Granzyme A causes detachment of alveolar epithelial A549 cells accompanied by promotion of interleukin-8 release

Granzyme A (GrA) is a serine protease produced in cytotoxic lymphocytes, lung epithelial cells (alveolar type-II cells), and alveolar macrophages. In the present study, recombinant rat GrA (rGrA) was found to cause rounding and detachment of an alveolar type-II epithelial cell line, A549. Also, rGrA stimulated release of a neutrophil chemoattractant, interleukin-8, from the cells, via a mechanism involving microtubule disruption, probably resulting from reduction of cell adhesion to culture dishes. These findings suggest that GrA might be involved in the pathogenesis of certain lung diseases characterized by loss of alveolar wall structures, neutrophil accumulation, and chronic inflammation.     

Granzyme A and thrombin differentially promote the release of interleukin-8 from alveolar epithelial A549 cells

Some of extracellular serine proteases with trypsin-like specificity of cleavage have been known to increase the release of inflammatory mediators from various cell types. For instance, two well-known trypsin-like serine proteases circulating in blood, granzyme A (GrA) and thrombin, have been found to promote interleukin (IL)-8 release from an alveolar epithelial A549 cell line. However, the mechanisms by which the proteases promote IL-8 release from the cells are not fully understood. In the present study, using A549 cells we found that (1) thrombin promoted IL-8 release from the cells via a mechanism partially involving activation of protease-activated receptor-1, a G-protein coupled receptor, whereas a recombinant form of GrA (rGrA) did it via a mechanism that does not involve the receptor activation; that (2) unlike rGrA, thrombin did not cause detachment and microtubule disruption of the cells; and that (3) the release of IL-8 induced by rGrA was inhibited in the presence of taxol, a microtubule-stabilizing reagent, whereas that induced by thrombin was not. These findings suggest that rGrA and thrombin promote the release of IL-8 from A549 cells through distinct mechanisms.     

Pulsed Low-Frequency Magnetic Fields Induce Tumor Membrane Disruption and Altered Cell Viability

Tumor cells express a unique cell surface glycocalyx with upregulation of sulfated glycosaminoglycans and charged glycoproteins. Little is known about how electromagnetic fields interact with this layer, particularly with regard to harnessing unique properties for therapeutic benefit. We applied a pulsed 20-millitesla (mT) magnetic field with rate of rise (dB/dt) in the msec range to cultured tumor cells to assess whether this affects membrane integrity as measured using cytolytic assays. A 10-min exposure of A549 human lung cancer cells to sequential 50- and 385-Hz oscillating magnetic fields was sufficient to induce intracellular protease release, suggesting altered membrane integrity after the field exposure. Heparinase treatment, which digests anionic sulfated glycan polymers, before exposure rendered cells insensitive to this effect. We further examined a non-neoplastic human primary cell line (lung lymphatic endothelial cells) as a typical normal host cell from the lung cancer microenvironment and found no effect of field exposure on membrane integrity. The field exposure was also sufficient to alter proliferation of tumor cells in culture, but not that of normal lymphatic cells. Pulsed magnetic field exposure of human breast cancer cells that express a sialic-acid rich glycocalyx also induced protease release, and this was partially abrogated by sialidase pretreatment, which removes cell surface anionic sialic acid. Scanning electron microscopy showed that field exposure may induce unique membrane “rippling” along with nanoscale pores on A549 cells. These effects were caused by a short exposure to pulsed 20-mT magnetic fields, and future work may examine greater magnitude effects. The proof of concept herein points to a mechanistic basis for possible applications of pulsed magnetic fields in novel anticancer strategies.     

Expression of functional TRPA1 receptor on human lung fibroblast and epithelial cells

