Human Lung Epithelial Cell Line Research Articles

Highly Stable Positively Charged Dendron-Encapsulated Gold Nanoparticles

We report the development of a novel cationic dendron (TAG1-PCD) and a positively charged gold nanoparticle-dendron conjugate (PCD-AuNP). TAG1-PCD was designed by considering the reactivity, hydrophilicity, and cationic nature that is required to yield a stable gold conjugate in aqueous media. The PCD-AuNPs, nominally 10 nm in size, were synthesized by reduction of chloroauric acid in the presence of TAG1-PCD. The physicochemical properties of PCD-AuNPs were characterized by dynamic light scattering, transmission electron microscopy, UV-vis absorbance, and X-ray photoelectron spectroscopy for investigation of size distribution, shape uniformity, surface plasmon resonance bands, and Au-dendron bonding. Asymmetric-flow field flow fractionation was employed to confirm the in situ size, purity, and surface properties of the PCD-AuNPs. Additionally, the stability of PCD-AuNPs was systematically evaluated with respect to shelf life determination, stability in biological media and a wide range of pH values, chemical resistance against cyanide, redispersibility from lyophilized state, and stability at temperatures relevant to biological systems. Dose dependent cell viability was evaluated in vitro using the human lung epithelial cell line A549 and a monkey kidney Vero cell line. Observations from in vitro studies are discussed. Overall, the investigation confirmed the successful development of stable PCD-AuNPs with excellent stability in biologically relevant test media containing proteins and electrolytes, and with a shelf life exceeding 6 months. The excellent aqueous stability and apparent lack of toxicity for this conjugate enhances its potential use as a test material for investigating interactions between positively charged NPs and biocellular and biomolecular systems, or as a vehicle for drug delivery.

Monophosphoryl Lipid a as an adjuvant for immune therapy? A detailed in vitro comparison to LPS

Monophosphoryl lipid A (MPL) is a non-toxic TLR4 ligand, derived from Salmonella minnesota R595 (Re) lipopolysaccharide (LPS) by chemical modification. It is clinically used as an adjuvant for cancer treatment (Fendrix®, Ceravix®) and allergen specific immunotherapy (Pollinex® Quattro, ORALVAC®). Nevertheless, reports on the mechanism of adjuvant activity are limited. The aim of this study was to compare the immune modulating capacities of MPL and LPS in vitro. In both human and murine lung epithelial cell lines (LA-4, A549) LPS induced a higher CCL2 secretion than MPL. In murine BM-derived myeloid dendritic cells (mDC), LPS as well as MPL stimulation resulted in the same pattern of cytokine secretion (IL-1β, IL-6, IL-10 and TNF-α). At high concentrations of MPL, IL-1β secretion was 4-fold higher compared to LPS, whereas LPS stimulation resulted in higher secretion of IL-6, IL-10 and TNF-α, respectively. Moreover, mDC stimulation with both adjuvants resulted in a pronounced cell activation pattern characterized by CD40 and CD69 upregulation, at which LPS proved to be more potent than MPL (thresholds for mDC activation: MPL: 100 ng/ml, LPS: 1 ng/ml). In MyD88-/- and Trif-/- mDC, MPL-induced cytokine secretion was absent in MyD88- but only reduced in Trif-deficient mDC. LPS induced cytokine secretion was mostly unchanged in Trif-/- mDC. Furthermore, the co-administration of MPL and Ova resulted in enhanced IFN-γ and IL-5 secretion from OVA-specific DO11.10 CD4+ T cells co-cultured with BALB/c mDC which was not observed for LPS controls. In line with this result, stimulation with a covalent fusion protein of MPL and Ova (MPL:Ova) resulted in enhanced cytokine secretion from both mDC (IL-1β, IL-6, TNF, IL-10, IL-12) and CD4 T cells (IL-5, IL-13, IL-2, IFN-γ, IL-17) compared to equimolar concentrations of MPL and Ova provided individually or as a mixture. Interestingly, Ova induced IL-9 secretion from CD4+ T cells was dose-dependently repressed when fused to MPL. In summary, using in vitro assay systems we observed similar but attenuated immune responses induced by MPL in comparison to LPS. MPL applied together with allergen (either mixed or covalently fused) on CD4+ T cells boosted allergen-specific TH1-, TH2-, and TH17-adaptive responses. Although considered safe in humans, further studies should critically assess the adjuvant capacity of MPL in order to evaluate potential non-desired immunological effects.

