Human Epithelial Cell Line A549 Research Articles

Transforming growth factor-beta1 is a potent inhibitor of glutathione synthesis in the lung epithelial cell line A549: transcriptional effect on the GSH rate-limiting enzyme gamma-glutamylcysteine synthetase.

Glutathione (GSH) is an essential antioxidant tripeptide that protects mammalian cells against oxidants and xenobiotics. Patients with fibrotic lung disorders have very low levels of GSH in their alveolar epithelial lining fluid (ELF), whereas transforming growth factor (TGF)-beta is overexpressed in their alveolar epithelial cells. We observed that TGF-beta1 increased susceptibility of the human alveolar epithelial cell line A549 to H2O2-mediated cytotoxicity (P < 0.05), decreased the activities of the antioxidant enzymes glutathione reductase and catalase by 31%, and markedly decreased GSH content in A549 cells (P < 0.01). GSH depletion was associated with an equivalent decrease in the activity of the rate-limiting enzyme in GSH synthesis, gamma-glutamylcysteine synthetase (gamma-GCS) (P < 0.01). Western blot analysis confirmed that the loss of gamma-GCS activity was associated with a marked decrease in gamma-GCS heavy subunit (gamma-GCShs) protein. TGF-beta1 suppressed the steady-state level of messenger RNA (mRNA) for the gamma-GCShs gene, with a maximal effect at 24 h. The half-life of gamma-GCShs mRNA was not affected by TGF-beta1, but transcription of the gene was downregulated as determined with nuclear run-on assays. Our findings indicate for the first time that TGF-beta1 is a potent inhibitor of GSH synthesis in human lung epithelial cells, and that the inhibition is mediated, at least in part, by a transcriptional effect on the gene encoding gamma-GCShs. Regulation of gamma-GCShs gene expression by TGF-beta1 is likely to play an important role in lower respiratory tract GSH homeostasis, and may represent a novel target for therapeutic efforts in lung fibrosis.

Pre-transplant anti-epithelial cell antibodies and graft failure after single lung transplantation

Eighty-five patients undergoing single lung transplantation have been studied to determine the presence of anti-epithelial cell antibodies (AECA) prior to transplantation using the human lung carcinoma epithelial cell line A549 in a microcytotoxicity assay. In addition, 29 healthy volunteers were also assayed for the presence of AECA. Twenty-seven of the 85 recipients exhibited AECA prior to transplantation compared to none of the 29 control subjects ( p = 0.0001). Actuarial graft survival at 1 year was 78% for the AECA negative group compared to 56% for AECA positive recipients ( p = 0.01). No correlation was seen between the presence of AECA and graft rejection as determined by transbronchial biopsy. However, there was an association between AECA and post-transplant infection ( p = NS) where 16 (64%) of the AECA positive recipients had postoperative infection episodes compared to 25 (47%) of the negative recipients. Sodium dodecylsulphate polyacrylamide gel electrophoresis and Western blotting was also performed for 68 of the recipients and antibody reactivity was detected in 22 patients compared to 26 patients exhibiting AECA detectable by microcytotoxicity. The presence of AECA demonstrable by Western blotting did not correlate with graft survival, rejection or infection. In conclusion, AECA detectable prior to single lung transplantation are associated with a decrease in graft survival and with postoperative infections.

Oxidative stress induces NF kappa B DNA binding and inducible NOS mRNA in the human epithelial cell line A549.

Oxidative stress induces NF kappa B DNA binding and inducible NOS mRNA in the human epithelial cell line A549.

The pneumotoxicant paraquat induces IL-8 mRNA in human mononuclear cells and pulmonary epithelial cells

Paraquat (PQ) is a herbicide which is highly pneumotoxic by generating reactive oxygen intermediates (ROI). Pro-inflammatory cytokines, particularly IL-1 and TNF, have been implicated in some ROI-mediated pathologies, including bleomycin toxicity and ischaemia/reperfusion injury. We have studied the effect of PQ on the expression of the neutrophil chemotactic cytokine, IL-8, by human peripheral blood mononuclear cells (PBMC). While almost no IL-8 mRNA was detected in unstimulated cells, PQ (100 μM) induced high mRNA expression with a maximum at 24 h of incubation. While PQ did stimulate the appearance of IL-8 mRNA, no significant production of IL-8 protein was detected. However, PQ potentiated the production of IL-8 in the presence of 1 ng/ml of endotoxin (lipopolysaccharide, LPS). This was paralleled by an increased production of chemotactic activity for neutrophils, indicating that the IL-8 was actually bioactive. Stimulation of IL-8 mRNA by PQ was suppressed by IL-4 and by free radical scavengers (dimethylsulfoxide, mannitol). Increased IL-8 expression by PQ was also observed in the human pulmonary epithelial cell line A549 indicating that the effect of PQ was not specific for PBMC. These findings suggest that IL-8 might be involved in the pulmonary effects of PQ and that its production might be stimulated following an oxidative insult, and might clarify the pathogenetic mechanisms of adult respiratory distress syndrome (ARDS) or oxidant-induced pulmonary fibrosis.

