Aeroallergen Der p 2 induces apoptosis of bronchial epithelial BEAS-2B cells via activation of both intrinsic and extrinsic pathway
© Lin et al. 2015
Received: 18 September 2015
Accepted: 14 December 2015
Published: 21 December 2015
Excessive apoptosis of airway epithelium is reported to induce airway remodeling and inhibited airway epithelium repair is highly associated with development of asthma and chronic obstructive pulmonary disease. Der p 2 is a major allergen derived from Dermatophagoides pteronyssinus and commonly causes airway hypersensitiveness and asthma; however, the connection between Der p 2 and epithelial apoptosis remains unclear. This study was aimed to explore whether Der p 2 induces apoptosis of airway epithelial cells and the underlying mechanisms.
Our results showed that recombinant Der p 2 (rDP2) inhibited cell growth and induced apoptosis of human bronchial epithelial cell BEAS-2B. Further investigation revealed that rDP2 increased intracellular reactive oxygen species, level of cytosolic cytochrome c and cleavage of caspase-9 and caspase-3. rDP2 also induced activation of p38 mitogen-activated protein kinase (P38) and c-Jun N-terminal kinase (JNK), and triggered proapoptotic signals including decrease of Bcl-2, increase of Bax and Bak, and upregulation of Fas and Fas ligand. In parallel, rDP2 inhibited glycogen synthase kinase 3beta and consequently enhanced degradation of cellular (FADD-like IL-1β-converting enzyme)-inhibitory protein (c-FLIP). Involvement of toll-like receptor (TLR)2 in rDP2-induced apoptosis was also demonstrated using specific small inhibitory RNA.
Our findings indicate that rDP2 suppresses cell growth and trigger apoptosis of BEAS-2B cells, which may attribute to induction of both intrinsic and extrinsic pathway via TLR2 and P38/JNK signaling and c-FLIP degradation. It suggests that Der p 2 may aggravate respiratory disorders through enhancement of apoptosis and the consequent airway injury.
Respiratory epithelium is a primary physical barrier which prevents invasion of pathogens and environmental factors such as allergens and air pollutants. In patients with chronic obstructive pulmonary disease and asthma, the epithelial barrier exhibits detachment of columnar ciliated cells, presence of epithelial cell aggregates in sputum, increased permeability to allergens and disturbed expression of the junction molecule at sites of epithelial detachment . These structural changes and functional disorders of airway epithelium are highly associated with increased cell apoptosis and enhanced interactions between the epithelial and immune and/or somatic cells underneath, which putatively induce airway remodeling and irreversible airway damages [2, 3]. In addition, it is evident that level of epithelial damage is correlated with degree of severity of airway hyperresponsiveness .
House dust mite (HDM) is one of the most important sources of indoor allergens and has been known as a predominant causative of respiratory disorders such as airway hypersensitiveness, asthma and exacerbation of lung function. HDM derived major allergens are categorized into two groups, proteolytic group-1 and non-proteolytic group-2 on the basis of IgE affinity . Recently, non-proteolytic group-2 HDM allergens including Der p 2 and Der f 2 have received increased interest in the role in activating immune response in asthma patients. Der p 2 has been demonstrated to share not only structural homology but also functional similarity with MD-2 protein that confers responsiveness to lipopolysaccharide in association with toll-like receptor (TLR) 4 . It has also been reported that Der p 2 aggravates respiratory airway disorder by induction of inflammatory cytokines and up-regulation of intercellular adhesion molecule-1 . However, association between Der p 2 and epithelial apoptosis has been rarely explored.
Apoptosis of airway epithelial cells plays a pivotal role in pathogenesis of chronic respiratory disorders . The increase in loss of epithelial integrity in the airway of asthmatics has been suggested as an imbalance between proliferation and apoptosis of epithelial cell, and the resultant decreased adhesion of the epithelial cells to the basement contributes to the epithelial layer shedding in airway . Previous studies have also implicated modulation of cell survival through apoptosis in the pathogenesis of chronic airway diseases [10, 11]. Although apoptosis is postulated as a critical cellular process in the development and progression of chronic airway diseases, influences of aeroallergens on epithelial apoptosis and the underlying signaling cascades remain sketchy. In the present study, we aimed to investigate effects of Der p 2 on airway epithelial cells with emphasis on apoptosis and the underlying mechanisms.
