A549 Human Airway Epithelial Cells Research Articles

Influence of cetirizine and levocetirizine on granulocyte-macrophage colony-stimulating factor and interleukin-8 secretion by A549 human airway epithelial cells stimulated with interleukin-1beta

Influence of cetirizine and levocetirizine on granulocyte-macrophage colony-stimulating factor and interleukin-8 secretion by A549 human airway epithelial cells stimulated with interleukin-1beta

Motorcycle Exhaust Particles Induce IL-8 Production Through NF-κB Activation in Human Airway Epithelial Cells

Motorcycle exhaust particles (MEP) are among the major air pollutants, especially in urban area of Taiwan. In our previous study, data showed that MEP induce proinflammatory and proallergic response profiles in BALB/c mice. Effects of MEP on interleukin (IL)-8 production in A549 human airway epithelial cells were further investigated in this study. It was found that MEP enhanced IL-8 protein and mRNA expression in human epithelial cells. Pretreatment with an NF-κB inhibitor (1 mM PDTC), extracellular signal-regulated kinase (ERK) inhibitor (50 μM PD98059), JNK inhibitor (25 μM SP600125), p38 inhibitor (2 μM SB203580), and three antioxidants (500 U/ml superoxide dismutase [SOD], 50 μM vitamin E, 10 mM N-acetylcysteine [NAC]) attenuated the MEP-induced increase in IL-8 production. Through further, direct detection of nuclear factor (NF)-κB activation in epithelial cells using immunoblotting of nuclear p65 and NF-κB reporter assay, data showed that MEP induced nuclear translocation of p65 and enhancement of NF-κB luciferase gene expression. MEP also induced activation of ERK, JNK, and p38 signaling pathways and produced an increase of oxidative stress in A549 cells. By using mitogen-activated protein kinase (MAPK) inhibitors and antioxidant, it was demonstrated that ERK inhibitor, JNK inhibitor, and antioxidants but not p38 inhibitor attenuated the MEP-induced increase in NF-κB reporter activity. In conclusion, evidence shows that filter-trapped particles emitted from unleaded gasoline-fueled, two-stroke motorcycle engines induce an increase in IL-8 production by activation of NF-κB in human airway epithelial cells.

Macrolides inhibit epithelial cell-mediated neutrophil survival by modulating granulocyte macrophage colony-stimulating factor release.

Macrolides have been shown to be effective in treating diffuse panbronchiolitis (DPB), although the precise modes of action remain unclear. At sites of airway inflammation, respiratory epithelium is considered an active participant in regulating neutrophil survival. We therefore examined the effect of erythromycin, clarithromycin, azithromycin, and josamycin on both neutrophil survival and on epithelial-derived factors, which influence neutrophil longevity. Media conditioned with transiently tumor necrosis factor (TNF)-alpha-stimulated A549 human airway epithelial cells prolonged neutrophil survival compared with control media. The presence of dexamethasone during neutrophil culture led to further prolongation of neutrophil survival. In contrast, none of the tested macrolides modulated neutrophil survival, suggesting a lack of direct effect of these drugs. On the other hand, pretreatment of TNF-alpha-stimulated A549 cells by erythromycin, clarithromycin, azithromycin, or dexamethasone, but not josamycin, decreased the neutrophil survival-enhancing effects in a dose-dependent manner. Neutralizing antibodies to granulocyte macrophage colony-stimulating factor (GM-CSF) dampened the prolonged neutrophil survival observed in TNF-alpha-stimulated A549 conditioned media. Erythromycin, clarithromycin, azithromycin, and dexamethasone inhibited TNF-alpha-induced GM-CSF expression in A549 cells at both the protein and messenger RNA levels. These results suggest that macrolides inhibit epithelial cell-mediated neutrophil survival by modulating GM-CSF release, which may, at least in part, explain the effectiveness of this family of drugs on DPB.

Effects by 8-bromo-cyclicAMP on basal and organic dust-induced release of interleukin-6 and interleukin-8 in A549 human airway epithelial cells