Comparative Study about Cytotoxicity with CuO Engineered Nanoparticles, TiO<sub>2</sub> Engineered Nanoparticles, CeO<sub>2</sub> Engineered Nanoparticles, Single-Walled Carbon Nanotubes

Engineered nanoparticles have been extensively used in electronics, energy, materials and biomedical areas. Engineered nanoparticles with unique magnetic, luminescent, and catalytic properties can not only benefit human society, but also cause potentially harmful interactions with living systems. This study was aimed at assessing the cytotoxicity of CuO ENPs, TiO2ENPs, CeO2ENPs, SWCNTs in human lung epithelial cell line (A549). Herein, we performed a comprehensive study on the cytotoxicity of four different ENPs by examining the viability of A549 cells. The results showed that CuO ENPs could cause a highest dose-dependent cytotoxicity, SWCNTs were more toxic than TiO2ENPs and CeO2ENPs to A549 cells, TiO2ENPs induced a slight loss of cell viability at 100 mg/L and CeO2ENPs had no obvious cytotoxicity. These effects were dose related, and should be considered in the application of different areas of ENPs.

Microarray-Based Gene Expression Profiling Suggests adaptation of Lung Epithelial Cells Subjected to Chronic Cyclic Strain

Background/Aims: Mechanical strain of the lung tissue is a physiological process that affects the behavior of lung cells. Since recent evidence also suggests alterations in the expression of certain genes as a consequence of mechanotransduction, our study aimed at the analysis of the gene expression profile in lung epithelial cells subjected to chronic cyclic strain. Methods: Various human lung epithelial cell lines (A549 as principal adherent cell line and four others) were subjected to cyclic strain (16 % surface distension, 12 min^-1) in a Strain Cell Culture Device for 24 h. In comparison to static controls, expression analyses were performed by gene microarray and qPCR. Results: Microarray analysis revealed many differences in the gene expression but at moderate levels. Altogether 25 genes were moderately down-regulated (0.86-fold ± 0.06) and 26 genes were up-regulated (1.18-fold ± 0.10) in A549 and the others. Strain-regulated genes often code for transcription factors, such as E2F4 and SRF. qPCR analyses confirmed the up-regulation of both transcription factors and further genes, such as PLAU (urokinase-type plasminogen activator) and S100A4 (S100 protein A4). Moreover, we showed the down-regulations of AGR2 (anterior gradient 2) and LCN2 (lipocalin 2). Conclusions: We identified many genes of which the expression was moderately altered in lung epithelial cells subjected to chronic cyclic strain. Although many moderate changes in the gene expression profile might affect cellular behavior, it also suggests an effective adaptation of cells to mechanical forces in long-term conditions.

MUC18 Differentially Regulates Pro-Inflammatory and Anti-Viral Responses in Human Airway Epithelial Cells.

MUC18 or CD146, a transmembrane glycoprotein, is mainly expressed by endothelial cells and smooth muscle cells where it serves as a cell-cell adhesion molecule. We have found MUC18 up-regulation in airway epithelial cells of patients with asthma and chronic obstructive pulmonary disease (COPD). However, the function of MUC18 in airway epithelial cells remains unclear. In the present study, we tested the hypothesis that MUC18 exerts a pro-inflammatory function during stimulation with a viral mimic polyI:C or human rhinovirus infection. Normal human primary airway epithelial cells were transduced with lentivirus encoding MUC18 cDNA to over-express MUC18 or with GFP (control), and treated with polyI:C or HRV for detection of pro-inflammatory cytokine IL-8 and anti-viral gene IFN-β. Additionally, we performed cell culture of human lung epithelial cell line NCIH292 cells to determine the mechanisms of MUC18 function. We found that MUC18 over-expression promoted IL-8 production, while it inhibited IFN-β expression following polyI:C stimulation or HRV infection. Increased phosphorylation of MUC18 serines was observed in MUC18 over-expressing cells. Reduction of MUC18 serine phosphorylation by inhibiting ERK activity was associated with less production of IL-8 following polyI:C stimulation. Our results for the first time demonstrate MUC18's pro-inflammatory and anti-viral function in human airway epithelial cells.

