TNF-alpha-induced up-regulation of intercellular adhesion molecule-1 is regulated by a Rac-ROS-dependent cascade in human airway epithelial cells

Abstract

Up-regulation of intercellular adhesion molecule-1 (ICAM-1) in the lung airway epithelium is associated with the epithelium-leukocyte interaction, critical for the pathogenesis of various lung airway inflammatory diseases such as asthma. However, little is known about how ICAM-1 is up-regulated in human airway epithelial cells. In this study, we show that tumor TNF-alpha induces monocyte adhesion to A549 human lung airway epithelium and also up-regulation of ICAM-1 expression. These effects were significantly diminished by pre-treatment with diphenyliodonium (DPI), an inhibitor of NADPH oxidase-like flavoenzyme. In addition, the level of reactive oxygen species (ROS) was increased in response to TNF-alpha in A549 cells, suggesting a potential role of ROS in the TNF-alpha-induced signaling to ICAM-1 expression and monocyte adhesion to airway epithelium. Further, we found out that expression of RacN17, a dominant negative mutant of Rac1, suppressed TNF-alpha-induced ROS generation, ICAM-1 expression, and monocyte adhesion to airway epithelium. These findings suggest that Rac1 lies upstream of ROS generation in the TNF-alpha-induced signaling to ICAM-1 expression in airway epithelium. Finally, pretreatment with pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-kappaB, reduced TNF-alpha-induced ICAM-1 expression and both DPI and RacN17 significantly diminished NF-kappaB activation in response to TNF-alpha. Together, we propose that Rac1-ROS-linked cascade mediate TNF-alpha-induced ICAM-1 up-regulation in the airway epithelium via NF-kappaB-dependent manner.

Figure 1

TNF-α induced monocyte adhesion to A549 lung epithelium. A549 cells were grown to confluence in 35-mm plates and stimulated with 10 ng/ml TNF-α or control buffer for 18 h. Then, Calcein-AM-prelabled U937 monocytes were added and we incubated them for additional 15 min. Adhesion of monocytes in response to TNF-α was observed using fluorescence microscopy (A) or counted after the indicated treatment times (B). Cells were counted from six different fields. Results are expressed as means ± SD of three independent experiments.

Figure 2

ROS are involved in the TNF-α-induced monocyte adhesion to lung epithelium. (A) A549 cells were stimulated with 10 ng/ml TNF-α for the indicated times, or (B) cells were pretreated with DPI for 30 min for the indicated concentration before stimulation with TNF-α (10 ng/ml) or control buffer (PBS) for 12 h. Quantification of ICAM-1 band intensity was performed by Image J version 1.24 software (developed at the National Institutes of Health, USA) and expressed in terms of fold increase of control (α-tubulin) for three independent experiments. Statistical significance of quantification was assessed using unpaired Student's T test (P < 0.05). (C) A549 cells were grown to confluence in 35-mm plates and stimulated with 10 ng/ml TNF-α or control buffer for 18 h in the absence or presence of DPI 5 µM, then we added Calcein-AM-prelabled U937 monocyte and incubated them for additional 15 min. Adhesion of monocytes was observed using fluorescence microscopy. These results shown are representative of at least three independent experiments.

Figure 3

TNF-α induces ROS generation via NADPH oxidase-like flavoprotein in A549 cells. (A) A549 cells were grown to confluence on cover slips for 2 days and serum-starved with 0.5% FBS-supplemented RPMI for additional 2 days. Then, 20 ng/ml TNF-α was applied for the indicated times. DCF fluorescence, reflecting relative levels of ROS (arbitrary units), was imaged using a confocal laser-scanning microscope and quantified. Statistical significance of ROS measurements was assessed using unpaired Student's test (^*P < 0.05, ^**P < 0.01). (B) Serum-starved A549 cells were stimulated with 20 ng/ml TNF-α for 30 min in the presence or absence of DPI (5 µM). Data are expressed as means ± SD (n = 30 cells) relative to untreated control A549 cells of three independent experiments. Statistical significance of ROS measurements was assessed using unpaired Student's test (^*P < 0.05, ^**P < 0.01).

Figure 4

TNF-α induces ROS generation via Rac1-dependent cascade. (A) A549 cells (2 × 10⁵) were plated in 60 mm plates, maintained in 10% FBS-supplemented RPMI for 48 h after which cells were transiently transfected with pcDNA-Rac^N17 utilizing Lipofectamine-Plus/DNA complex in non-supplemented RPMI. After 3 h, cells were washed and recovered in complete medium, and then cells were exposed to 20 ng/ml TNF-α for 30 min with DCFDA (5 µg/ml). The relative intensity of DCF fluorescence was quantified. (B) Rac1 activation was evaluated in response to TNF-α (10 ng/ml) by the PAK-binding assay as described in Materials and methods. The PAK-bound Rac1-GTP was resolved by SDS-PAGE and transferred on to PVDF. Activated Rac protein, Rac1-GTP, was detected using an anti-Rac1 antibody and, as a control, total Rac protein was also determined by immunoblotting with Rac1 antibody. The relative intensity of Rac activation was measured and expressed as percentages ± SD of three independent experiments.

Figure 5

Rac1 is required for TNF-α-induced ICAM-1 expression and adhesiveness. (A) A549 cells and Rac^N17-transiently-transfected cells were stimulated with 10 ng/ml TNF-α for 12 h. The cell lysates were subjected to immunoblot for detecting the expressions of ICAM-1, Rac1, and HA-Rac^N17. The relative intensity of ICAM-1 band was measured and expressed as percentages ± SD of three independent experiments. (B) Adhesion assays were conducted with A549 and A549 cells transfected with Rac^N17 as described in Figure 1 in the presence or absence of 10 ng/ml TNF-α. Data are expressed as means ± SD (n = 30 cells) of three independent experiments.

Figure 6

Rac1-ROS-linked cascade regulates TNF-α-induced ICAM-1 expression via a stimulation of NF-κB. A549 cells were exposed to TNF-α (10 ng/ml) for 10 min (A) or 12 h (C) in the presence or absence of DPI (A) or PDTC (C) with the indicated concentrations, respectively. Western blotting for IκBα (A) or ICAM-1(C) was carried out to determine the expression levels, respectively. (B) A549 cells were transiently transfected with a reporter gene (pNF-κB-Luc) and DPI was added for 30 min prior to the application of TNF-α (10 ng/ml). The relative luciferase activity was measured. (D) A549 cells were transiently co-transfected with a reporter gene (pNF-κB-Luc) along with pcDNA3 or pcDNA3-Rac^N17 and the relative luciferase activity was measured using luminometer. These results shown are representative of at least three independent experiments. Statistical significance of NF-κB activation was assessed using unpaired Student's test (^*P < 0.05).