Effect of matrigel on development of A549 lung epithelial cells

Extracellular matrix components are known to influence the growth and differentiation of cultured cells, often causing them to behave more like their in vivo counterparts than cells grown on plastic. To develop an in vitro model in which to study the interaction of pathogenic microorganisms with the lung epithelium, we have undertaken a morphological study of the effects of extracellular matrix components on the human lung alveolar epithelial cell line A549. A549 cells were grown for varying amounts of time on plastic; Costar polycarbonate membrane filter inserts; or on Matrigel coated polycarbonate filter inserts. Cells were fixed for 1 h in 2% glutaraldehyde in 0.1 M cacodylate buffer, post-fixed in 1 % OsO4 for 45 min, dehydrated in ascending ethanol, and embedded in Spurr’s resin.A549 cells grown on a plastic slide formed an even monolayer with cells connected by wellformed junctional complexes or separated by interdigitating microvilli. The cytoplasm showed many polyribosomes, rough endoplasmic reticulum, small golgi complexes, mitochondria, occasional lysosomes and bundles of microfilaments.

Neutrophil-activating peptide ENA-78.

Neutrophil-activating peptide ENA-78 was recently isolated from a human type II-like epithelial cell line A549 (1). ENA-78 is a peptide of 78 amino acids and belongs to the C-X-C supergene family of the chemotactic cytokines (now called chemokines). The C-X-C family contains five other peptides with neutrophil-activating properties, interleukin-8 (IL-8) (2), three closely related members of the GRO family, GROα, GROβ and GROγ (3,4) and neutrophil-activating peptide 2 (NAP-2) (5). Two other members of the same subfamily, platelet factor 4 and interferon-γ induced peptide 10 (γ-IP10) have been shown not to be active on human neutrophils (6,7).KeywordsIdiopathic Pulmonary FibrosisHuman NeutrophilAdult Respiratory Distress SyndromeChemotactic CytokineNeutrophil Chemotactic FactorThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Physiological actions of taurine

Physiological actions of taurine

Detection of piluslike structures on clinical and environmental isolates of Vibrio vulnificus

Twenty clinical isolates of Vibrio vulnificus were compared with 10 environmental strains by using electron microscopy and agglutination assays with human erythrocytes, guinea pig erythrocytes, and Saccharomyces cerevisiae. In addition, the isolates were tested for ability to adhere to the human epithelial cell lines HEp-2 and A549. When examined by electron microscopy, 16 (80%) of the 20 clinical isolates demonstrated the presence of piluslike structures; the composition of the bacterial populations ranged from 0 to 68% piliated cells. In contrast, only 3 (30%) of the 10 environmental isolates were piliated, with a range from 0 to 16% piliated cells. A significant association between the presence of piliated cells and the isolate source was found (P less than 0.05). None of the 30 strains agglutinated erythrocytes or yeast cells. V. vulnificus adherence results obtained with HEp-2 cells showed 10 (50%) of 20 clinical isolates and 0 (0%) of 10 environmental isolates with averages of greater than 10 adherent bacteria per cell, demonstrating a correlation between attachment and the isolate source (P less than 0.05). Selected strains were tested to determine whether methyl alpha-D-mannopyranoside, fructose, or alpha-L-(-)-fucose would inhibit bacterial adherence to HEp-2 cells. Multiple patterns of adherence inhibition were observed. Adherence to A549 cells showed 8 (40%) of 20 clinical isolates and 0 (0%) of 10 environmental strains with averages of greater than 10 adherent bacteria per cell. A statistical association between attachment and the isolate source was demonstrated (P less than 0.05). These data suggest that the presence of piluslike structures and the ability to adhere to human epithelial cell lines may be more closely associated with V. vulnificus isolates from clinical specimens than with environmental strains.