Type I Interferon Imposes a TSG101/ISG15 Checkpoint at the Golgi for Glycoprotein Trafficking during Influenza Virus Infection

Several enveloped viruses exploit host pathways, such as the cellular endosomal sorting complex required for transport (ESCRT) machinery, for their assembly and release. The influenza A virus (IAV) matrix protein binds to the ESCRT-I complex, although the involvement of early ESCRT proteins such as Tsg101 in IAV trafficking remain to be established. We find that Tsg101 can facilitate IAV trafficking, but this is effectively restricted by the interferon (IFN)-stimulated protein ISG15. Cytosol from type I IFN-treated cells abolished IAV hemagglutinin (HA) transport to the cell surface in infected semi-intact cells. This inhibition required Tsg101 and could be relieved with deISGylases. Tsg101 is itself ISGylated in IFN-treated cells. Upon infection, intact Tsg101-deficient cells obtained by CRISPR-Cas9 genome editing were defective in the surface display of HA and for infectious virion release. These data support the IFN-induced generation of a Tsg101- and ISG15-dependent checkpoint in the secretory pathway that compromises influenza virus release.

Iron Oxide Nanoparticle-induced Oxidative Stress and Genotoxicity in Human Skin Epithelial and Lung Epithelial Cell Lines

Iron oxide (Fe₃O₄) nanoparticles (IONPs) have received much attention for their utility in biomedical applications such as magnetic resonance imaging, drug delivery and hyperthermia. Recent studies reported that IONPs induced cytotoxicity in mammalian cells. However, little is known about the genotoxicity of IONPs following exposure to human cells. In this study, we investigated the cytotoxicity, oxidative stress and genotoxicity of IONPs in two human cell lines; skin epithelial A431 and lung epithelial A549. Prepared IONPs were polygonal in shape with a smooth surface and had an average diameter of 25 nm. IONPs (25-100 μg/ml) induced dose-dependent cytotoxicity in both types of cells, which was demonstrated by cell viability (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide) and lactate dehydrogenase leakage assays. IONPs were also found to induce oxidative stress in a dose-dependent manner, evident by depletion of glutathione and induction of reactive oxygen species (ROS) and lipid peroxidation. Comet assay revealed that level of DNA damage was higher with concentration of IONPs in both types of cells. Quantitative real-time PCR analysis showed that following the exposure of cells to IONPs, the expression levels of mRNA of caspase-3 and caspase-9 genes were higher. We also observed the higher activity of caspase-3 and caspase-9 enzymes in IONPs treated cells. Moreover, western blot analysis showed that protein expression level of cleaved caspase-3 was up-regulated by IONPs in both types of cells. Taken together, our data demonstrates that IONPs have potential to induce genotoxicity in A431 and A549 cells, which is likely to be mediated through ROS generation and oxidative stress. This study suggests that genotoxic effects of IONPs should be further investigated at in vivo level.

Anti-apoptotic mechanism of Bacoside rich extract against reactive nitrogen species induced activation of iNOS/Bax/caspase 3 mediated apoptosis in L132 cell line

Nitric oxide is a highly reactive free radical gas that reacts with a wide range of bio-molecules to produce reactive nitrogen species and exerts nitrative stress. Bacopa monniera is a traditional folk and ayurvedic medicine known to alleviate a variety of disorders. Aim of the present study is to evaluate the protective propensity of Bacopa monniera extract (BME) through its oxido-nitrosative and anti-apoptotic mechanism to attenuate sodium nitroprusside (SNP)-induced apoptosis in a human embryonic lung epithelial cell line (L132). Our results elucidate that pre-treatment of L132 cells with BME ameliorates the mitochondrial and plasma membrane damage induced by SNP as evidenced by MTT and LDH leakage assays. BME pre-treatment inhibited NO generation by down-regulating inducible nitric oxide synthase expression. BME exhibited potent antioxidant activity by up-regulating the antioxidant enzymes. SNP-induced damage to cellular, nuclear and mitochondrial integrity was also restored by BME, which was confirmed by ROS estimation, comet assay and mitochondrial membrane potential assays respectively. BME pre-treatment efficiently attenuated the SNP-induced apoptotic biomarkers such as Bax, cytochrome-c and caspase-3, which orchestrate the proteolytic damage of the cell. By considering all these findings, we report that BME protects L132 cells against SNP-induced toxicity via its free radical scavenging and anti-apoptotic mechanism.

