulated the expression of ICAM-1 in BECs, while ET-1 and CGRP up-regulated ICAM-1 expression. NF-kappaB inhibitor MG132 blocked ICAM-1 expression induced by ET-1 and CGRP. Furthermore, in electric mobility shift assay (EMSA), VIP and EGF restrained the binding activity of NF-kappaB to the NF-kappaB binding site within the ICAM-1 promoter in ozone-stressed BECs, while CGRP and ET-1 promoted this binding activity. IkappaB degradation was consistent with NF-kappaB activation. These observations indicate that VIP and EGF inhibit inflammation, while ET-1 and CGRP enhance the inflammation reaction.

We have previously shown that the binding of integrins with extracellular matrix component fibronectin (Fn) can improve the ability of bronchial epithelial cells (BECs) in resisting oxidant injury by up-regulating the activity of catalase and increasing the content of GSH. However, the molecular mechanism or its signaling pathway of this protection is still unclear. In order to examine the intracellular signaling mechanism activated by Fn-integrin binding reaction, the present study investigated the mRNA expression of catalase in primary cultured rabbit BECs using RT-PCR based on a cell-injury model made with ozone exposure. The product bands of target gene CAT were checked with Southern blot and oligonucleotide probe hybridization. The results showed that Fn (10 microg/ml) promoted the catalase mRNA transcription (P<0.01). This effect was abolished either by protein-tyrosine kinase inhibitor genistein or calmodulin inhibitor W(7) (P<0.01). These results indicate that the promotion of catalase activity induced by Fn-integrin reaction is partly due to the elevation of catalase mRNA transcription, and that its signalling are possibly relevant to tyrosine phosphorylation or calmodulin pathway.

Intercellular adhesion molecule-1 (ICAM-1) is an important adhesion molecule leading to adhesion between cells; NF-kappaB, being universally distributed in the organism, is an important nuclear transcription factor leading to a rapid response to the stimuli. Line of evidence have shown that ICAM-1 transcription and NF-kappaB activation is an important step of inflammatory reaction. To testify that intrapulmonary regulatory peptides modulate inflammatory lesion of bronchial epithelial cells (BECs) through their effect on ICAM-1 expression and nuclear factor kappaB (NF-kappaB) activation, we used immunocytochemistry, RT-PCR, and electrophoretic mobility-shift assay (EMSA) to determine the ICAM-1 expression and NF-kappaB activity in BECs. The effects of NF-kappaB inhibitor MG-132 on ICAM-1 expression were also observed. The results showed that vasoactive intestinal peptide (VIP) and epidermal growth factor (EGF) decreased ICAM-1 expression in O(3)-stressed BECs, while endothelin-1 (ET-1) and calcitonin gene-related peptides (CGRP) increased ICAM-1 expression in resting BECs. MG-132 blocked ICAM-1 expression induced by O(3), ET-1 and CGRP. The results obtained by using EMSA confirmed that VIP and EGF restrained the activation of NF-kappaB in O(3)-stressed BECs; CGRP and ET-1 promoted activation of NF-kappaB. These observations indicate that VIP and EGF abated the injury by means of down-regulatory effects on ICAM-1 transcription and NF-kappaB activation, while ET-1 and CGRP enhanced the inflammation reaction by an up-regulatory effect. It is suggested that a developing and intensive airway inflammation correlates closely with a persistent expression of ICAM-1 and repeated activation of NF-kappaB.

Objective: To determine into the inflammation process of bronchial epithelial cells (BECs) and observe the modulatory effect of some protective factors on BECs., Methods: We built the cell injury model with ozone and observed the effect of ozone stress on polymorphonuclear loeukocyte (PMNs) and eosinophil (EOS) adhesion to BECs. The effect of intercellular adhesion molecule (ICAM-1) antibody on the adhesion and ICAM-1 protein expression of BECs was observed at the same time., Results: O3 increased the adhesion of BECs to PMNs and EOS. Adhesion could be blocked by ICAM-1 antibody, suggesting ICAM-1 was the principal molecule leading PMNs and EOS adhesion to BECs. The immunocytochemistry assay indicated that O3 increased the expression of ICAM-1 protein of BECs., Conclusion: Ozone stress leads to inflammation, and the principal adhesion molecule is ICAM-1 resulting in adhesion between BECs and PMNs, and between BECs and EOS.