The transient receptor potential subfamily A member 1 (TRPA1) is a non-selective cation channel implicated in the pathogenesis of several airway diseases like asthma and chronic obstructive pulmonary disease (COPD). Most of the research on TRPA1 focuses on its expression and function in neuronal context; studies investigating non-neuronal expression of TRPA1 are lacking. In the present study, we show functional expression of TRPA1 in human lung fibroblast cells (CCD19-Lu) and human pulmonary alveolar epithelial cell line (A549). We demonstrate TRPA1 expression at both mRNA and protein levels in these cell types. TRPA1 selective agonists like allyl isothiocyanate (AITC), 4-hydroxynonenal (4-HNE), crotonaldehyde and zinc, induced a concentration-dependent increase in Ca⁺² influx in CCD19-Lu and A549 cells. AITC-induced Ca⁺² influx was inhibited by Ruthenium red (RR), a TRP channel pore blocker, and by GRC 17536, a TRPA1 specific antagonist. Furthermore, we also provide evidence that activation of the TRPA1 receptor by TRPA1 selective agonists promotes release of the chemokine IL-8 in CCD19-Lu and A549 cells. The IL-8 release in response to TRPA1 agonists was attenuated by TRPA1 selective antagonists. In conclusion, we demonstrate here for the first time that TRPA1 is functionally expressed in cultured human lung fibroblast cells (CCD19-Lu) and human alveolar epithelial cell line (A549) and may have a potential role in modulating release of this important chemokine in inflamed airways.     

House dust mite major allergens Der p 1 and Der p 5 activate human airway-derived epithelial cells by protease-dependent and protease-independent mechanisms

House dust mite allergens (HDM) cause bronchoconstriction in asthma patients and induce an inflammatory response in the lungs due to the release of cytokines, chemokines and additional mediators. The mechanism how HDM components achieve this is largely unknown. The objective of this study was to assess whether HDM components of Dermatophagoides pteronissinus with protease activity (Der p 1) and unknown enzymatic activity (Der p 2, Der p 5) induce biological responses in a human airway-derived epithelial cell line (A549), and if so, to elucidate the underlying mechanism(s) of action. A549 cells were incubated with HDM extract, Der p 1, recombinant Der p 2 and recombinant Der p 5. Cell desquamation was assessed by microscopy. The proinflammatory cytokines, IL-6 and IL-8, were measured by ELISA. Intracellular Ca²⁺ levels were assessed in A549 cells and in mouse fibroblasts expressing the human protease activated receptor (PAR)1, PAR2 or PAR4. HDM extract, Der p 1 and Der p 5 dose-dependently increased the production of IL-6 and IL-8. Added simultaneously, Der p 1 and Der p 5 further increased the production of IL-6 and IL-8. The action of Der p 1 was blocked by cysteine-protease inhibitors, while that of Der p 5 couldn't be blocked by either serine- or cysteine protease inhibitors. Der p 5 only induced cell shrinking, whereas HDM extract and Der p1 also induced cell desquamation. Der p 2 had no effect on A549 cells. Der p 1's protease activity causes desquamation and induced the release of IL6 and IL-8 by a mechanism independent of Ca²⁺ mobilisation and PAR activation. Der p 5 exerts a protease-independent activation of A549 that involves Ca²⁺ mobilisation and also leads to the production of these cytokines. Together, our data indicate that allergens present in HDM extracts can trigger protease-dependent and protease-independent signalling pathways in A549 cells.     

Proteomic analysis of the effect of Antrodia camphorata extract on human lung cancer A549 cell

Antrodia camphorata (niu-chang-chih) is a fungus native to Taiwan that is believed to be effective in preventing diseases. This study demonstrates that 0.2-2% v/v ethanol extracts of A. camphorata cultivated by solid-state fermentation (SACE) can effectively impede the proliferation of human non-small cell lung carcinoma A549 cells but not primary human fetal lung fibroblast MRC-5. The results of apoptotic analyses implicate that SACE might trigger the apoptosis in the A549 cells by inducing endoplasmic reticulum stress. Two-dimensional gel maps of non-treated and treated A549 cells were compared using PDQUEST analytical software to discover five statistically significant twofold or above-twofold differentially-expressed protein spots. The five protein spots that were significantly de-regulated were chosen for subsequent identification by high performance liquid chromatography electro-spray tandem mass spectrometry. The five proteins were later identified as human galectin-1, human eukaryotic translation initiation factor 5A, human Rho GDP dissociation inhibitor alpha, human calcium-dependent protease small subunit and human annexin V. All five proteins were confirmed to be down-regulated by Western blotting. The analytical results of this study help to provide insight into the effect of SACE on the gene expression of the tumor cells.     