Piceatannol Modulates Lung Epithelial Cellular Responses to Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen, which is the major cause of severe chronic lung infection in cystic fibrosis patients. It is also responsible for systemic infections in immunocompromised individuals and those presenting with significant pulmonary conditions in intensive care units. This microorganism has the capacity to initiate severe inflammation in infected lungs resulting in detrimental tissue damage. We have hypothesized that Syk protein tyrosine kinase mediates lung epithelial cellular responses to P. aeruginosa infection, and that a naturally occurring non-toxic Syk inhibitor piceatannol can protect infected human cells against the deleterious effects associated with this infection. We infected Syk-positive H292 or Syk-negative A549 human lung epithelial cell lines with P. aeruginosa and assessed the resulting cellular responses, i.e. production of proinflammatory cytokines, adhesion molecule expression, generation of reactive oxygen species, and apoptosis of infected cells, utilizing a multiplex bead-based immunoassay and flow cytometry. We also studied the internalization of P. aeruginosa using the gentamicin exclusion assay. We found that the piceatannol treatment significantly suppressed inflammation, oxidative stress and apoptosis in H292, but not in A549 cells implicating Syk participation in the regulation of the pathological processes induced by P. aeruginosa infection. Intriguingly, piceatannol was able to down-regulate the internalization of P. aeruginosa by both Syk-positive and Syk-negative cell lines, implying that the mechanisms of action of this compound extend beyond Syk inhibition. As piceatannol can interfere with several mechanisms of bacterial pathogenesis this natural compound deserves further study as a potential therapeutic option in P. aeruginosa infection.

Epithelial-to-mesenchymal transition in fibrosis: Collagen type I expression is highly upregulated after EMT, but does not contribute to collagen deposition

The hallmark of fibrosis is an accumulation of fibrillar collagens, especially of collagen type I. There is considerable debate whether in vivo type II epithelial-to-mesenchymal transition (EMT) is involved in organ fibrosis. Lineage tracing experiments by various groups show opposing data concerning the relative contribution of epithelial cells to the pool of myofibroblasts. We hypothesized that EMT-derived cells might directly contribute to collagen deposition. To study this, EMT was induced in human epithelial lung and renal cell lines in vitro by means of TGF-β1 stimulation, and we compared the collagen type I (COL1A1) expression levels of transdifferentiated cells with that of myofibroblasts obtained by TGF-β1 stimulation of human dermal and lung fibroblasts. COL1A1 expression levels of transdifferentiated epithelial cells appeared to be at least one to two orders of magnitude lower than that of myofibroblasts. This was confirmed at immunohistochemical level: in contrast to myofibroblasts, collagen type I deposition by EMT-derived cells was not or hardly detectable. We postulate that, even when type II EMT occurs in vivo, the direct contribution of EMT-derived cells to collagen accumulation is rather limited.

Reduction of Lectin Valency Drastically Changes Glycolipid Dynamics in Membranes but Not Surface Avidity

Multivalency is proposed to play a role in the strong avidity of lectins for glycosylated cell surfaces and also in their ability to affect membrane dynamics by clustering glycosphingolipids. Lectins with modified valency were designed from the β-propeller fold of Ralstonia solanacearum lectin (RSL) that presents six fucose binding sites. After identification of key amino acids by molecular dynamics calculations, two mutants with reduced valency were produced. Isothermal titration calorimetry confirmed the loss of three high affinity binding sites for both mutants. Crystal structures indicated that residual low affinity binding occurred in W76A but not in R17A. The trivalent R17A mutant presented unchanged avidity toward fucosylated surfaces, when compared to hexavalent RSL. However, R17A is not able anymore to induce formation of membrane invaginations on giant unilamellar vesicules, indicating the crucial role of number of binding sites for clustering of glycolipids. In the human lung epithelial cell line H1299, wt-RSL is internalized within seconds whereas the kinetics of R17A uptake is largely delayed. Neolectins with tailored valency are promising tools to study membrane dynamics.

