Pulmonary Type II-like Epithelial Cells Research Articles

Toxic effect of cooking oil fumes in primary fetal pulmonary type II-like epithelial cells

Epidemiological studies indicated that there is an increased risk of respiratory tract cancer among cooks and bakers. The cooking oil fumes are believed to conduct this risk, and many studies have focused on evaluating the mutagenicity and finding the mutagenic components in oil fumes. COFs contains two major classes of compounds. One class consists of polycyclic aromatic hydrocarbons (PAHs), such as benzo[a]pyrene, benzo[b]fluoranthene, fluoranthene, and benzo[g,h,i]perylene. BaP is a known immunosuppressant. It can also alter cell cycle progression, induce inflammation, and impair DNA repair and apoptotic processes leading to aberrant cellular functioning. This study investigates the effect of toxicity of cooking oil fumes (COFs) in primary ICR mice’ fetal lung type II-like epithelium cells (AEC II). The cells were cultured in different concentrations (0, 12.5, 25, 50, 100, and 200μg/ml) of COFs for different time periods. The results showed that cell viability decreased in a dose- and time- dependent manner, which is accompanied by increased malondialdehyde (MDA) level and decreased superoxide dismutase (SOD) and glutathione (GSH) activities. Moreover, comet assay suggested DNA damage, as well as increased production of DNA adducts induced by PAHs. The present study also shows that COFs may disturb cell cycles even at a very low dose. In summary, the present study indicates that COFs may lead to toxicity in AEC II cells.

Comparative in vitro cytotoxicity assessment of selected gaseous compounds in human alveolar epithelial cells

Exposure to airborne contaminants is significantly associated with human health risks, ranging from bronchial reactivity to morbidity and mortality due to acute intense or long term low level repeated exposures. However, the precise mechanisms that derive such effects are not always understood. Although inhalation studies are technologically complicated, correct hazard characterisation is essential for comparable risk assessment of inhaled materials. The aim of this study was to investigate the comparative in vitro cytotoxicity of selected gaseous contaminants in human lung cells. The cytotoxicity of nitrogen dioxide (NO 2), sulphur dioxide (SO 2) and ammonia (NH 3) was investigated in A549- human pulmonary type II-like epithelial cell lines cultured on porous membranes in Snapwell inserts. A dynamic direct exposure method was established by utilizing the horizontal diffusion chamber system (Harvard Apparatus Inc, USA) for delivery of test atmospheres. Test atmospheres were generated using a dynamic direct dilution method and the concentration monitored by appropriate analytical methods. A diversified battery of in vitro assays including the MTS (tetrazolium salt; Promega), NRU (neutral red uptake; Sigma) and ATP (adenosine triphosphate; Promega) assays was implemented. Airborne IC 50 (50% inhibitory concentration) values were calculated based on the most sensitive assay for each test gas including NO 2 (IC 50 = 11 ± 3.54 ppm; NRU) > SO 2 (IC 50 = 48 ± 2.83 ppm; ATP)> and NH 3 (IC 50 = 199 ± 1.41 ppm; MTS). However, all in vitro assays revealed similar toxicity ranking for selected gaseous contaminants. Identical toxicity ranking was achieved using both in vitro and published in vivo data. This comparison suggests that results of in vitro methods are comparable to in vivo data and may provide greater sensitivity for respiratory toxicity studies of gaseous contaminants.

An experimental in vitro model for dynamic direct exposure of human cells to airborne contaminants

The aim of this study was to establish a dynamic in vitro model for direct exposure of human cells to gaseous contaminants to investigate the cellular responses to airborne chemical exposures. Nitrogen dioxide (NO 2) was selected as a model gas compound. Standard test atmospheres were generated (2.5–10 ppm), using a dynamic direct dilution method. Human cells including: A549 pulmonary type II-like epithelial cell lines and skin fibroblasts were grown on porous membranes. Human cells on snapwell inserts were placed in horizontal diffusion chambers and exposed to various airborne concentrations of NO 2 directly at the air/liquid interface for 1 h at 37 °C. Cytotoxicity of the test gas was investigated using the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium), NRU (neutral red uptake) and ATP (Adenosine triphosphate) assays. Dose-dependent effects of NO 2 were observed in human cells tested which resulted in a significant reduction of cell viability at concentrations normally encountered in workplace environments ( p < 0.05). Our findings suggest that the dynamic direct exposure method can be used for in vitro inhalational and dermal toxicity studies and potentially as an advanced technology for biomonitoring of airborne contaminants in future occupational and environmental toxicity assessments.

Asbestos induction of nuclear transcription factors and interleukin 8 gene regulation.