Induction of monocyte chemoattractant protein-1 release from A549 cells by agonists of protease-activated receptor-1 and -2

Hypersecretion of cytokines and serine proteases has been observed in asthma. However, the influence of proteases and protease-activated receptors (PARs) on monocyte chemoattractant protein-1 (MCP-1) release from airway epithelial cells remains largely unknown. In the present study, A549 cells were challenged with agonists of PARs, and levels of MCP-1 released in the supernatant and mRNA expression were examined by ELISA and real time polymerase chain reaction (PCR), respectively. The results show that thrombin, tryptase, elastase and trypsin induced an up to 6.5-, 1.8-, 1.6-, and 3.1-fold increase in MCP-1 release from A549 cells, respectively, following a 16-h incubation period. The protease-induced secretion of MCP-1 can be abolished by specific protease inhibitors. Agonist peptides of PAR-1 and PAR-2 stimulate MCP-1 secretion up to 15- and 12.7-fold, respectively. Real-time PCR showed that MCP-1 mRNA is up-regulated by the serine proteases tested and by agonist peptides of PAR-1 and PAR-2. In conclusion, serine proteases can stimulate MCP-1 release from A549 cells possibly through a PARs-related mechanism, suggesting that they are likely to contribute to MCP-1-related airway inflammatory disorders in man.     

Effect of halothane on lung carcinoma cells A 549

The halogenated hydrocarbons, such as halothane, are widely used as anesthetics in clinical practice; however their application is often accompanied with metabolic, cardiovascular and respiratory complications. One of the possible factors for this negative outcome might be the severe toxicity of these agents. In this paper, we investigate in vitro effects of halothane on human lung carcinoma A 549 cells, namely on their cytotoxicity, adhesive properties and metabolic activity. The cytotoxicity response of lung carcinoma A 549 cells to halothane was determined by lactate dehydrogenase (LDH) assay (for cytotoxicity), by detachment assay after adhesion to type IV collagen (for cell adhesive properties) and by surface tension measurements of culture medium (for cell metabolic activity). Regarding the cytotoxicity, the determined maximal non-toxic concentration of halothane on A 549 cells, given here as volume percentages (vol.%) was 0.7 vol.% expressed as aqueous concentration in the culture medium. Direct measurement of the actual halothane concentration in the culture medium showed that 0.7 vol.% corresponds to 1.05 mM and 5.25 aqueous-phase minimum alveolar concentration (MAC). Concentrations equal or higher than 1.4 vol.% (2.1 mM; 10.5 MAC) of halothane provoked complete detachment (cell death), or reduction of initial adhesion to collagen IV in half of the cell population. Surfactant production of A 549 cells, registered up to 48 h after halothane treatment, was inhibited by halothane concentrations as low as 0.6 vol.% (0.9 mM; 4.5 MAC). Our results demonstrate that sub toxic halothane concentrations of 0.6 vol.% inhibits surfactant production; concentrations in the range 0.8-1.4 vol.% induce membrane damages and concentrations equal and higher than 1.4 vol.%--cell death of approximately 50% of the cells.     

Pulmonary epithelial CCR3 promotes LPS-induced lung inflammation by mediating release of IL-8

Interleukin (IL)-8 from pulmonary epithelial cells has been suggested to play an important role in the airway inflammation, although the mechanism remains unclear. We envisioned a possibility that pulmonary epithelial CCR3 could be involved in secretion and regulation of IL-8 and promote lipopolysaccharide (LPS)-induced lung inflammation. Human bronchial epithelial cell line NCI-H292 and alveolar type II epithelial cell line A549 were used to test role of CCR3 in production of IL-8 at cellular level. In vivo studies were performed on C57/BL6 mice instilled intratracheally with LPS in a model of acute lung injury (ALI). The activity of a CCR3-specific inhibitor (SB-328437) was measured in both in vitro and in vivo systems. We found that expression of CCR3 in NCI-H292 and A549 cells were increased by 23% and 16%, respectively, 24 h after the challenge with LPS. LPS increased the expression of CCR3 in NCI-H292 and A549 cells in a time-dependent manner, which was inhibited significantly by SB-328437. SB-328437 also diminished neutrophil recruitment in alveolar airspaces and improved LPS-induced ALI and production of IL-8 in bronchoalveolar lavage fluid. These results suggest that pulmonary epithelial CCR3 be involved in progression of LPS-induced lung inflammation by mediating release of IL-8. CCR3 in pulmonary epithelia may be an attractive target for development of therapies for ALI.     