Comet assay and air–liquid interface exposure system: A new combination to evaluate genotoxic effects of cigarette whole smoke in human lung cell lines

Over the past three decades, the genotoxic effects of cigarette smoke have generally been evaluated in non-human cell models after exposure to particulate phase, gas phase, or cigarette smoke condensate, rather than the whole smoke aerosol itself. In vitro setups using human cell lines and whole smoke exposure to mimic actual aerosol exposure should more accurately reflect human cigarette smoke exposure. We investigated the VITROCELL® 24 air–liquid interface exposure system in combination with the comet assay to assess DNA damage in two different human lung epithelial cell lines exposed to whole smoke. Results showed a repeatable and reproducible dose–response relationship between DNA damage and increased whole smoke dose in both cell lines. Thus, the combination of the comet assay with the VITROCELL® 24 represents a valuable new in vitro test system to screen and assess DNA damage in human lung cells exposed to whole smoke.

Stenotrophomonas maltophilia Encodes a Type II Protein Secretion System That Promotes Detrimental Effects on Lung Epithelial Cells

The Gram-negative bacterium Stenotrophomonas maltophilia is increasingly identified as a multidrug-resistant pathogen, being associated with pneumonia, among other infections. Despite this increasing clinical problem, the genetic and molecular basis of S. maltophilia virulence is quite minimally defined. We now report that strain K279a, the first clinical isolate of S. maltophilia to be sequenced, encodes a functional type II protein secretion (T2S) system. Indeed, mutants of K279a that contain a mutation in the xps locus exhibit a loss of at least seven secreted proteins and three proteolytic activities. Unlike culture supernatants from the parental K279a, supernatants from multiple xps mutants also failed to induce the rounding, detachment, and death of A549 cells, a human lung epithelial cell line. Supernatants of the xps mutants were also unable to trigger a massive rearrangement in the host cell's actin cytoskeleton that was associated with K279a secretion. In all assays, a complemented xpsF mutant behaved as the wild type did, demonstrating that Xps T2S is required for optimal protein secretion and the detrimental effects on host cells. The activities that were defined as being Xps dependent in K279a were evident among other respiratory isolates of S. maltophilia. Utilizing a similar type of genetic analysis, we found that a second T2S system (Gsp) encoded by the K279a genome is cryptic under all of the conditions tested. Overall, this study represents the first examination of T2S in S. maltophilia, and the data obtained indicate that Xps T2S likely plays an important role in S. maltophilia pathogenesis.

Potential link between cysteinyl-leukotriene receptors and release of bioactive amino acids in regulation of lung function. Focus on “Volume-sensitive release of organic osmolytes in the human lung epithelial cell line A549: role of the 5-lipoxygenase”

transient changes in extracellular concentrations of solutes and the rates of transmembrane transport of ions and nutrients cause alterations in cell volume, which are particularly pronounced in various types of epithelia. With very few exceptions, animal cells are capable of normalizing their

Biological effects induced by BSA-stabilized silica nanoparticles in mammalian cell lines

Much of the concerns regarding engineered nanoparticle (NP) toxicity are based on knowledge from previous studies on particles in ambient air or occupational situations. E.g., the effects of exposure to silica dust particles have been studied intensely due to the carcinogenicity of crystalline silica. However, the increasing usage of engineered amorphous silica NPs has emphasized the need for further mechanistic insight to predict the consequences of exposure to the amorphous type of silica NPs. The present study focused on the in vitro biological effects following exposure to well-dispersed, BSA-stabilized, amorphous silica NPs whereas unmodified silica NPs where included for reasons of comparison. The cytotoxicity of the silica NPs was investigated in six different cell lines (A549, THP-1, CaCo-2, ASB-XIV, J-774A.1, and Colon-26) selected to explore the significance of organ and species sensitivity in vitro. Viability data demonstrated that macrophages were most sensitive to silica NP and interestingly, murine cell lines were generally found to be more sensitive than comparable human cell lines. Further studies were conducted in the human epithelial lung cell line, A549, to explore the molecular mechanism of silica toxicity. Generation of reactive oxygen species, one of the proposed toxicological mechanisms of NPs, was investigated in A549 cells by the dichlorofluorescin (DCF) assay to be significantly induced at NP concentrations above 113μg/mL. However, induction of oxidative stress related pathways was not found after silica NP exposure for 24h in gene array studies conducted in A549 cells at a relatively low NP concentration (EC20). Up-regulated genes (more than 2-fold) were primarily related to lipid metabolism and biosynthesis whereas down-regulated genes included several processes such as transcription, cell junction, extra cellular matrix (ECM)-receptor interaction and others. Thus, gene expression data proposes that several cellular processes other than oxidative stress could be affected by exposure to silica NPs.