To explore the role of regulatory peptides in the secretion of bronchial epithelial cells (BECs), we observed the effects of four peptides, i.e.vasoactive intestinal peptide (VIP), epidermal growth factor (EGF), endothelin-1 (ET-1), and calcitonin gene-related peptide (CGRP), on the secretion of ILs from unstimulated or O3-stressed BECs. The results of the experiments showed that VIP exerted an inhibitory effect on the secretion of IL-1 and IL-8 from unstimulated and O3-stressed BECs, VIP also decreased the secretion of IL-5 from O3-stressed BECs; EGF promoted secretion of IL-1 and IL-8 from unstimulated BECs, but decreased the secretion of ILs from O3-stressed BECs; ET-1 and CGRP enhanced the secretion of IL-1, IL-5, and IL-8 from unstimlated BECs, CGRP also increased the secretion of ILs from O3-stressed BECs. The results obtained demonstrate that intrapulmonary regulatory peptides modulate the secretion of ILs from BECs, and may play an important part in transduction of inflammatory signals.

To investigate the influence of vasoactive intestinal peptide (VIP) on chemotaxis of bronchial epithelial cells (BECs). Rabbit chemotactic migration of primary BEC was assessed in a blind-well Boyden chamber. Radioimmunoassay and radio-ligand affinity analysis were used for determining VIP secretion and vasoactive intestinal peptide receptor (VIPR) expression. The results showed: (1) the method for determining chemotaxis of BECs by using insulin as chemotactic factor was stable and reproducible (r=0.9703, P<0.01). (2) VIP (0.001-1 micromol/L) elicited chemotaxis of BECs which was substantial and concentration-dependent. The effects of VIP were inhibited by W-7 and H-7 (P<0.01). (3) Heat stress enhanced the secretion of VIP (P<0.01) and upregulated the expression of VIPR on BECs (P<0.05). These results indicate that VIP in the lungs may play an important role in the repair of damaged epithelium, accelerating restoration of the airway to its normal state. Calmodulin and protein kinase C may be involved in the signal transduction of VIP effects.

To explore the roles of regulatory peptides in the process of various anaphylactic inflammation of the airway, we observed the influence of four peptides, i.e., vasoactive intestinal peptide (VIP), epidermal growth factor (EGF), endothelin-1 (ET-1), and calcitonin gene-related peptide (CGRP), on the adhesion of eosinophil (EOS) to unstimulated and O(3)-stressed bronchial epithelial cells (BEC). From the experiments we observed that VIP and EGF decreased EOS adherence to O(3)-stressed BEC and downregulated airway inflammation; ET-1 and CGRP increased the adhesion of EOS to BEC in the inflammatory process; and CGRP aggravated O(3)-stressed reactions. The effects of ET-1 and CGRP were inhibited by W(7)and H(7). Anti-ICAM-1 antibody inhibited the adhesion of EOS to BEC, which brings to light that EOS adherence to BEC may be related to the expression of ICAM-1 of BEC.

Neonatal respiratory system disease is closely associated with embryonic lung development. Our group found that integrin β4 (ITGB4) is downregulated in the airway epithelium of asthma patients. Asthma is the most common chronic respiratory illness in childhood. Therefore, we suspect whether the deletion of ITGB4 would affect fetal lung development. In this study, we characterized the role of ITGB4 deficiency in bronchopulmonary dysplasia (BPD). ITGB4 was conditionally knocked out in CCSP‐rtTA, Tet‐O‐Cre and ITGB4f/f triple transgenic mice. Lung tissues at different developmental stages were collected for experimental detection and transcriptome sequencing. The effects of ITGB4 deficiency on lung branching morphogenesis were observed by fetal mouse lung explant culture. Deleting ITGB4 from the airway epithelial cells results in enlargement of alveolar airspaces, inhibition of branching, the abnormal structure of epithelium cells and the impairment of cilia growth during lung development. Scanning electron microscopy showed that the airway epithelial cilia of the β4ccsp.cre group appear to be sparse, shortened and lodging. Lung‐development‐relevant factors such as SftpC and SOX2 significantly decreased both mRNA and protein levels. KEGG pathway analysis indicated that multiple ontogenesis‐regulating‐relevant pathways converge to FAK. Accordingly, ITGB4 deletion decreased phospho‐FAK, phospho‐GSK3β and SOX2 levels, and the correspondingly contrary consequence was detected after treatment with GSK3β agonist (wortmannin). Airway branching defect of β4ccsp.cre mice lung explants was also partly recovered after wortmannin treatment. Airway epithelial‐specific deletion of ITGB4 contributes to lung developmental defect, which could be achieved through the FAK/GSK3β/SOX2 signal pathway. [ABSTRACT FROM AUTHOR]