Expression and purification of recombinant Der p 2 (rDP2)
rDP2 was expressed in a pGEX-T vector with the N-terminal glutathione S-transferase (GST) moiety followed by Der p 2 in E. coli and purified using glutathione chromatography (Sigma-Aldrich, St. Louis, MO, USA) according to manufacturer’s instructions. Control protein GST was expressed and purified as same as rDP2. After filtrated with 0.22-μm sterile filter (Millipore, Bedford, MA), the purified proteins were quantitated using BCA protein assay kit (Pierce Biotechnology, Rockford, IL, USA) according to the manufacturer’s instructions and used for the following treatments.
Cell culture and treatments
Immortalized, nontumorigenic human bronchial epithelial cell line BEAS-2B was obtained from ATCC (CRL-2503) and cultured in complete growth medium consisting of RPMI1640 supplemented with 10 % fetal bovine serum (FBS). Cells were maintained at 37 ℃ in a humidified atmosphere with 5 % CO2 and subcultured according to resources’ instructions.
For protein expression, cells at a density of 5 × 105/mL were incubated with rDP2 at serial concentrations for 24 h. For kinase signaling analysis, cells at a density of 1 × 106/mL were incubated with rDP2 (20 μg/mL) for 30 min. Inhibition of p38 mitogen-activated protein kinase (P38) or c-Jun N-terminal kinase (JNK) was performed by pretreating cells with 10 μM SB203580 or SP600125 (Sigma-Aldrich) for 30 min, respectively. Inhibition of Akt activation was performed by pretreating cells with wortmannin at 200 nM for 30 min.
Cell viability assay
Cell viability was determined using 3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Cells were seeded at a density of 4 × 104 cells/well in a 24-well plate and cultured for 24 h. Then, the cells were treated with rDP2 at 5, 10, 20 and 40 μg/mL for 24 h. After the treatments, cells were washed with phosphate-buffered saline (PBS), and then incubated with 5 mg/mL MTT solution for 4 h. After removing the supernatant, isopropanol was added to solubilize the resulting formazan. Absorbance at 563 nm was measured for cell viability. Three independent experiments were performed for statistical analysis.
Cell cycle distribution analysis
Cells were seeded in 6-well plates at a density of 1 × 105 cells and treated with varying concentrations of rDP2 (10, 20, 40 μg/mL), GST (40 μg/mL) or vehicle for 24 h. After incubation, cells were trypsinized, washed twice with ice-cold PBS, and fixed overnight in 70 % ethanol at 4 ℃. Cells were then resuspended in PBS and treated with 1 mg/mL of RNase A and 1 mg/mL of propidium iodide in PBS for 45 min. Cell-cycle distribution was evaluated using flow cytometer (FACS Calibur, version 2.0, BD Biosciences, Franklin Lakes, NJ, USA) using CellQuest software. Quantitative data were obtained from three independent experiments.
Detection of apoptotic cells
The apoptotic cells were detected and quantitated by using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) system (Promega, Madison, WI, USA) and propidium iodide (PI)/annexin V method (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. For TUNEL system, 4′,6-diamidino-2-phenylindole (DAPI) was used for nuclei staining. Signal detection was performed using a fluorescence microscopy (LSM 710, Carl-Zeiss, Oberkochen, Germany). The index of apoptosis was the average of the percentages of positive nuclei including apoptotic bodies per total number of nuclei from five random observation fields at 200× magnitudes. For PI/annexin V system, cells were washed with PBS and resuspended in medium containing GST (40 μg/mL) or rDP2 (5 and 20 μg/mL). Cells staining with PI and binding fluorescein isothiocyanate (FITC)-labeled annexin V were determined by flow cytometer (FACS Calibur, version 2.0, BD Biosciences, Franklin Lakes, NJ, USA) using CellQuest software. Quantitative data were obtained from three independent experiments.