MMP-12 induces IL-8/CXCL8 secretion through EGFR and ERK1/2 activation in epithelial cells

Macrophage metalloelastase (MMP-12) is described to be involved in pulmonary inflammatory response. To determine the mechanisms linking MMP-12 and inflammation, we examined the effect of recombinant human MMP-12 (rhMMP-12) catalytic domain on IL-8/CXCL8 production in cultured human airway epithelial (A549) cells. Stimulation with rhMMP-12 resulted in a concentration-dependent IL-8/CXCL8 synthesis 6 h later. Similar results were also observed in cultured BEAS-2B bronchial epithelial cells. In A549 cells, synthetic matrix metalloproteinase (MMP) inhibitors prevented rhMMP-12-induced IL-8/CXCL8 release. We further demonstrated that in A549 cells, rhMMP-12 induced transient, peaking at 5 min, activation of ERK1/2. Selective MEK inhibitors (U0126 and PD-98059) blocked both IL-8/CXCL8 release and ERK1/2 phosphorylation. IL-8/CXCL8 induction and ERK1/2 activation were preceded by EGF receptor (EGFR) tyrosine phosphorylation, within 2 min, and reduced by selective EGFR tyrosine kinase inhibitors (AG-1478 and PD168393) by a neutralizing EGFR antibody and by small interfering RNA oligonucleotides directed against EGFR, implicating EGFR activation. In addition, we observed an activation of c-Fos in A549 cells stimulated by rhMMP-12, dependent on ERK1/2. Using small interfering technique, we showed that c-Fos is involved in rhMMP-12-induced IL-8/CXCL8 production. From these results, we conclude that one mechanism, by which MMP-12 induces IL-8/CXCL8 release from the alveolar epithelium, is the EGFR/ERK1/2/activating protein-1 pathway.     

The plasminogen-like molecule apically secreted by epithelial thyroid cells is sulfated

Plasminogen (Pl), a circulating protease synthesized in the liver, is also present in several tissues. In the thyroid gland a Pl-like protease was found in the apical lumen where it is involved, through its proteolytic activity, in luminal degradation of thyroglobulin (Tg). Here, we showed for the first time that the Pl-like protease apically secreted by epithelial thyroid cells is sulfated, both on tyrosine residue(s) and on oligosaccharide side chains. The Pl molecule is composed of a large N-terminal moiety made of five distinct Kringle domains (K1-K5) separated by small peptidic fragments, and of a C-terminal domain with serine protease activity. Using a software tool able to predict tyrosine sulfation sites in protein sequences we localized the potential tyrosine sulfation sites of Pl. Then, we became aware that, whatever the species considered, at least three of the four potential tyrosine sulfation sites of Pl were located on Kringle sites, and more precisely, for K1, on the highly conserved binding domain of K1. We determined with the same software tool which potential sulfation sites were the most likely to be really sulfated. We hypothesize that the sulfation of these sites modulates the binding properties of Pl.     

Thiazolidinedione inhibits production of RANTES in a cytokine-treated human lung epithelial cell line.

The chemokine RANTES is a potent chemoattractant for eosinophils. RANTES is produced by lung epithelial cells during eosinophil-rich inflammatory diseases such as asthma. In this study, we examined the effects of thiazolidinediones (TZD) on RANTES expression in a human lung epithelial cell line, A549. In A549 cells, interleukin-1beta and tumor necrosis factor-alpha induced endogenous RANTES protein secretion, mRNA expression, and promoter activity. The TZD inhibited these effects. Our data indicate that the suppression of the expression of RANTES can be accomplished by TZD treatment, raising the possibility that TZD might be of therapeutic value in diseases such as asthma.     

Oroxylin A sensitizes non-small cell lung cancer cells to anoikis via glucose-deprivation-like mechanisms: c-Src and hexokinase II

BackgroundCellular metabolism, particularly glycolysis, is altered during the metastatic process and is highly associated with tumor progression and apoptosis resistance. Oroxylin A, a natural plant flavonoid, exhibits chemopreventive and therapeutic anti-inflammatory and anticancer potential. However, the anticancer effects of oroxylin A on non-small cell lung carcinoma (NSCLC) remain poorly understood.MethodsIn vitro studies were performed using 2D and 3D conditions. The effects on anoikis-sensitization and glycolysis-inhibition of oroxylin A in human non-small cell lung cancer A549 cells were examined. In vivo murine lung metastasis experiments were utilized to assess the anti-metastatic capacity of oroxylin A.ResultsROS-mediated activation of c-Src following detachment caused anoikis resistance in A549 cells. Oroxylin A sensitized A549 cells to anoikis by inactivating the c-Src/AKT/HK II pathway in addition to inducing the dissociation of HK II from mitochondria. Prior to sensitizing A549 cells to anoikis, oroxylin A decreased the ATP level and inhibited glycolysis. Furthermore, oroxylin A inhibited lung metastasis of A549 cells in vivo in nude mice.ConclusionsOroxylin A sensitized anoikis, which underlies distinct glucose-deprivation-like mechanisms that involved c-Src and HK II.General significanceThe findings in this study indicated that oroxylin A could potentially be utilized in the development of improved metastatic cancer treatments.     