Oxidative stress induced by hypoxia/ischemia resulted in the excessive reactive oxygen species (ROS) and the relative inadequate antioxidants. As the initial barrier to environmental pollutants and allergic stimuli, airway epithelial cell is vulnerable to oxidative stress. In recent years, the antioxidant effect of hydrogen sulfide (H2S) has attracted much attention. Therefore, in this study, we explored the impact of H2S on CoCl2-induced cell injury in 16HBE14o- cells. The effect of CoCl2 on the cell viability was detected by Cell Counting Kit (CCK-8) and the level of ROS in 16HBE14o- cells in response to varying doses (100–1000 μmol/L) of CoCl2 (a common chemical mimic of hypoxia) was measured by using fluorescent probe DCFH-DA. It was shown that, in 16HBE14o- cells, CoCl2 acutely increased the ROS content in a dose-dependent manner, and the increased ROS was inhibited by the NaHS (as a donor of H2S). Moreover, the calcium ion fluorescence probe Fura-2/AM and fluorescence dye Rh123 were used to investigate the intracellular calcium concentration ([Ca2+]i) and mitochondria membrane potential (MMP) in 16HBE14o- cells, respectively. In addition, we examined apoptosis of 16HBE14o- cells with Hoechst 33342. The results showed that the CoCl2 effectively elevated the Ca2+ influx, declined the MMP, and aggravated apoptosis, which were abrogated by NaHS. These results demonstrate that H2S could attenuate CoCl2-induced hypoxia injury via reducing ROS to perform an agonistic role for the Ca2+ influx and MMP dissipation. [ABSTRACT FROM AUTHOR]

Abstract: Epidemiological data indicate an increasing incidence of asthma in the obese individuals recent decades, while very little is known about the possible association between them. Here, we compared the roles of adipocyte-derived factors, including leptin, adiponectin and resistin on proliferation, wound repair and apoptosis in human bronchial epithelial cells (HBECs) which play an important role in the pathogenesis of asthma. The results showed that exogenous globular adiponectin (gAd) promoted proliferation, cell-cycle and wound repair of HBECs. This effect may be relevant to Ca2+/calmodulin signal pathway. Besides, gAd inhibited apoptosis induced by ozone and release of lactate dehydrogenase (LDH) of HBECs via regulated adipoR1 and reactive oxygen species. No effects of leptin or resistin on proliferation, wound repair and apoptosis of HBECs were detectable. These data indicate that airway epithelium is the direct target of gAd which plays an important role in protecting HBECs from mechanical or oxidant injuries and may have therapeutic implications in the treatment of asthma. [Copyright &y& Elsevier]

Abstract: In the present study, we investigated the effects of vasoactive intestinal peptide (VIP) on wound healing of bronchial epithelium. Wound healing of the mechanical damaged human bronchial epithelial cells (HBEC) was observed in the absence or presence of VIP. Effects of VIP on chemotactic migration, cell proliferation of HBEC were also tested. HBEC chemotaxis was assessed by the blind well chamber technique, the cell cycle was determined by flow cytometry, and cell proliferation was determined by measuring the expression of proliferating cell nuclear antigen Ki67. Effects of VIP on epithelial E-cadherins protein and mRNA were also measured by immunohistochemistry and RT-PCR. The results showed that VIP accelerated the recovery of wound area of HBEC. VIP increased the migration and proliferation of HBEC, and these effects were blocked by a VPAC1 receptor antagonist. VIP also increased the expression of E-cadherin mRNA and protein in HBEC, suggesting that protective effects of VIP on wound healing may be related to its ability to increase the expression of E-cadherin. In conclusion, VIP has protective effects against human bronchial epithelial cell damage, and the beneficial effects of VIP might be mediated, at least in part, by VPAC1, and associated with increased expression of E-cadherin. [Copyright &y& Elsevier]