Pro-inflammatory cytokines increase reactive oxygen species through mitochondria and NADPH oxidase in cultured RPE cells

Abstract

Reactive oxygen species (ROS) generated during inflammation are believed to play critical roles in various ocular diseases. However, the underlying mechanisms remain poorly understood. We investigated if pro-inflammatory cytokines, tumor necrosis factor (TNF)-α, interleukin-1β (IL-1β), and interferon-γ (IFN-γ), induce ROS in human retinal pigment epithelial (RPE) cells. TNF-α, IL-1β and IFN-γ increased both intracellular and extracellular ROS production in a time- and dose-dependent manner. Thenoyltrifluoroacetone (TTFA), an inhibitor of mitochondrial respiratory chain, blocked TNF-α- and IFN-γ-, but not IL-1β-induced ROS, whereas other two mitochondrial respiratory chain inhibitors, rotenone and antimycin A, had no effect. NADPH oxidase inhibitor (diphenylene iodinium) abolished the ROS production induced by IL-1β or IFN-γ, but not by TNF-α, whereas 6-aminonicotinamide (6AN), an inhibitor of the hexose monophosphate shunt (HMS), had no significant effects on the ROS induced by all three cytokines. ROS scavengers, pyrrolidinedithiocarbamate (PDTC) and N-acetyl-cysteine (NAC), reduced the levels of ROS induced by TNF-α, IL-1β and IFN-γ (P < 0.05). Collectively, these results demonstrate that TNF-α, IL-1β and IFN-γ increase mitochondrial- and NADPH oxidase-generated ROS in human RPE cells.

Introduction

Reactive oxygen species (ROS) are ubiquitous, highly reactive, diffusible molecules, including superoxide anions, hydrogen peroxide, hydroxyl radical, and nitric oxide (Fridovich, 1997). Cells generate ROS intracellularly and may release them extracellularly (Karlsson and Dahlgren, 2002, Kopprasch et al., 2003). While intracellular ROS serve mainly for host defense against infectious agents, redox-sensitive signal transduction, and other cellular processes, the extracellular release of ROS may damage surrounding tissues, potentially promoting inflammatory processes (Duval et al., 2003, Kopprasch et al., 2003). ROS are involved in aging and many diseases such as atherosclerosis, angiogenesis, cancer, diabetes mellitus, neurological degeneration, and tumor invasion (Harris and Shi, 2003, Wu, 2004). ROS also promote oxidative damage in eye disorders including age-related macular degeneration (AMD), cataracts, and uveitis (Rao, 1990, Winkler et al., 1999, Beatty et al., 2000, Cai et al., 2000, Truscott, 2000).

The retinal pigment epithelium (RPE) separates the neural retina from its blood supply in the choroid. In this strategic position, the RPE helps maintain an appropriate environment for photoreceptor function by transporting fluid, ions, and metabolites into and out of the space surrounding the photoreceptor outer segments. The RPE is an important factor in the development of AMD; central vision decreases when RPE cells cease to function properly, causing photoreceptor degeneration or damage in the macula, the portion of the retina used for central vision (Green et al., 1985, Young, 1987, Hageman et al., 2001, Penfold et al., 2001). Several studies have indicated that RPE cells are capable of producing ROS under certain conditions (Dorey et al., 1989, Miceli et al., 1994, Tate et al., 1995a, Wu and Rao, 1999, Yoshida et al., 2003, Kannan et al., 2004, Kindzelskii et al., 2004). RPE cells may therefore be an important source of ROS in the eye. Indeed, focused light (Dorey et al., 1990), high oxygen tension in the macula (Alder and Cringle, 1985), and photoreceptor outer segment phagocytosis and degradation (Tate et al., 1995b, Higgins et al., 2003) all promote RPE oxidative stress (Tso, 1987, Gottsch et al., 1990, Cai et al., 1999, Wassell et al., 1999, Higgins et al., 2003, Kindzelskii et al., 2004). Godley et al. (2005) showed that ROS levels were strikingly higher in macular RPE compared to peripheral RPE. Usually, RPE oxidative stress is reduced by endogenous RPE antioxidants and antioxidant enzymes, such as superoxide dismutase (SOD), catalase and glutathione peroxidase (GPX) (Newsome et al., 1990, Tate et al., 1993, Tate et al., 1995a, Edge et al., 1997, Fukuzawa et al., 1998, Jarrett et al., 2006). With aging, however, these RPE defenses decrease (Tate et al., 1993, Liles et al., 1991, Samiec et al., 1998), permitting ROS to ultimately overwhelm RPE cell defenses, leading to apoptotic damage (Ballinger et al., 1999).

AMD is a degenerative eye disease, common in people over 65 years of age, in which low-grade inflammation is now recognized to play a role. The inflammatory response in AMD lesions is characterized by an infiltration of the blood–retina barrier including RPE layer, by macrophages and lymphocytes (Seregard et al., 1994, Reddy et al., 1995, Penfold et al., 2001, Grossniklaus et al., 2002). In AMD, reactive, migrating or proliferating RPE cells are found adjacent to newly formed vessels in the subretinal space of wet AMD lesions (Miller et al., 1986, Sakamoto et al., 1995). Activation of RPE, inflammatory and endothelial cells may result in the release of a plethora of inflammatory mediators that individually or in concert may induce pathological changes in the retina. Pro-inflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukin-1β (IL-1β), and interferon-γ (IFN-γ) may be among the primary components responsible for the inflammatory response observed in the AMD, as TNF-α and IL-1β are secreted by macrophages and vescular endothelial cells while IFN-γ is secreted by lymphocytes (Oh et al., 1999). Furthermore, TNF-α, IL-1β, and IFN-γ are linked with pro-inflammatory RPE cell functions (Elner et al., 1990, Elner et al., 1991, Elner et al., 1992, Elner et al., 1997, Hollborn et al., 2001).

TNF-α, IL-1β, and IFN-γ are polypeptides that exert pleiotropic actions on multiple cell functions regulating gene expression, host defense reactions, and the immune response. TNF-α increases mitochondrial ROS production in tumor cells, endothelial cells, and hepatocytes (Schulze-Osthoff et al., 1993, Corda et al., 2001). IL-1β stimulates ROS production in various cell types (Mendes et al., 2003, Brigelius-Flohe et al., 2004, Hwang et al., 2004, Kaur et al., 2004). IFN-γ induces an immediate and marked augmentation of intracellular ROS in transformed lymphoblast cell lines (HSC536/N and PD149L) (Pearl-Yafe et al., 2003, Pearl-Yafe et al., 2004). IFN-γ also induces ROS and endoplasmic reticulum stress during IFN-γ-induced apoptosis of hepatocytes (Watanabe et al., 2003). However, the abilities of TNF-α, IL-1β, or IFN-γ to stimulate ROS production in RPE cells have not yet been reported.

In this study, therefore, we assessed: (1) whether human RPE cells produce ROS when stimulated by pro-inflammatory cytokines, TNF-α, IL-1β, or IFN-γ; and (2) if so, the cellular source of ROS production induced by these cytokines.