Reactive oxygen species are required for the phagocytosis of myelin by macrophages
Introduction
Multiple sclerosis (MS) is characterized by the occurrence of inflammatory demyelinating lesions in the central nervous system (CNS). In experimental allergic encephalomyelitis (EAE), an animal model for multiple sclerosis (MS), it has been shown that blood-derived macrophages play a crucial role in the development of clinical symptoms (Huitinga et al., 1990). Macrophages contribute to demyelination by their ability to phagocytose and degrade myelin. Macrophages present in the lesions are filled with myelin, according to histological staining of lesion material with antibodies against myelin proteins (Brück et al., 1995). The exact mechanism of the phagocytosis of myelin by macrophages is not yet fully understood (Smith, 1993; Mosley and Cuzner, 1996; Van der Laan et al., 1996).
ROS are produced both in MS and in EAE (Glabinsky et al., 1993; Guy et al., 1993; Lin et al., 1993). Furthermore, scavengers of ROS have been shown to decrease the severity of clinical symptoms in EAE (Hartung et al., 1988; Guy et al., 1989; Ruuls et al., 1995). The cells responsible for production of these ROS are thought to be the macrophages or activated microglia cells present in the lesions (Griot et al., 1989; Mayer et al., 1991; Okuda et al., 1995; Ruuls et al., 1995). Apart from the direct measurement of ROS in MS and EAE, also their consequences, i.e., the damage of ROS on lipid membranes by lipid peroxidation have been demonstrated in MS and EAE (Toshniwal and Zarling, 1992; Brett and Rumsby, 1993; Glabinsky et al., 1993). It has been shown that ROS can damage both the myelin sheath (Chia et al., 1983; Arduini et al., 1985; Konat and Wiggins, 1985) and the blood-brain barrier (Rubanyi, 1988). Here, we describe the role of ROS in the phagocytosis of myelin by macrophages.
We used an in vitro myelin phagocytosis assay described earlier (Van der Laan et al., 1996). Fluorescent-labeled myelin was fed to macrophages in the presence or absence of various ROS scavengers. One of the scavengers used in this study is Lipoic acid (LA). LA is a known scavenger of various ROS, such as hydroxyl radicals (OH.) and hypochlorous acid (HOCl) (Suzuki et al., 1991; Yan et al., 1996). LA is used as a therapeutic agent in the treatment of polyneuropathy of diabetes (Nickander et al., 1996). Furthermore, ROS production by macrophages after myelin phagocytosis was determined with either a nitrobluetetrazolium (NBT) assay or a dihydrorhodamine (DHR) assay.
From our results we hypothesize that myelin can trigger the NADPH oxidase complex of macrophages, which results in the production of ROS. These ROS can not only damage the myelin sheath, but can also activate the macrophage to phagocytose the myelin. Scavengers of ROS can prevent the phagocytosis of myelin, which might lead to a decrease of macrophage activation and damage to myelin.
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Animals
Adult male WagRij rats (Dutch Cancer Institute, Amsterdam, Netherlands) were kept under conventional circumstances. Rats were sacrificed and the resting peritoneal macrophages were isolated by rinsing the peritoneal cavity 3× with 10 ml RPMI 1640 (Gibco). The yield was approximately 12.5×106 cells per rat. Fresh normal rat serum (NRS), a source of complement, was prepared by centrifugation of clotted peripheral blood of the rats.
Preparation and labelling of myelin
Myelin was prepared from brain tissue of adult WagRij according to
Nitrobluetetrazolium (NBT) assay
In order to measure the effect of myelin on ROS production by macrophages, the NBT assay was performed. In absence of myelin only an occasional peritoneal macrophage was weakly positive for the blue formazan NBT reduction product (NBT-positive) (Fig. 1A). Following incubation of peritoneal macrophages with myelin in the presence of 2% NRS, the cells (70–80%) became NBT-positive (Fig. 1B). Peritoneal macrophages incubated with PMA all were NBT-positive (data not shown). In case of myelin (but
Discussion
ROS are thought to be involved in the pathogenesis of EAE and MS (Hartung et al., 1988; Guy et al., 1989; Glabinsky et al., 1993; Hansen et al., 1995; Malfroy et al., 1997). ROS are not only able to damage CNS tissue, such as endothelial cells of the blood-brain barrier (Rubanyi, 1988), but they can also damage the myelin sheath (Chia et al., 1983; Arduini et al., 1985; Konat and Wiggins, 1985). In this study we show that ROS are involved in the phagocytosis of myelin by macrophages.
Several
Acknowledgements
The authors wish to thank Christa Homburg and Petra Hilarius for their help with the NBT assay, and Prof. A. Bast for valuable discussion concerning LA. This work was supported financially by the Dutch foundation `Vrienden MS Research', project MS 95-202.
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