Elsevier

Toxicology Letters

Volume 168, Issue 1, 10 January 2007, Pages 1-6
Toxicology Letters

The effect of oxidative stress on macrophages and lung epithelial cells: The role of phosphodiesterases 1 and 4

https://doi.org/10.1016/j.toxlet.2006.10.016Get rights and content

Abstract

Reactive oxygen species (ROS) have been implicated in various pulmonary diseases by causing direct injury to lung epithelial cells. Signalling activity of cells through transcription factors such as nuclear factor kappa B (NF-κB) and AP-1 have been shown to be regulated by ROS, and the release of pro-inflammatory cytokines demonstrated in the study of inflammatory disease. In this study, we examined the effect of the oxidant tert-butylhydroperoxide (tBHP) on mouse J774 macrophages and its ability to cause the release of the pro-inflammatory cytokine tumour necrosis factor alpha (TNF-α). The role of calcium as a signalling molecule was studied using various calcium antagonists. The role of the signalling molecule cAMP was also investigated using phosphodiesterase inhibitors PDE1 and PDE4 families. Oxidative stress was investigated in lung epithelial (A549) cells with and without calcium antagonists and PDE inhibitors with regard to their ability to modulate release of the neutrophil chemoattractant interleukin 8 (IL-8). The oxidant tBHP significantly increased the cytosolic calcium concentration in J774 macrophages, which was prevented by the PDE1 inhibitor. The production of TNF-α protein by J774 macrophages was mediated by a pathway involving calcium as addition of calcium antagonists inhibited the tBHP stimulated increase in the cytokine. Inhibitors of both PDE1 and PDE4 completely prevented the tBHP stimulated TNF-α release suggesting that the cAMP pathway may be important in the oxidant induced signalling pathway leading to gene expression of pro-inflammatory cytokines. In the presence of oxidant alone, A549 epithelial cells released significant amounts of IL-8, which was inhibited by both calcium antagonist treatment and PDE inhibition treatment. These data suggest that ROS-mediated lung inflammation could be mediated at least in part by calcium and elevated PDE activity associated with decreased cAMP in both macrophages and epithelial cells. Inhibition of these pathways may provide a route for treatment of inflammatory lung diseases.

Introduction

Reactive oxygen species (ROS) have been implicated in a variety of pulmonary diseases including acute respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease (COPD), asthma and interstitial pulmonary fibrosis (Hoidal, 2001). In addition to causing direct injury to lung epithelial cells, ROS are involved in the regulation of signalling activity, in particular through transcription factors such as nuclear factor kappa B (NF-κB) and activating protein 1 (AP-1) thus enhancing inflammation (Sen and Packer, 1996). The pro-inflammatory cytokine tumour necrosis factor alpha (TNF-α) is a key protein in the regulation of inflammation and the activation of pro-inflammatory cell signalling has been widely investigated in the study of inflammatory disease (Haddad, 2002).

Cyclic AMP (cAMP) is an important second messenger in the cell, the degradation of which occurs through cAMP phosphodiesterase (PDE) activity (Hoffmann et al., 1999). Cyclic AMP phosphodiesterases therefore have a key function in the regulation of cellular processes by altering cAMP levels and the modulation of a variety of physiological responses (Hoffmann et al., 1999). Increased phosphodiesterase activity has been observed in mononuclear cells (monocytes and macrophages) in response to several pro-inflammatory stimuli, including histamine, LPS and cytokines such as interferon (IFN)-γ and interleukin 4 (IL-4) (Souness et al., 2000). Marx et al. (2002) confirmed that type 4 PDE inhibitors were effective in inhibiting TNF-α cytokine release from human blood and from nasal polyp cells in a dose-dependent manner. PDE 4 inhibitors have also been shown to suppress arachidonic acid breakdown, phagocytosis and production of reactive oxygen species in mononuclear phagocytes (Souness et al., 2000). The generation of TNF-α by both monocytes and macrophages is sensitive to the inhibitory effects of PDE 4 inhibitors. The mechanism by which PDE 4 inhibitors suppress TNFα release is uncertain although it is thought that an effect on mRNA expression occurs (Probhakar et al., 1994). Studies have also reported that PDE 4 inhibitors have the ability to block leukocyte adhesion as well as expression of cell adhesion molecules such as vascular cell adhesion molecule 1 (VCAM 1) (Silva et al., 2001).