Abstract 627: Fiber-induced DNA damage response in human lung epithelial cells.

Abstract Asbestos, a group of naturally occurring mineral fibers, is a significant lung carcinogen. Exposure to asbestos fibers induces the formation of reactive oxygen species in cells and, subsequently, DNA double-strand breaks (DSBs), the most serious form of DNA damage related to transformation of cells. As a part of the DNA damage response, H2AX histone variant molecules are phosphorylated at ser139, producing γH2AX foci at the site of DSBs, and these serve as a docking site for DNA repair molecule complexes. Thereby, γH2AX foci are considered a sensitive marker for DSBs. DNA damage response has not thoroughly been investigated in particle carcinogenesis. Here, we studied fiber-induced DSB formation in two human lung epithelial cell lines, the cancer cell line A549 and the transformed cell line BEAS-2B to provide further knowledge on DNA damage associated with fiber exposure as a risk factor for lung cancer.A sub-lethal asbestos fiber concentration of 2 μg /cm2 was determined, by cell viability tests, as suitable for working with the A549 and BEAS-2B cells. Then, we studied the kinetics of fiber induced DSB formation in terms of H2AX phosphorylation. Foci of γH2AX can be detected using specific antibody in immunocytochemistry assays. A549 and BEAS-2B cells were exposed to asbestos fibers for 4, 24 and 48 h, and γH2AX foci were counted. Concurrent non-exposed control cultures were settled at each time point. At 4 h, the control group displayed remarkable endogenous DSB formation, possibly due to replication stress. In exposed cultures, the formation of γH2AX foci peaked at 24 h. We also explored whether it is possible, by using γH2AX antibody in a chromatin immunoprecipitation followed by next generation sequencing, to find out the distribution of DSBs at 24 h in the fiber-exposed A549 cells. The sequences involved in γH2AX foci were genome-widely distributed. To conclude, the kinetics of fiber-induced γH2AX foci formation in the A549 and BEAS-2B cells was partly interfered by endogenously formed DSBs. This study revealed sequences that may be implicated within γH2AX foci for further elucidation on their contribution to lung tumor progression in particle carcinogenesis. Citation Format: Eeva Kettunen, Päivi Tuominen, Satu Suhonen, Julia Catalán, Sisko L. Anttila, Penny Nymark, Hannu Norppa. Fiber-induced DNA damage response in human lung epithelial cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 627. doi:10.1158/1538-7445.AM2013-627

Volume-sensitive release of organic osmolytes in the human lung epithelial cell line A549: role of the 5-lipoxygenase

Pathophysiological conditions challenge cell volume homeostasis and perturb cell volume regulatory mechanisms leading to alterations of cell metabolism, active transepithelial transport, cell migration, and death. We report that inhibition of the 5-lipoxygenase (5-LO) with AA861 or ETH 615-139, the cysteinyl leukotriene 1 receptor (CysLT₁) with the antiasthmatic drug Zafirlukast, or the volume-sensitive organic anion channel (VSOAC) with DIDS blocks the release of organic osmolytes (taurine, meAIB) and the concomitant cell volume restoration following hypoosmotic swelling of human type II-like lung epithelial cells (A549). Reactive oxygen species (ROS) are produced in A549 cells upon hypotonic cell swelling by a diphenylene iodonium-sensitive NADPH oxidase. The swelling-induced taurine release is suppressed by ROS scavenging (butylated hydroxytoluene, N-acetyl cysteine) and potentiated by H₂O₂. Ca²⁺ mobilization with ionomycin or ATP stimulates the swelling-induced taurine release whereas calmodulin inhibition (W7) inhibits the release. Chelation of the extracellular Ca²⁺ (EGTA) had no effect on swelling-induced taurine release but prevented ATP-induced stimulation. H₂O₂, ATP, and ionomycin were unable to stimulate the taurine release in the presence of AA861 or Zafirlukast, placing 5-LO and CysLT₁ as essential elements in the swelling-induced activation of VSOAC with ROS and Ca²⁺ as potent modulators. Inhibition of tyrosine kinases (genistein, cucurbitacin) reduces volume-sensitive taurine release, adding tyrosine kinases (Janus kinase) as regulators of VSOAC activity. Caspase-3 activity during hypoxia is unaffected by inhibition of 5-LO/CysLT₁ but reduced when swelling-induced taurine loss via VSOAC is prevented by DIDS excess extracellular taurine, indicating a beneficial role of taurine under hypoxia.