Intracellular ROS determination
Production of intracellular reactive oxygen species (ROS) was determined by spectrofluorometrical method using 2′,7′-dihydrodichlorofluorescein diacetate (DCFH-DA) assay with modification . DCFH-DA diffuses through the cell membrane and is enzymatically hydrolyzed by intracellular esterases to the nonfluorescent DCFH, which can be rapidly oxidized to the highly fluorescent DCF, the fluorescent product, in the presence of ROS. After exposure to GST or rDP2, DCFH-DA was added to the culture plates at a final concentration of 5 μM and incubated for 40 min at 37 ℃ in darkness. DCF fluorescence intensity was detected with emission wavelength at 530 nm and excitation wavelength at 485 nm using a SpectraMax Plus microplate reader (Molecular Devices Corporation, Sunnyvale, CA, USA). The values were expressed as the mean absorbance normalized to the ratio of control value.
Cells were washed with PBS and incubated with lysis buffer (10 mM HEPES, pH 7.6; containing 15 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, 1 mM dithiothreitol, 0.05 % Igepal CA-630 and 1 mM PMSF, 1 mM Na3VO4, 50 mM NaF, 10 μg/mL leupeptin/aprotinin) for 10 min. Cell lysates were collected by a centrifugation at 2500 g for 10 min at 4 ℃. The supernatant containing the cytosol was further centrifuged at 20,000g for 15 min at 4 ℃, namely cytosolic fraction. The pellets containing nuclei were washed with PBS, resuspended in nuclear buffer (25 mM HEPES, pH 7.6, 0.1 % Igepal CA-630, 1 M KCl, 0.1 mM EDTA, 1 mM PMSF, 1 mM Na3VO4, 2 mM NaF, 10 μg/mL leupeptin/aprotinin), and centrifuged at 10,000g for 15 min at 4 ℃. The resulting supernatants were collected, namely nuclear fraction.
Cells were collected and lysed for protein extraction and the followed immunoblotting as previously described . Phosphorylation and level of protein was demonstrated by using antibodies against human cellular proteins, including caspase-3, caspase-9, caspase-8, cytochrome c, Poly (ADP-ribose) polymerase (PARP), Bcl-2, Bak, Bax, Fas, FasL, phosphorylated P38 (p-P38), total P38, phosphorylated JNK (p-JNK), total JNK, phosphorylated glycogen synthase kinase 3beta (GSK3β) (Ser9), cellular FLICE inhibitory protein (c-FLIP), and α-tubulin (Cell signaling, Beverly, MA). Detection of antigen–antibody complex was performed by using ECL reagent (Millipore, Bedford, MA, USA) and luminescence image system (LAS-4000 mini; Fujifilm, Tokyo, Japan). Semi-quantitation of reacted signals was determined using Multi Gauge software version 2.2 (FujiFilm, Tokyo, Japan). Three independent analyses were performed for statistical analysis.
RNA extraction and quantitative real-time PCR (qPCR)
Total RNA extraction was performed by using RNeasy mini kit (Qiagen, Hilden, Germany). cDNA was synthesized from total RNA by reverse transcription using RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific Inc, Waltham, MA). qPCR was performed using the ABI PRISM 7700 sequence detection system (Applied Biosystems, Foster City, CA). For mRNA quantitation, FastStart Universal SYBR Green Master (Roche Applied Science, Mannheim, Germany) was used for Taqman PCR. Relative gene expressions were calculated by using the 2−ΔΔCt method . Expression of GADPH gene was used an internal endogenous control. All qPCR experiments were performed in duplicates for each sample. The correct size of the PCR products was confirmed by agarose gel electrophoresis.
Inhibition of TLR2 expression with small interfering RNA (siRNA)
To specifically inhibit TLR2 expression, a combination of three different siRNAs (3 target sequence for TLR2; UUC UCA UCU CAC AAA AUU G, CUU GUG ACC GCA AUG GUA U, and UCU UUA UGU CAC UAG UUA U) was used for transfection. Transfection was performed according to the manufacture’s instruction. Briefly, cells were seeded in a 96-well plate at 2 × 104 cells/well and incubated at 37 ℃ and 5 % CO2 for 16 h. The siRNA solution was prepared in RNase-free buffered solution and the final concentration of each siRNA was 1 μM per well (Dharmacon® AccellTM siRNA reagents, Thermo scientific, Hudson, NH, USA). After 72 h, the transfected cells were treated with rDP2 for 24 h.