Effect of proinflammatory cytokines on interleukin-8 mRNA expression and protein production by isolated human alveolar epithelial cells type II in primary culture

Alveolar epithelial cells type II (AEC-II) are ideally situated to regulate the recruitment and activation of different types of cells through the production of chemokines in response to inflammatory stimulation from the alveolar space. We hypothesized that these cells are important producers of interleukin-8 (IL-8) in the lung. This lead us to investigate the capacity of isolated human AEC-II cells to release IL-8 and whether this IL-8 release is regulated by proinflammatory cytokines, i.e. IL-1 beta, TNF-alpha and IFN-gamma. We isolated AEC-II from tumor-free sections of human lungs obtained by pneumectomy and purified the cells by magnetic activated cell sorting. For control experiments the AEC-II-like cell line A549 was used. IL-8 concentration was measured by ELISA in supernatants of unstimulated and LPS-, IL-1 beta-, TNF-alpha- and IFN-gamma- stimulated cells. IL-8 mRNA expression was evaluated by RT-PCR. Spontaneous IL-8 mRNA expression and protein secretion by AEC-II were significantly higher in comparison with A549 cells. TNF-alpha increased both IL-8 mRNA expression and protein production, whereas IL-1 beta slightly increased IL-8 release but did not change mRNA expression in AEC-II. LPS and IFN-gamma did not influence IL-8 expression in AEC-II and A549 cells. These results show considerable differences between A549 cell and AEC-II. The latter are capable of producing IL-8 under the control of proinflammatory cytokines. Our findings demonstrate that the modulation of IL-8 release in AEC-II may have an important impact on the immunoreactivity of these cells during pulmonary inflammation in vivo.     

Ethyl pyruvate modulates adhesive and secretory reactions in human lung epithelial cells

AimsEthyl pyruvate (EtP) may prolong survival and ameliorate organ dysfunction in a variety of models of critical illness, e.g. severe sepsis and acute respiratory syndrome, by modulation of the expression of inflammatory mediators. Here, we studied the effects of EtP on the reactions in and between human neutrophils and lung epithelial (A549) cells in vitro.Main methodsNeutrophil adhesion to, surface expression of ICAM-1 and VCAM-1 on, and release of IL-8 and G-CSF from A549 cells were measured by ELISA after stimulation with IL-1β or TNFα.Key findingsAfter treatment of A549 cells with EtP, a substantial reduction in the cytokine-induced adhesion of neutrophils to monolayers was noted, whereas sodium pyruvate (NaP) conferred no reduction. Likewise, treatment with 2.5–10 mM EtP (but not NaP) reduced ICAM-1 and VCAM-1 expression in a dose-dependent fashion. The generation of cytokines of significance for adhesive and proliferative events in host defense, IL-8 and G-CSF, was also potently impaired by EtP.SignificanceExposure of lung epithelial cells to 2.5–10 mM EtP inhibited the generation of inflammatory-regulating cytokines IL-8 and G-CSF, reduced ICAM-1 and VCAM-1 expression and impeded the adhesiveness of neutrophils to lung epithelial cells. These are reactions of significance for early inflammatory responses in the lung, suggesting a role for EtP as a treatment for acute pulmonary conditions.     

Synthesis and release of sulfated glycoproteins by cultured glial cells

Both primary cultured glial cells and cloned (C-6) glioma cells have been shown to synthesize and release sulfated glycoproteins. It was found that N-linked tri- and tetra-antennary glycopeptides recovered from the glycoproteins contained most of the (35S) sulfate label. C-6 glial cells showed a higher rate of oligosaccharide sulfation than the primary glial cultures. Both cell types exhibited a high rate of release of sulfated glycoproteins into the medium. The ratio of 35S/3H incorporated from (35S) sulfate and (3H) glucosamine in the released material was higher than that of the glycoproteins associated with the cell, indicating an enrichment of sulfated glycoproteins in the secreted materials. Monensin inhibited both the synthesis and the release of sulfated glycoproteins.