Proinflammatory cytokines and chemotactic peptides are strongly implicated as mediators of the pathophysiologic responses of asbestosis and other chronic inflammatory lung diseases. Recent studies in our laboratory have demonstrated that asbestos fibers stimulate lung epithelial cells to produce interleukin-8 (IL-8), the major neutrophil chemoattractant in the lung. The mechanisms by which asbestos regulates IL-8 expression were studied using the pulmonary type II-like epithelial cell line A549. Membrane permeable hydroxyl scavengers inhibited asbestos induced IL-8 expression. Using A549 cells transfected with the -546 IL-8 construct linked to a chloramphenicol acetyl transferase reporter gene, we have shown that these antioxidants directly inhibited asbestos-stimulated IL-8 promoter-dependent transcription. Asbestos fibers as well as reactive oxygen species generating systems hypoxanthine-xanthine oxidase and hydrogen peroxide stimulated DNA binding activity to the regulatory elements in the IL-8 promoter, binding sites of nuclear factor (NF)-kappaB- and NF-IL-6-like transcription factors. Asbestos-inducible DNA binding activity was partially inhibited by tetramethylthiourea, a hydroxyl radical scavenger. IL-8 secretion was also suppressed by staurosporine, an inhibitor of protein kinase C, and by inhibitors of tyrosine kinase such as herbimycin A and genistein. The suppression paralleled the effect of these inhibitors on asbestos-induced DNA binding to the NF-kappaB- and NF-IL-6-like binding sites of the IL-8 promoter. Taken together, the results suggest that asbestos-induced redox changes and phosphorylation events, mediated by staurosporine-sensitive and tyrosine kinase(s), activate nuclear proteins which recognize the NF-kappaB/NF-IL-6 binding sites of the IL-8 promoter and contribute to the regulation of IL-8 gene expression.

Cytotoxicity of NO 2 gas to cultured human and murine cells in an inverted monolayer exposure system

We report the development of an optimised exposure system for the exposure of inverted cell cultures to NO 2, which presents several advantages over conventional, right-side-up exposure systems. Firstly, the cells may be directly exposed to NO 2 in the gas phase for up to 1 h, without the interposition of an aqueous layer. Secondly, the chamber system allows simple and precise control of the gas concentration during the exposure. Finally, the system allows the simultaneous exposure of large numbers of cells under sterile conditions, facilitating further culture of the cells after the exposure period. We report the application of this system to a comparative study of the toxicity of NO 2 in three different cell types involved in the circuit of the inflammatory response, the IC-21 murine macrophage line, the A-549 human pulmonary type II-like epithelial cell line and human umbilical vein endothelial cells. As little as 2 ppm NO 2 for 20 min reduced colony-forming efficiency of HUVE cells and A-549 cells to 35% and 78% of their air controls, respectively. Exposure to 5 ppm NO 2 for 1 h increased lactate dehydrogenase release of HUVE cells, IC-21 macrophages and A-549 cells from 7.9% to 21.6%, 5.7% to 10.9% and 2.0% to 3.4%, respectively, whilst 10 ppm NO 2 for 1 h lowered cellular glutathione in HUVE cells, IC-21 cells and A-549 cells from 35.2 nmol/mg to 23.3 nmol/mg, from 45.0 nmol/mg to 31.0 nmol/mg and from 86.4 nmol/mg to 69.2 nmol/mg, respectively. Of the cell types tested it was shown that HUVE cells and IC-21 cells were equally sensitive to the toxicity of NO 2 whilst A-549 cells displayed considerable resistance, perhaps due to the considerably higher levels of glutathione in this cell line. Further, a comparison of the sensitivity of HUVE cells to NO 2 using several modes of exposure (inverted and right-side-up (either rocked or static)) and the assay of lactate dehydrogenase and [ 3H]deoxyglucose release, revealed that the present inverted exposure technique potentiated the acute cytotoxicity of the gas.

Interleukin-8 is a potent mediator of eosinophil chemotaxis through endothelium and epithelium

Interleukin-8 (IL-8), a potent pro-inflammatory cytokine, has been shown to have chemotactic activity for neutrophils, lymphocytes, and basophils. Effects of IL-8 on eosinophil chemotaxis are unresolved. Because eosinophils accumulate at the site of allergic inflammation and may play a role in the pathogenesis of asthma, we investigated the eosinophilotactic capacity of IL-8. We examined the ability of IL-8 to induce human eosinophil migration across 3-microns pore naked filters, and human umbilical vein endothelial cell and human pulmonary type II-like epithelial cell (A549) monolayers cultured on these filters. IL-8 induced similar dose-related eosinophil migration through all three barriers. Kinetic experiments indicated more rapid migration through noncellular barriers but equivalent migration through all barriers by 3 h. Chemotactic/chemokinetic data show that IL-8-induced eosinophil migration is chemotactic. We also determined that the ability of IL-8 to induce transcellular migration was unique in comparison with other cytokines and was not dependent on the use of fresh vs. passaged monolayer cells as barriers. Therefore our data indicate that IL-8 may play a significant role in tissue eosinophilia observed in allergic respiratory diseases.

Interleukin-8-induced transcellular neutrophil migration is facilitated by endothelial and pulmonary epithelial cells.

