Involvement of the extracellular signal regulated kinase pathway in hydrocarbon-induced reactive oxygen species formation in human neutrophil granulocytes

Abstract

In the present study we have examined the effects of hydrocarbons on the formation of reactive oxygen species (ROS) in human neutrophil granulocytes in vitro. We found that hydrocarbons induce ROS formation in a concentration-dependent manner and that the ROS-inducing potency increases with increasing number of carbon atoms in the structure. In general, aromatic hydrocarbons were less potent inducers of ROS than aliphatic and cyclic hydrocarbons. The most potent compound in each group, t-butylcyclohexane, n-decane, and n-butylbenzene, were chosen for mechanistic studies. ROS formation was inhibited by the MEK1/2 inhibitor U0126, the tyrosine kinase inhibitor erbstatin-A, and the phosphatidylinositol-3 kinase inhibitor wortmannin. The involvement of the ERK1/2 pathway was confirmed by Western blot analysis of phosphorylated ERK1/2. The study revealed only small differences in the mechanisms involved for the three compounds. The responses were not affected by Pertussis toxin, indicating that G_i-protein coupled receptors are not involved in neutrophil activation after hydrocarbon exposure. Based on these findings we propose a mechanism involving tyrosine kinases, PI3 kinase, and the ERK1/2 pathway, leading to activation of the NADPH oxidase and production of ROS in neutrophils stimulated by organic solvents.

Introduction

Exposure to organic solvents has been associated with chronic toxic encephalopathy and brain damage, but less is known about the effects on the immune system. There is, however, limited but sufficient evidence that exposure to some organic solvents can have adverse effects on the immune system (Snyder, 1994). Immunological effects have been shown after exposure to n-hexane and its metabolites both in man and laboratory animals Kannan et al 1985, Upreti and Shanker 1987, Karakaya et al 1996. Immunosuppressive effects have also been demonstrated in mice exposed to benzene and toluene Hsieh et al 1988, Hsieh et al 1989, Hsieh et al 1991. Increased respiratory burst in neutrophils after hydrocarbon exposure has been demonstrated by fluorescence spectroscopy, hydroxylation of 4-hydroxybenzoic acid, and electron paramagnetic resonance spectroscopy Myhre et al 1999, Myhre et al 2000. The mechanisms for reactive oxygen species (ROS) production were found to involve several intracellular signaling pathways, including phospholipases and protein kinase C, leading to activation of the NADPH oxidase complex and ROS formation (Myhre et al., 2000).

Neutrophil granulocytes are an important part of the immune system, and they constitute the first line of defense against invading pathogens Edwards 1996, Babior 2000. These cells react with a strong response toward chemoattractants, with activation of functions such as chemotaxis, release of degrading enzymes, and production and release of superoxide anion radicals. The superoxide production is catalyzed by the NADPH oxidase complex (Babior, 1999). This complex consists of several components that reside in the cytosol and in the membrane in resting cells, and is assembled to a functional complex when the cells are activated. Superoxide is rapidly converted to hydrogen peroxide (H₂O₂), either spontaneously or enzymatically by superoxide dismutase. H₂O₂ is then reduced to water by catalase or converted to hypochlorous acid (HOC1) by myeloperoxidase (Burg and Pillinger, 2001). H₂O₂ can also be transformed to hydroxyl radicals in the presence of transition metal ions (Halliwell and Gutteridge, 1999). Neutrophils stimulated by the cyclic hydrocarbon 1,2,4-trimethylcyclohexane have been shown to produce mainly superoxide, but also small amounts of the hydroxyl radical (Myhre et al., 2000).

The agents that stimulate neutrophils to microbicidal actions do so by binding to membrane receptors. These receptors can be seven-spanning membrane receptors that exert their effects through trimeric G-proteins, or they can function through tyrosine kinase activity, either by intrinsic tyrosine kinase activity (e.g., growth factor receptors like EGF-R) or through association with cytosolic tyrosine kinases (Downey et al., 1995). Activation of these receptors can stimulate several intracellular signaling systems eventually leading to activation of the NADPH oxidase complex, including tyrosine kinases, phosphatidylinositol-3-kinase (PI3 kinase), phospholipase C, phospholipase D, phospholipase A₂, and mitogen-activated protein (MAP) kinases Morel et al 1991, Downey et al 1995, Prossnitz and Ye 1997, Burg and Pillinger 2001.

The MAP kinase family include the stress response-activated kinases Jun kinase and p38 and the extracellular signal regulated kinases (ERK) 1 and 2, which traditionally have been implicated in the control of cell division and differentiation (Derijard et al., 1995). These kinases phosphorylate a wide range of targets in the cells, including several different transcription factors, but have also been shown to be involved in the activation of the NADPH oxidase complex after stimulation of neutrophils with different chemoattractants like N-formyl-methionyl-leucine-phenylalanine (fMLP), TNF-α, and opsonized zymosan Torres et al 1993, Hazan et al 1997, Downey et al 1998, Zu et al 1998.

The involvement of these signaling pathways in neutrophil activation after physiological stimulation lead us to suspect that neutrophil activation after hydrocarbon exposure could involve similar mechanisms in addition to those shown previously (Myhre et al., 2000). The aim of the present study was to investigate whether signaling through tyrosine kinases, heterotrimeric G-proteins, and MAP kinases could be involved in activation of ROS production in neutrophils after hydrocarbon exposure. Furthermore, we wanted to investigate the possible correlations between hydrocarbon structure and ROS-inducing potency in neutrophil granulocytes.