Data were expressed as means ± SEMs of the three independent experiments. Statistical significance analysis was determined by using 1-way ANOVA followed by Dunnett for multiple comparisons with the control or the impaired 2-tailed Student t test. The differences were considered significant for p values less than 0.05.
rDP2 reduced viability and induced apoptosis of BEAS-2B cell
rDP2 induced increase of intracellular ROS and activation of intrinsic apoptosis cascades in BEAS-2B cell
rDP2 triggered intrinsic apoptotic signaling in BEAS-2B cell via activation of P38 and JNK
rDP2 promoted expression of Fas and FasL in BEAS-2B cell through P38 and JNK activation
rDP2 enhanced caspase-8 activation via Akt/GSK3β-mediated down-regulation of c-FLIP in BEAS-2B cell
Involvement of TLR2 signaling in DP2-induced apoptosis of BEAS-2B cell
Apoptosis is commonly observed in alveolar epithelial cells in pulmonary fibrosis, a condition that predisposes to severe asthma . Asthmatic bronchial epithelium is characteristically damaged with loss of columnar epithelial cells that is highly relative to unregulated apoptosis . The present study indicates that non-proteolytic aeroallergen rDP2 reduces viability of BEAS-2B cell via inducing apoptosis, suggesting that rDP2 not only evokes immune responses but also directly causes damage of airway epithelium. Previous studies have indicated roles for ROS in the pathology of asthma both in terms of increased stress and decreased antioxidant protection . In addition, airway responses have been shown to correlate with oxidant generation by eosinophils after antigen challenge in vivo  and neutrophil superoxide generation correlates with bronchial hyperreactivity . Our results demonstrate that rDP2 promotes intracellular ROS level, which may contribute to the increase of Bax/Bcl-2 ratio and the cytochrome c release in BEAS-2B cell. These findings provide evidences that rDP2 may contributes to airway hyperreactivity via enhancing intracellular ROS and the consequent pathogenic and pro-apoptotic signaling in epithelial cells.
Cellular apoptosis is mediated by a fine balance of pro- and anti-apoptotic proteins in cells. Particularly, the mitochondrial apoptosis pathway is regulated by early translocation of anti-apoptotic (Bcl-2 and Bcl-XL) and pro-apoptotic (Bak and Bax) members of the Bcl-2 family of proteins to or from mitochondria , which modulate the release of pro-apoptotic components such as cytochrome c that activate the apoptosome, effecter caspases and DNases resulting in DNA fragmentation and cleaved PARP. Furthermore, the presence of cleaved PARP (p85) has been reported as an important marker of early apoptosis in asthmatic bronchial epithelium . Our results reveal that rDP2 decreases Bcl-2 but increases Bax and Bak level in BEAS-2B cells, as well as increases p85 PARP, indicating that rDP2 triggers intrinsic apoptosis of human bronchial epithelial cells attributing to imbalance of pro-apoptotic and anti-apoptotic members.
A recent study reports that Dermatophagoides pteronyssinus (DP) extract can inhibit apoptosis of human neutrophil . The DP extract, generally, consists of more than ten types of allergens that have been identified and characterized . Among the identified DP allergens, group 1 and group 3 is known to possess protease activity, and the others are non-proteolytic . In this study, our findings indicate that rDP2 evokes apoptosis of epithelial cell BEAS-2B. It suggests that the discrepancy may result from different cell types in response to multiple allergens and single allergen. However, it is interesting that mite allergens can prolong the neutrophil by inhibiting its apoptosis and meanwhile, induce the apoptosis of airway epithelial cell, which synergically enhances inflammatory response and airway remodeling that further exacerbates the airway injury. Further investigation is needed to clarify the underlying mechanism.