The release of pro-inflammatory mediators such as the cytokine TNF-α is driven by intracellular calcium-related signalling pathways in diseases such as sepsis (Sayeed, 1996, Drouet et al., 1991, Rhoades et al., 1992). Calcium is released from the endoplasmic reticulum stores on stimulation of the cell, leading to a calcium influx across the plasma membrane via calcium channels (Hoyal et al., 1998). Various pathogenic particles have been shown to produce changes in calcium flux within the cell (Faux et al., 1994, Lim et al., 1997, Stone et al., 2000) and a large number of physiological and pathological cellular functions could be stimulated via calcium signalling. The intracellular signalling molecule calcium has also been shown to play an important role in the induction of TNF-α cytokine protein release from macrophages exposed to ROS derived from ultrafine or nanoparticles (particles less than 100 nm diameter) (Brown et al., 2004). The PDE1 family of enzymes have also been shown to be stimulated by the calcium/calmodulin complex, providing a link between calcium and cAMP signalling (Yan, 2005).

In this study, we have examined the effect of the oxidant tert-butylhydroperoxide (tBHP) and its ability to cause TNF-α release in J774 mouse macrophages. We also examined the role of calcium by including a range of calcium antagonists such as verapamil (calcium channel blocker), BAPTA-AM (intracellular calcium chellator) and W7 (calmodulin inhibitor). The role of the signalling molecule cAMP was investigated using the PDE 1 inhibitor 8-Me-IBMX and the PDE 4 inhibitor RO-20-1724. We have also studied the effect of oxidative stress using the human alveolar epithelial cell line (A549) with and without calcium antagonists and PDE's with regard to their ability to release the neutrophil chemoattractant IL-8.

Section snippets

J774 cell culture

The murine macrophage cell line J774 was cultured in RPMI-1640 medium supplemented with 10% heat inactivated foetal bovine serum (FBS), 2 mM l-glutamine, 0.06 U/ml penicillin and 30 μg/ml streptomycin (all obtained from Life Technologies) at 37 °C with an atmosphere of 5% CO2.

J774 cell treatment with tBHP and phosphodiesterase (iPDE) inhibitors

Cells were seeded in 24-well plates at a density of 0.5 × 106 cells/ml (1 ml/well) and incubated overnight at 37 °C, 5% CO2 prior to treatment. The medium was removed and the cells were treated with 250 μl of RPMI medium containing

TNF-α production by J774 cells after iPDE 1 and iPDE 4 treatment

Tert-butylhydroperoxide alone significantly increased TNF-α protein release compared to the control (p < 0.01), however, both iPDE 1 and iPDE 4 significantly reduced the tBHP-induced TNF-α protein release by J774 cells (Fig. 1). Fig. 1 also shows the effect of the calcium channel blocker verapamil, the calcium chelator BAPTA-AM, the calmodulin antagonist W7 and the phosphodiesterase inhibitors iPDE 1 and iPDE 4 on tBHP induced TNF-α protein release by J774 macrophages. Both verapamil and BAPTA-AM

Discussion

Signalling pathways linked to the generation of reactive oxygen species are believed to constitute a vital component of cellular oxygen signalling mechanisms which integrate the expression of genes involved in energy production, oxygen transfer, cellular differentiation and free radical scavenging (Haddad, 2002). Activation of NF-κB has also been shown in human peripheral blood mononuclear cells, airway epithelial cells and lung tissue in response to oxidants (Rahman and MacNee, 1998). Many

Acknowledgement

This work was generously funded by the Colt Foundation.

References (21)

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