N-acetylcysteine attenuates TNF-alpha-induced p38 MAP kinase activation and p38 MAP kinase-mediated IL-8 production by human pulmonary vascular endothelial cells

Abstract

1. We have previously shown that tumour necrosis factor-alpha (TNF-alpha) activates p38 mitogen-activated protein (MAP) kinase to produce interleukin-8 (IL-8) by human pulmonary vascular endothelial cells. Reactive oxygen species (ROS) including H(2)O(2) generated by TNF-alpha can act as signalling intermediates for cytokine induction; therefore, scavenging ROS by anti-oxidants is important for the regulation of cytokine production. However, the effect of N-acetylcysteine (NAC), which acts as a precursor of glutathione (GSH) synthesis, on TNF-alpha-induced activation of p38 MAP kinase pathway and p38 MAP kinase-mediated IL-8 production by human pulmonary vascular endothelial cells has not been determined. To clarify these issues, we examined the effect of NAC on TNF-alpha-induced activation of p38 MAP kinase, MAP kinase kinase (MKK) 3 and MKK6 which are upstream regulators of p38 MAP kinase, and p38 MAP kinase-mediated IL-8 production. 2. Human pulmonary vascular endothelial cells that had been preincubated with NAC were stimulated with TNF-alpha and then the activation of p38 MAP kinase and MKK3/MKK6 in the cells and IL-8 concentrations in the culture supernatants were determined. 3. Intracellular GSH levels increased in NAC-treated cells. 4. NAC attenuated TNF-alpha-induced activation of p38 MAP kinase and MKK3/MKK6. 5. NAC attenuated p38 MAP kinase-mediated IL-8 production by TNF-alpha-stimulated cells. 6. These results indicate that the cellular reduction and oxidation (redox) regulated by intracellular GSH is critical for TNF-alpha-induced activation of p38 MAP kinase pathway and p38 MAP kinase-mediated IL-8 production by human pulmonary vascular endothelial cells, and we emphasize that anti-oxidant therapy is an important strategy for the treatment of acute lung injury.

Figure 1

Cellular redox state. The intracellular GSH levels in HPAECs were measured at 1 h after incubation with various concentrations of NAC (a). The intracellular H₂O₂ levels in HPAECs that had been pretreated with or without 10 mM of NAC were measured at 5 min after TNF-α (10 ng ml⁻¹) stimulation (b). Relative fluorescence intensity was calculated using unstimulated control cells as standard. The results are expressed as mean±s.d.mean in three different experiments. ^*1 P<0.05 compared with the intracellular GSH levels in the cells cultured with medium. ^*2 P<0.01 compared with the intracellular GSH levels in the cells cultured with medium. ^*3 P<0.01 compared with the intracellular H₂O₂ levels in the cells cultured with medium. ^*4 P<0.05 compared with the intracellular H₂O₂ levels in the cells cultured with TNF-α.

Figure 2

TNF-α activates p38 MAP kinase. HPAECs were stimulated with TNF-α (10 ng ml⁻¹) for the desired times as indicated. The HPAEC lysates were separated by a 15% SDS – PAGE, transferred to membranes, and probed with a specific antibody directed against the phosphorylated threonine and tyrosine of p38 MAP kinase (phospho-p38 MAP kinase; upper panel). These blots were then stripped and reprobed using a phosphorylation state-independent p38 MAP kinase-specific antibody to determine the amounts of p38 MAP kinase blotted (p38MAP kinase; lower panel). P: positive control, protein prepared from C-6 glioma cells stimulated with anisomycin to phosphorylate the threonine and tyrosine of p38 MAP kinase; N: negative control, protein prepared from C-6 glioma cells not stimulated with anisomycin. Blots are representative of three identical experiments independently performed. The amounts of phosphorylated p38 MAP kinase were quantitated by National Institutes of Health (NIH) image analyzer (National Institute of Health, Bethesda, MD, USA) and are presented as the amounts of phosphorylated p38 MAP kinase relative to control cells treated without agonist (1.0). Fold increase in amounts of phosphorylated p38 MAP kinase proteins as indicated as below are expressed as mean±s.d.mean in three different experiments.

