1992;1:145C153. al., 1998) and bacterial lipopolysaccharide (LPS) (Castano et al., 1998). In all such models, reactive microglia are extremely prominent (Le et al., 1995a, 1996; Chen et al., 1998). The key question is whether activated microglia can initiate or amplify injury to the nigral dopaminergic neurons, or is their role merely phagocytic. Furthermore, do immune/inflammatory processes participate in the oxidative stress known to be present in many of these models as well as in PD? To characterize the potential roles of PD IgG and microglia in dopaminergic nigral cell injury, we have developed ansystem in which PD IgG, in the presence of DA-quinone (DA-Q) and H2O2-modified dopaminergic cell membranes, can activate microglia and target a free radical-mediated injury to dopaminergic cells. MATERIALS AND METHODS for 10 min at 4C to remove the crude nuclear fractions. The supernatants were again centrifuged at 100,000 for 60 min at 4C. The precipitates were homogenized and suspended in culture medium and used as the neuronal membrane fractions. = 7), and high-dose DA-Q-M membranes (150 g/ml) for 2 d, and medium was collected for measurement of TNF- and IL-1 levels. Some of the microglial cultures were stimulated with 1 g/ml PMA for 2 hr before assaying the released levels of O2?, H2O2, and NO. Incubation with LPS, PD IgG, and DA-Q-M membranes also elevated microglial-immunoreactive iNOS and NADPH oxidase (Fig.?(Fig.3),3), with corresponding increases of NO and O2?(Fig. ?(Fig.22). Open in a separate window Fig. 3. iNOS (indicate three isoforms of NADPH oxidase reacting with antibodies of 0.05 and ** 0.01 compared with DC IgG +DA-Q-M. The specificity of membrane modification by DA-Q versus H2O2 and the specificity of modified MES 23.5 cell membranes versus modified non-DAergic cell membranes in microglial activation To determine whether microglial activation was specific for the method of cell membrane modification and the dopaminergic or cholinergic phenotype of the cell, we contrasted modification by DA-Q or H2O2 in membranes isolated from dopaminergic or cholinergic cell lines. Both the dopaminergic cell line (MES 23.5) and the CGP60474 cholinergic cell line (SN56) (Wainer et al., 1991) are derived from the same parental neuroblastoma line. Both MES 23.5 cells and SN56 cells were separately incubated with CGP60474 10 m H2O2 or 50 m DA-Q, and the isolated membrane fractions were incubated with primary rat microglia in the presence and absence of low-dose PD IgG. Both high-dose (150 g/ml) SN56 and MES 23.5 cell membranes had significant microglial activating effects, whether modified by DA-Q or by H2O2. At low doses (15 g/ml), neither activated microglia. Low-dose DA-Q- or H2O2-modified MES 23.5 cell membranes incubated with low-dose PD IgG were able to activate microglia to a similar extent. However, low-dose DA-Q- or H2O2-modified SN56 cell membranes CGP60474 incubated with low-dose PD IgG had minimal activating potential (Fig. ?(Fig.55). Open in a separate window Fig. 5. The specificity of microglial activation induced by DA-Q-M- or H2O2-M membranes from MES 23.5 cells as compared with SN 56 cells. The cells were treated with DA-Q (50 m) or H2O2 (10 m) for 24 hr, and the cell membranes alone or in combination with PD IgG were incubated with rat microglia for 2 Rabbit Polyclonal to OR5W2 d. TNF- levels in the culture medium were determined by ELISA. ** 0.01.