The Human Antimicrobial Peptide LL-37, but Not the Mouse Ortholog, mCRAMP, Can Stimulate Signaling by Poly(I:C) through a FPRL1-dependent Pathway

LL-37 is an antimicrobial peptide produced by human cells that can down-regulate the lipopolysaccharide-induced innate immune responses and up-regulate double-stranded (ds) RNA-induced innate responses through Toll-like receptor 3 (TLR3). The murine LL-37 ortholog, mCRAMP, also inhibited lipopolysaccharide-induced responses, but unlike LL-37, it inhibited viral-induced responses in mouse cells. A fluorescence polarization assay showed that LL-37 was able to bind dsRNA better than mCRAMP. In the human lung epithelial cell line BEAS-2B, LL-37, but not mCRAMP, colocalized with TLR3, and the colocalization was increased in the presence of dsRNA. The presence of poly(I:C) increased the accumulation of LL-37 in Rab5 endosomes. Signaling by cells induced with both LL-37 and poly(I:C) was sensitive to inhibitors that affect clathrin-independent trafficking, whereas signaling by poly(I:C) alone was not, suggesting that the LL-37-poly(I:C) complex trafficked to signaling endosomes by a different mechanism than poly(I:C) alone. siRNA knockdown of known LL-37 receptors identified that FPRL1 was responsible for TLR3 signaling induced by LL-37-poly(I:C). These results show that LL-37 and mCRAMP have different activities in TLR3 signaling and that LL-37 can redirect trafficking of poly(I:C) to effect signaling by TLR3 in early endosomes in a mechanism that involves FPRL1.

Role of aberrant WNT signalling in the airway epithelial response to cigarette smoke in chronic obstructive pulmonary disease

BackgroundWNT signalling is activated during lung tissue damage and inflammation. We investigated whether lung epithelial expression of WNT ligands, receptors (frizzled; FZD) or target genes is dysregulated on cigarette smoking...

Genetic polymorphisms in catalase and CYP1B1 determine DNA adduct formation by benzo(a)pyrene ex vivo

Genetic polymorphisms can partially explain the large inter-individual variation in DNA adduct levels following exposure to polycyclic aromatic hydrocarbons. Effects of genetic polymorphisms on DNA adduct formation are difficult to assess in human studies because exposure misclassification attenuates underlying relationships. Conversely, ex vivo studies offer the advantage of controlled exposure settings, allowing the possibility to better elucidate genotype-phenotype relationships and gene-gene interactions. Therefore, we exposed lymphocytes of 168 non-smoking volunteers ex vivo to the environmental pollutant benzo(a)pyrene (BaP) and BaP-related DNA adducts were quantified. Thirty-four genetic polymorphisms were assessed in genes involved in carcinogen metabolism, oxidative stress and DNA repair. Polymorphisms in catalase (CAT, rs1001179) and cytochrome P450 1B1 (CYP1B1, rs1800440) were significantly associated with DNA adduct levels, especially when combined. Moreover, reverse transcription-polymerase chain reaction (RT-PCR) analysis in a subset of 30 subjects revealed that expression of catalase correlated strongly with expression of CYP1B1 (R = 0.92, P < 0.001). To further investigate the mechanism by which catalase influences CYP1B1 and how they simultaneously affect BaP-related DNA adduct levels, catalase expression was transiently knocked down in the human lung epithelial cell line A549. Although catalase knockdown did not immediately change CYP1B1 gene expression, recovery of catalase expression 8 h after the knockdown coincided with a 2.2-fold increased expression of CYP1B1 (P < 0.05). We conclude that the genetic polymorphism in the promoter region of CAT may determine the amount and activity of catalase, which may subsequently regulate the expression of CYP1B1. As a result, both genetic polymorphisms modulate DNA adduct levels in lymphocytes by BaP ex vivo.