MAPK signaling cascades were revealed in a myriad of fundamental cellular processes including proliferation, motility, stress reaction and apoptosis. Among MAPKs, JNK and P38 activation are generally pro-apoptotic in response to stress and cellular damage [25, 26]. JNK/P38 activation has been reported to contribute to hyperphosphorylation of c-Jun and promote transcription of FasL, leading to apoptosis of ovarian carcinoma cell . In addition, active P38 phosphorylates Bcl-xL and Bcl-2 and prevents the accumulation of these anti-apoptotic components within the mitochondria, contributing to loss of mitochondrial membrane potential and the release of cytochrome c . P38 is also known to mediate Fas-induced mitochondrial death pathway in CD8+ T cells . Interestingly, our results reveal that rDP2 up-regulates both mRNA expression and protein level of Fas and FasL via JNK and P38 activation, indicating that rDP2 not only triggers intrinsic pathway but also reinforces extrinsic apoptosis via up-regulation of death-inducing receptor and its ligand through JNK/P38 pathway.
c-FLIP is a death effector domain (DED)-containing family member that inhibits one of the most proximal steps of death receptor (DR)-mediated apoptosis. After being recruited to the death-induced signaling complex (DISC), c-FLIP suppresses pro-caspase-8 activation and inhibits DR-mediated apoptosis via different mechanisms [29, 30]. A recent study demonstrates that a linkage between GSK3 inhibition and c-FLIP down-regulation, thus highlighting a new mechanism by which GSK3 modulates the extrinsic apoptotic pathway . Similar with the previous findings, the present study reveals that rDP2 inhibits GSK3β by Akt-mediated phosphorylation at serine-9, consequently down-regulating c-FLIP and enhancing extrinsic apoptotic pathway.
Der p 2 has been reported to induce inflammatory responses via activation of TLR2/4 and the downstream MyD88/NF-kB pathway [6, 7, 32]. The capability of TLR activation links Der p 2 to be regarded as an innate immunity activator. A very recent study shows that Der p 2 can be internalized by epithelial cell that promotes TLR-mediated proinflammatory signaling . These findings demonstrates that TLRs play a crucial role in Der p 2-triggered cellular signaling and inflammatory responses. Similarly, our results indicates that TLR2 partially involves in rDP2-induced epithelial apoptosis. However, the TLR2-independent apoptotic signaling in response to Der p 2 needs further investigation.
In summary, this study shows a novel mechanism by which rDP2 induces mitochondrial pathway and promotes extrinsic pathway by up-regulation of Fas/FasL and down-regulation of c-FLIP through Akt-dependent phosphorylation of GSK3β (Ser9). Through this study, we are able to show, for the first time, that the non-proteolytic aeroallergen rDP2 directly triggers apoptosis of airway epithelial cell via both intrinsic and extrinsic pathway and highlights the role of JNK/P38 and TLR2 signaling in the rDP2-induced apoptosis, thus providing a new reasonable explanation for how rDP2 exacerbates airway damage in chronic respiratory diseases.
recombinant Der p 2
glycogen synthase kinase 3beta
c-Jun N-terminal kinase
p38 mitogen-activated protein kinase
3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
reactive oxygen species
cellular (FADD-like IL-1β-converting enzyme)-inhibitory protein
toll-like receptor 2
terminal deoxynucleotidyl transferase dUTP nick end labeling
CHL carried out the analysis of cell viability, flow cytometric analysis, immunoblotting, and gene inhibition by siRNA, and helped to draft the manuscript. YCH carried out the TUNEL assay and quantitation of gene expression. SHK conceived of the study, performed the statistical analysis, participated in its design and coordination and drafted the manuscript. All authors read and approved the final manuscript.
We thank the support of quantitative real-time PCR machine by the Instrument of Center, Chung Shan Medical University, which is partially supported by National Science Council, Ministry of Education and Chung Shan Medical University. This work was supported by grant NSC99-2320-B-040-003-MY3 and MOST103-2632-B-040-002 from the Ministry of Science and Technology, Taiwan.
The authors declare that they have no competing interests.