Figure 3

NAC attenuates TNF-α-induced p38 MAP kinase activation. HPAECs that had been pretreated either with medium or NAC (10 mM) for 1 h were stimulated with TNF-α (10 ng ml⁻¹) for 5 min. The HPAEC lysates were separated by a 15% SDS – PAGE, transferred to membranes, and probed with a specific antibody directed against the phosphorylated threonine and tyrosine of p38 MAP kinase (phospho-p38 MAP kinase; upper panel). These blots were then stripped and reprobed using a phosphorylation state-independent p38 MAP kinase-specific antibody to show the amounts of p38 MAP kinase blotted (p38MAP kinase; lower panels). The cells were cultured with medium (lane 1), NAC 0.1 mM (lane 2), NAC 1.0 mM (lane 3), NAC 10 mM (lane 4), TNF-α (lane 5), TNF-α+NAC 0.1 mM (lane 6), TNF-α+NAC 1.0 mM (lane 7), and TNF-α+NAC 10 mM (lane 8). Lane P, lane N and fold were described as Figure 2 legend. Blots are representative of three identical experiments independently performed. The amounts of phosphorylated p38 MAP kinase were quantitated by National Institutes of Health (NIH) image analyzer (National Institute of Health, Bethesda, MD, U.S.A.) and are presented as the amounts of phosphorylated p38 MAP kinase relative to control cells treated without agonist (1.0). Fold increase in amounts of phosphorylated p38 MAP kinase proteins as indicated as below are expressed as mean±s.d.mean in three different experiments. Amounts of phosphorylated threonine and tyrosine of p38MAP kinase were significantly lower in NAC-pretreated cells than those in NAC-untreated cells (P<0.01).

Figure 4

NAC attenuates TNF-α-induced MKK3 and MKK6 activation. HPAECs that had been pretreated either with medium or NAC (10 mM) for 1 h were stimulated with TNF-α (10 ng ml⁻¹) for 5 min. The HPAEC lysates were separated by a 15% SDS – PAGE, transferred to membranes, and probed with a specific antibody directed against the phosphorylated serine of MKK3 and MKK6 (phospho-MKK3/MKK6; upper panel). These blots were then stripped and reprobed using a phosphorylation-state independent MKK3-specific antibody to determine total MKK3 levels (MKK3; lower panels). The cells were cultured with medium (lane 1), NAC 10 mM (lane 2), TNF-α (lane 3), TNF-α+NAC 10 mM (lane 4). Lane P: positive protein prepared from NIH3T3 cells stimulated with UV treatment for phosphorylated serine of MKK3 and MKK6; Lane N: negative protein prepared from NIH3T3 cells without UV treatment. Blots are representative of three identical experiments independently performed. The amounts of phosphorylated MKK3/MKK6 were quantitated by National Institutes of Health (NIH) image analyzer (National Institute of Health, Bethesda, MD, U.S.A.) and are presented as the amounts of phosphorylated MKK3/MKK6 relative to control cells treated without agonist (1.0). Fold increase in amounts of phosphorylated MKK3/MKK6 proteins as indicated as below are expressed as mean±s.d.mean in three different experiments. Amounts of phosphorylated threonine and tyrosine of p38MAP kinase were significantly lower in NAC-pretreated cells than those in NAC-untreated cells (P<0.01).

Figure 5

NAC attenuates TNF-α-induced IL-8 production. HPAECs that had been pretreated either with medium or various concentrations of NAC for 1 h were cultured with medium or TNF-α (10 ng ml⁻¹). Simultaneously, the cells that had been pretreated with SB 203580 (10 μM) were stimulated with TNF-α (10 ng ml⁻¹) to examine the effect of SB 203580 on TNF-α-induced IL-8 production. The concentrations of IL-8 in the culture supernatants were determined after 24 h of culture. The results are expressed as mean±s.d. in five different experiments. ^*1 P<0.05 compared with IL-8 concentrations in the cells cultured with TNF-α only. ^*2 P<0.01 compared with IL-8 concentrations in the cells cultured with TNF-α only.