Materials and methods

Reagents

The recombinant rat IL-10 (rrIL-10; R&D system, Minneapolis, MN). LPS were purchased from Sigma-Aldrich (St. Louis, MO). All the cell culture ingredients were obtained from Invitrogen (Carlsbad, CA). The [3H] DA (30Ci/mmol) was from Perkin-Elmer Life Sciences (Boston, MA), The fluorescence probe DCFH-DA was obtained from Calbiochem (La Jolla, CA).

Rat mesencephalic neuron-glia cultures

Primary mesencephalic neuron-glia cultures were prepared from the brains of embryonic day 14/15 Fischer 344 rats, following our previously described protocol (Liu et al., 2002). Briefly, the ventral mesence-phalic tissues were removed and dissociated by a mild mechanical trituration. Cells were seeded at 5 x 105/well to 24-well culture plates pre-coated with poly-D-lysine (20ng/ml) and maintained at 37°C in a humidified atmosphere of 5% CO2 and 95% air in 0.5 ml/well maintenance medium. The medium consisted of minimum essential medium containing 10% heat-inactivated fetal bovine serum and 10% heat-inactivated horse serum, 1 g/l glucose, 2 mM [SCAP]L-glutamine, 1mM sodium pyruvate, 100 pM nonessential amino acids, 50U/ml penicillin, and 50ng/ml streptomycin. Three days after the initial seeding, 0.5 ml of fresh maintenance medium was added to each well. Seven-day-old cultures were used for treatment. The composition of the cultures at the time of treatment was approximately 48% astrocytes, 11% microglia, 40% neurons, and 1 to 1.5% TH-immunoreactive (ir) neurons.

Primary microglia-enriched cultures

Rat microglia-enriched cultures, with a purity of >98%, were prepared from whole brains of 1 -day-old Fischer 344 rat pups, following our described protocol (Liu et al., 2002). For superoxide assays, 105 cells/well/

0.2 ml medium were grown overnight in 96-well culture plates before use.

Uptake assay

[3H]DA uptake assays were performed as described previously (Liu et al., 2002). Cultures were incubated for 20 min at 37°C with 1 mM [3H]DA in Krebs-Ringer buffer (16 mM sodium phosphate, 119 mM NaCl, 4.7 mM KCl, 1.8mMCaCl2, 1.2 mM MgSO4, 1.3 mM EDTA, and 5.6 mM glucose; pH 7.4). After washing three times with ice-cold Krebs-Ringer buffer, cells were collected in 1N NaOH. Radioactivity was determined by liquid scintillation counting. Nonspecific DA uptake observed in the presence of mazindol (10 mM) was subtracted.

Nitrite and TNFa assays

The production of NO was determined by measuring the accumulated levels of nitrite in the supernatant with the Griess reagent, and release of TNFa was measured with a rat TNFa enzyme-linked immunosorbent assay kit from R&D Systems (Minneapolis, MN).

Superoxide assay

The production of superoxide was determined by measuring the superoxide dismutase (SOD)-inhibita-ble reduction of the tetrazolium salt WST-1. Micro-glia-enriched cultures in 96-well culture plates were washed twice with Hanks' balanced salt solution without phenol red (HBSS). Cultures were then incubated at 37°C for 30 min with vehicle control (water) or IL-10 in HBSS (50 ml/well). Afterward, to each well was added 50 ml of HBSS with and without SOD (50U/ml, final concentration), 50 ml of WST-1 (1 mM) in HBSS, and 50 ml of vehicle or LPS (10 ng/ml). Thirty minutes later, absorbance at 450 nm was read with a SpectraMax Plus microplate spectrophotometer (Molecular Devices Corp., Sunnyvale, CA). The difference in absorbance observed in the absence and presence of SOD was considered to be the amount of superoxide produced, and results were expressed as percentage of vehicle-treated control cultures.

Statistical analysis

The data were expressed as the mean ± S.E.M. Statistical significance was assessed with an analysis of variance followed by Bonferroni's t test using the Stat View program (Abacus Concepts, Berkeley, CA). A value of p < 0.05 was considered statistically significant.

Results

Effect of IL-10 on LPS-induced degeneration of DA neurons

Mesencephalic neuron-glia cultures were pretreated with IL-10 for 1 h and then stimulated with LPS for 7days. The degeneration of DA neurons was then determined by [3H] DA uptake assay. The [3H] DA uptake assay showed that LPS treatment reduced the capacity of the cultures to take up DA to approximately 40% of the vehicle control (Fig. 1A). At 30ng/ml IL-10, the LPS-induced decrease in DA uptake was completely restored, and IL-10 alone at this concentration range did not affect DA uptake levels in the cultures.

IL-10 treatment inhibits LPS-induced production of NO, TNFa and intracellular and extracellular reactive oxygen species

The LPS-stimulated activation of microglia was suppressed by pretreatment with IL-10 in neuron-glia cultures. Accumulation of nitrite, an indicator of LPS stimulated production of NO, was determined 24hrs and 48hrs after LPS stimulation. As shown in Fig. 1B, pretreatment with 30ng/ml IL-10, completely blocked LPS-stimulated NO production. As shown in Fig. 1C, pretreatment with 30ng/ml IL-10 significantly reduced LPS-induced production of TNFa determined at 3 h after LPS stimulation.

To test the effect of IL-10 on the microglial generation of ROS, enriched-microglial

IL-10+LPS IL-10

control

IL-10+LPS IL-10

control

ol f

100 0

control

IL-10+LPS IL-10

□ Superoxide

control

LPS IL-10+LPS

Fig. 1. IL-10 is neuroprotective against LPS-induced neurotoxicity and inhibits LPS-induced microglia activation. Rat primary mesencephalic neuron-glia cultures seeded in a 24-well culture plate at 5 x 105 cells were pretreated with IL-10 (30ng/ml) for 1 h before the addition of 10ng/ml LPS. Eight days later, the LPS-induced dopaminergic neurotoxicity was quantified by the [3H] DA uptake assay (A); Effects of IL-10 on LPS-induced production of nitrite oxide (B); TNF-a (C); superoxide and iROS (D) as % of control. The results are the mean ± SE of 4 individual experiments in triplicate in each experiment. *P < 0.05, **P < 0.01, compared with LPS culture cultures were pretreated with IL-10, then exposed to LPS. IL-10 significantly inhibited intracellular ROS production and microglial superoxide response to nearly control levels. (Fig. 1D). Based on our previous data that indicates a central role for ROS in micro-glial-mediated destruction of DA neurons, it appears that the neuroprotective effect of IL-10 is at least partially due to a reduction in LPS-induced oxidative stress.

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