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- de Boer WI, Sharma HS, Baelemans SM, Hoogsteden HC, Lambrecht BN, Braunstahl GJ. Altered expression of epithelial junctional proteins in atopic asthma: possible role in inflammation. Can J Physiol Pharmacol. 2008;86:105–12.PubMedView ArticleGoogle Scholar
- Locke NR, Royce SG, Wainewright JS, Samuel CS, Tang ML. Comparison of airway remodeling in acute, subacute, and chronic models of allergic airways disease. Am J Respir Cell Mol Biol. 2007;36:625–32.PubMedView ArticleGoogle Scholar
- James AL, Wenzel S. Clinical relevance of airway remodelling in airway diseases. Eur Respir J. 2007;30:134–55.PubMedView ArticleGoogle Scholar
- Tillie-Leblond I, Gosset P, Tonnel AB. Inflammatory events in severe acute asthma. Allergy. 2005;60:23–9.PubMedView ArticleGoogle Scholar
- Thomas WR, Smith WA, Hales BJ, Mills KL, O’Brien RM. Characterization and immunobiology of house dust mite allergens. Int Arch Allergy Immunol. 2002;129:1–18.PubMedView ArticleGoogle Scholar
- Trompette A, Divanovic S, Visintin A, Blanchard C, Hegde RS, Madan R, et al. Allergenicity resulting from functional mimicry of a Toll-like receptor complex protein. Nature. 2009;457:585–8.PubMedPubMed CentralView ArticleGoogle Scholar
- Osterlund C, Gronlund H, Polovic N, Sundstrom S, Gafvelin G, Bucht A. The non-proteolytic house dust mite allergen Der p 2 induce NF-kappaB and MAPK dependent activation of bronchial epithelial cells. Clin Exp Allergy. 2009;39:1199–208.PubMedView ArticleGoogle Scholar
- Demedts IK, Demoor T, Bracke KR, Joos GF, Brusselle GG. Role of apoptosis in the pathogenesis of COPD and pulmonary emphysema. Respir Res. 2006;7:53.PubMedPubMed CentralView ArticleGoogle Scholar
- Yamauchi K. Airway remodeling in asthma and its influence on clinical pathophysiology. Tohoku J Exp Med. 2006;209:75–87.PubMedView ArticleGoogle Scholar
- Weng T, Karmouty-Quintana H, Garcia-Morales LJ, Molina JG, Pedroza M, Bunge RR, et al. Hypoxia-induced deoxycytidine kinase expression contributes to apoptosis in chronic lung disease. FASEB J. 2013;27:2013–26.PubMedPubMed CentralView ArticleGoogle Scholar
- Kuwano K. Epithelial cell apoptosis and lung remodeling. Cell Mol Immunol. 2007;4:419–29.PubMedGoogle Scholar
- Wang WC, Tsai JJ, Kuo CY, Chen HM, Kao SH. Non-proteolytic house dust mite allergen, Der p 2, upregulated expression of tight junction molecule claudin-2 associated with Akt/GSK-3beta/beta-catenin signaling pathway. J Cell Biochem. 2011;112:1544–51.PubMedView ArticleGoogle Scholar
- van der Vliet A. Nox enzymes in allergic airway inflammation. Biochim Biophys Acta. 2011;1810:1035–44.PubMedPubMed CentralView ArticleGoogle Scholar
- Tan C, Qian X, Jia R, Wu M, Liang Z. Matrine induction of reactive oxygen species activates p38 leading to caspase-dependent cell apoptosis in non-small cell lung cancer cells. Oncol Rep. 2013;30:2529–35.PubMedGoogle Scholar
- Shi L, Yu X, Yang H, Wu X. Advanced glycation end products induce human corneal epithelial cells apoptosis through generation of reactive oxygen species and activation of JNK and p38 MAPK pathways. PLoS One. 2013;8:e66781.PubMedPubMed CentralView ArticleGoogle Scholar
- Wang ZS, Luo P, Dai SH, Liu ZB, Zheng XR, Chen T. Salvianolic acid B induces apoptosis in human glioma U87 cells through p38-mediated ROS generation. Cell Mol Neurobiol. 2013;33:921–8.PubMedView ArticleGoogle Scholar
- Beurel E, Jope RS. The paradoxical pro- and anti-apoptotic actions of GSK3 in the intrinsic and extrinsic apoptosis signaling pathways. Prog Neurobiol. 2006;79:173–89.PubMedPubMed CentralView ArticleGoogle Scholar