Inhalation of organic dust from a swine-confinement building leads to an intense inflammatory reaction with an increased number of inflammatory cells and mediators in the upper and lower respiratory tract of previously unexposed subjects. In vitro the dust induces cytokine release from epithelial cells and alveolar macrophages. It is known that intracellular cyclic AMP (cAMP) contributes to the regulation of inflammatory responses. We therefore investigated whether 8-Bromo-cAMP, a cell membrane-permeable cAMP analogue, would influence release of the cytokines interleukin-6 (IL-6) and IL-8 in a human airway epithelial cell line, A549, exposed to a suspension of the organic dust, and to a supernatant prepared by centrifugation (at low g-force) of a suspension of dust. The large particulate matter was thus sedimented, leaving bacteria, whole and cell wall constituents in the supernatant. Cytokine release was measured with enzyme-linked immunosorbent assay (ELISA). The cytokine release induced by a supernatant was 23% (IL-6) and 27% (IL-8) of the release induced by a dust suspension. 8-Bromo-cAMP (1mM) doubled basal IL-6 release and IL-6 release induced by a dust supernatant ( P<0.01), and increased IL-6 release induced by a dust suspension by 19% ( P<0.05). 8-Bromo-cAMP did not affect basal IL-8 release, partially inhibited (28%) the release of IL-8 induced by a dust suspension ( P<0.01), but increased IL-8 release induced by a dust supernatant by 13% ( P<0.05). In summary, expression of the cytokines IL-6 and IL-8 is differentially regulated by 8-Bromo-cAMP, both with regard to basal and dust-induced release. The results indicate that 8-Bromo-cAMP attenuated IL-8 release by affecting signaling transductions induced by the particulate fraction.

Metabolic activation of A549 human airway epithelial cells by organic dust: A study based on microphysiometry

A Cytosensor™ microphysiometer, which measures extracellular acidification rate (ECAR), was used to study the early metabolic activation by organic dust from a swine confinement building in a human airway epithelial cell line, A549. The dust is known to cause an intense airway inflammatory reaction following inhalation in vivo and cytokine release in vitro. Dimethyl amiloride (DMA) was used to study sodium/proton exchanger (NHE) activity in cells growing at different cell densities. Exposing cells at low density to dust induced an initial release of acid not involving NHE, followed by a sustained DMA-sensitive NHE activation. In cells near high density, NHE was not activated during exposure resulting in a modest increase in ECAR. Exposing cells at high density resulted in a bi-phasic ECAR pattern; an initial increase in proton release followed by an inhibition of ECAR below baseline. Pretreatment with pertussis toxin (PTX), an inhibitor of receptor/G iα-coupled signal transductions did not affect ECAR in low and medium density cells, but abolished the inhibition of ECAR in high-density cells. The dust did not prevent forskolin-induced cAMP accumulation and PTX did not affect cAMP in near-confluent cells suggesting the PTX-effect to be cAMP-independent. The ECAR response to organic dust was similar to that of lipopolysaccharide (LPS) except for high-density cells where PTX did not influence the LPS-induced decrease in ECAR below baseline. In summary, the organic dust induces PTX-sensitive (cAMP independent) signalling in near-confluent A549 epithelial cells and, depending on cell density opposing effects on NHE activity during exposure.

The tyrosine kinase inhibitor genistein increases basal cAMP and potentiates forskolin-induced cAMP accumulation in A549 human airway epithelial cells.

Genistein is often used as an inhibitor of tyrosine kinases. A less studied side effect of genistein is an inhibition of cyclic AMP-phosphodiesterase (cAMP-PDE) activity resulting in increased cAMP accumulation. The effect of genistein on intracellular cAMP-levels, basal and forskolin-induced, was studied in A549 human airway epithelial cells and compared with the unspecific PDE inhibitor, isobutylmethylxanthine (IBMX). It was shown that genistein (50 microM) increased basal cAMP and potentiated forskolin-induced cAMP accumulation to the same extent as IBMX (100 microM). Thus, the use of genistein in studies on signaling transductions may result in erroneous conclusions since increased cAMP may cause or contribute to the observed effects.

Inhibition of inducible nitric oxide synthase expression by interleukin-4 and interleukin-13 in human lung epithelial cells.

Nitric oxide produced by the inducible enzyme, nitric oxide synthase (iNOS), is implicated in immunological and inflammatory processes. We determined the effects of T-helper (Th)2-derived cytokines on the induction of iNOS from an epithelial A549 cell line and human airway epithelial cells stimulated by a mixture of interleukin-1 beta (IL-1 beta), interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha). Interleukin-4 (IL-4) and interleukin-13 (IL-13) but not interleukin-10 (IL-10) inhibited both iNOS mRNA expression and nitrite release in A549 cells. On human airway epithelial cells, IL-4 and IL-13 reduced iNOS mRNA expression. Dexamethasone also inhibited both iNOS expression and nitrite release. Th2 cytokines, IL-4 and IL-13, inhibit iNOS upregulation by Th1 cytokines, indicating an important reciprocal role of Th1 and Th2 T-cell subsets on lung epithelial cells.