Interleukin-8 (IL-8) is an 8,000 D protein produced by many cells and has potent neutrophil chemoattractant and activating properties. Indeed, there is substantial data supporting a role for IL-8 in neutrophilic lung inflammatory responses. In vivo, neutrophils must adhere to and then migrate across endothelial and epithelial cell barriers in order to reach inflammatory foci. Therefore, we examined IL-8-induced neutrophil migration through naked filters and through human umbilical vein endothelial (HUVE) cells and human pulmonary type II-like epithelial cells (A549) cultured on these filters. IL-8 induced both dose- and time-dependent migration of neutrophils across all three barriers. At IL-8 concentrations greater than 10(-8) M, neutrophil migration across both endothelial and epithelial cell barriers was significantly greater than that observed across naked filters. In addition, time-course experiments indicated that neutrophil migration continued to occur for up to 3 h across both cellular barriers while neutrophil migration across naked filters plateaued by approximately 60 to 90 min. Migration of neutrophils through all barriers was completely inhibited by anti-IL-8 neutralizing antibody. The increased migration observed through both cellular barriers was not due to either changes in chemotactic gradients or the production of other soluble chemotactic factors by IL-8-stimulated HUVE and A549 cells versus naked filters. Furthermore, pretreatment of monolayers with actinomycin-D had no effect on the degree of transcellular migration. Thus, the facilitation of neutrophil migration through HUVE and A549 monolayers is not dependent upon new protein synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Alveolar macrophage-derived cytokines induce monocyte chemoattractant protein-1 expression from human pulmonary type II-like epithelial cells

Many acute and chronic lung diseases are characterized by the presence of increased numbers of activated macrophages. These macrophages are derived predominantly from newly recruited peripheral blood monocytes and may play a role in the amplification and perpetuation of an initial lung insult. The process of inflammatory cell recruitment is poorly understood, although the expression of inflammatory cell-specific chemoattractants and subsequent generation of chemotactic gradients is likely involved. Although immune cells such as macrophages and lymphocytes are known to generate several inflammatory cell chemoattractants, parenchymal cells can also synthesize and secrete a number of bioactive factors. We now demonstrate the generation of significant monocyte chemotactic activity from tumor necrosis factor (TNF)-alpha and interleukin (IL)-1 beta-treated pulmonary type II-like epithelial cells (A549). The predominant inducible monocyte chemotaxin had an estimated molecular mass of approximately 14-15 kDa and was neutralized by specific antibody to human monocyte chemotactic protein-1 (MCP-1). Induction of activity was accompanied by increases in steady-state mRNA level for MCP-1. These data are consistent with the induction of MCP-1 expression from A549 cells by TNF and IL-1. MCP-1 production from A549 cells could be induced by lipopolysaccharide (LPS)-stimulated alveolar macrophage (AM)-conditioned media, but not by LPS alone. The inducing activity in AM-conditioned media was neutralized with specific antibodies to IL-1 beta, but not TNF-alpha. Our findings suggest that the alveolar epithelium can participate in inflammatory cell recruitment via the production of MCP-1 and that cytokine networking between contiguous alveolar macrophages and the pulmonary epithelium may be essential for parenchymal cell MCP-1 expression.

Interleukin-8 gene expression by a pulmonary epithelial cell line. A model for cytokine networks in the lung.

Cellular constituents of the alveolar-capillary wall may be key participants in the recruitment of polymorphonuclear leukocytes to the lung through the generation of the novel neutrophil chemotactic peptide interleukin-8 (IL-8). This interaction appears to occur via the ability of human alveolar macrophage (AM)-derived monokines, tumor necrosis factor (TNF), and interleukin-1 (IL-1) to induce gene expression of IL-8 from pulmonary type II-like epithelial cells (A549). Northern blot analysis demonstrated that steady-state IL-8 mRNA expression, by either TNF- or IL-1 beta-treated A549 cells, occurred in both a dose- and time-dependent fashion. Similarly, extracellular antigenic IL-8, as assessed by specific ELISA, was expressed from TNF- or IL-1 beta-stimulated epithelial cells in a time-dependent fashion with maximal IL-8 antigen detected at 24 h poststimulation. Immunohistochemical staining utilizing rabbit anti-human IL-8 antibody identified immunoreactive, cell-associated IL-8 antigen as early as 8 h post-TNF or IL-1 beta stimulation. A549-generated neutrophil chemotactic bioactivity paralleled IL-8 steady-state mRNA levels. Signal specificity was demonstrated in this system as IL-8 mRNA or protein expression by lipopolysaccharide (LPS)-treated A549 cells was not different from unstimulated cells. Although LPS did not serve as a direct stimulus for the production of IL-8 by type II-like epithelial cells, the condition media from LPS-challenged AM induced a significant expression of IL-8 mRNA by the A549 cells. 24-h conditioned media from LPS-treated cells was as potent as either IL-1 beta or TNF in generating steady-state IL-8 mRNA by A549 cells. Preincubation of LPS-treated AM-conditioned media with anti-human TNF or IL-1 beta neutralizing antibodies resulted in significant abrogation of IL-8 gene expression by A549 pulmonary epithelial cells. These findings demonstrate potential cell-to-cell communication circuits that may be important between AMs and pulmonary epithelial cells during the recruitment phase of acute lung inflammation.