- Uhal BD, Joshi I, Hughes WF, Ramos C, Pardo A, Selman M. Alveolar epithelial cell death adjacent to underlying myofibroblasts in advanced fibrotic human lung. Am J Physiol. 1998;275:L1192–9.PubMedGoogle Scholar
- Bucchieri F, Puddicombe SM, Lordan JL, Richter A, Buchanan D, Wilson SJ, et al. Asthmatic bronchial epithelium is more susceptible to oxidant-induced apoptosis. Am J Respir Cell Mol Biol. 2002;27:179–85.PubMedView ArticleGoogle Scholar
- Morcillo EJ, Estrela J, Cortijo J. Oxidative stress and pulmonary inflammation: pharmacological intervention with antioxidants. Pharmacol Res. 1999;40:393–404.PubMedView ArticleGoogle Scholar
- Sedgwick JB, Calhoun WJ, Vrtis RF, Bates ME, McAllister PK, Busse WW. Comparison of airway and blood eosinophil function after in vivo antigen challenge. J Immunol. 1992;149:3710–8.PubMedGoogle Scholar
- Postma DS, Renkema TE, Noordhoek JA, Faber H, Sluiter HJ, Kauffman H. Association between nonspecific bronchial hyperreactivity and superoxide anion production by polymorphonuclear leukocytes in chronic air-flow obstruction. Am Rev Respir Dis. 1988;137:57–61.PubMedView ArticleGoogle Scholar
- Antonsson B. Mitochondria and the Bcl-2 family proteins in apoptosis signaling pathways. Mol Cell Biochem. 2004;256–257:141–55.PubMedView ArticleGoogle Scholar
- Kim EH, Lee JS, Lee NR, Baek SY, Kim EJ, Lee SJ, et al. Regulation of constitutive neutrophil apoptosis due to house dust mite allergen in normal and allergic rhinitis subjects. PLoS One. 2014;9:e105814.PubMedPubMed CentralView ArticleGoogle Scholar
- Xia Z, Dickens M, Raingeaud J, Davis RJ, Greenberg ME. Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science. 1995;270:1326–31.PubMedView ArticleGoogle Scholar
- Min L, He B, Hui L. Mitogen-activated protein kinases in hepatocellular carcinoma development. Semin Cancer Biol. 2011;21:10–20.PubMedView ArticleGoogle Scholar
- Mansouri A, Ridgway LD, Korapati AL, Zhang Q, Tian L, Wang Y, et al. Sustained activation of JNK/p38 MAPK pathways in response to cisplatin leads to Fas ligand induction and cell death in ovarian carcinoma cells. J Biol Chem. 2003;278:19245–56.PubMedView ArticleGoogle Scholar
- Farley N, Pedraza-Alva G, Serrano-Gomez D, Nagaleekar V, Aronshtam A, Krahl T, et al. p38 mitogen-activated protein kinase mediates the Fas-induced mitochondrial death pathway in CD8+ T cells. Mol Cell Biol. 2006;26:2118–29.PubMedPubMed CentralView ArticleGoogle Scholar
- Scaffidi C, Schmitz I, Krammer PH, Peter ME. The role of c-FLIP in modulation of CD95-induced apoptosis. J Biol Chem. 1999;274:1541–8.PubMedView ArticleGoogle Scholar
- Irmler M, Thome M, Hahne M, Schneider P, Hofmann K, Steiner V, et al. Inhibition of death receptor signals by cellular FLIP. Nature. 1997;388:190–5.PubMedView ArticleGoogle Scholar
- Chen S, Cao W, Yue P, Hao C, Khuri FR, Sun SY. Celecoxib promotes c-FLIP degradation through Akt-independent inhibition of GSK3. Cancer Res. 2011;71:6270–81.PubMedPubMed CentralView ArticleGoogle Scholar
- Chiou YL, Lin CY. Der p2 activates airway smooth muscle cells in a TLR2/MyD88-dependent manner to induce an inflammatory response. J Cell Physiol. 2009;220:311–8.PubMedView ArticleGoogle Scholar
- Yin SC, Liao EC, Chiu CL, Chang CY, Tsai JJ. Der p2 internalization by epithelium synergistically augments toll-like receptor-mediated proinflammatory signaling. Allergy Asthma Immunology Res. 2015;7:393–403.View ArticleGoogle Scholar