Zhao ML, Liu JS, He D, Dickson DW, Lee SC. in microglia was mediated via a signaling pathway involving protein-tyrosine kinase. These data support the hypothesis that gp41 induces astrocyte NO production indirectly by triggering upregulation of microglial cell IL-1 expression. The following reagents were purchased from the indicated sources: antibodies to SGC 0946 microglial cell CD68 antigen and astrocyte glial fibrillary acidic protein (GFAP) (Dako, Carpinteria, CA) and oligodendrocyte galactocerebroside (Polysciences, Warrington, PA); anti-digoxigenin-fluorescein antibody and propidium iodide (Boehringer SGC 0946 Mannheim, Indianapolis, IN); biotinylated SGC 0946 goat-anti rabbit IgG (Novacastra Laboratories, Burlingame, CA); cytokines (IL-1 and IFN-), anti-IL-1 antibodies, and IL-1 receptor antagonist protein (IRAP) (R & D Systems, Minneapolis, MN); anti-NOS2 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA); fetal bovine serum (FBS) (Hyclone Laboratories, Logan, UT); viral envelope protein gp41 (Intracel, Inc., Issaquah, WA); oligo-dT12C18 primer and dNTP mixture (Pharmacia, Piscataway, NJ); reverse transcription (RT) buffers and SuperScript II RNase reverse transcriptase SGC 0946 (Life Technologies, Gaithersburg, MD); DNA polymerase (Promega, Madison, WI); and culture reagents, including DMEM, HBSS, protein kinase C (PKC) inhibitor H7, protein-tyrosine kinase (PTK) inhibitor G103, pyrrolidinedithiocarbamate (PDTC), polymyxin B, the iNOS inhibitorHuman fetal brain tissue was obtained from 16- to 22-week-old aborted fetuses under a protocol approved by the Human Subjects Research Committee at our institution. The procedure for isolating highly enriched primary human fetal microglial cells has been previously described (Chao et al., 1996a). Briefly, brain tissues were dissociated after 30 min trypsinization (0.25%) and plated in 75 cm2 Falcon culture flasks in DMEM containing 10% heat-inactivated FBS, penicillin (100 U/ml), and streptomycin (100 g/ml). The medium was replenished 4 d after plating in medium containing 10% FBS only. Microglia were harvested 10C14 d later. Purified microglia were composed of a cell population of which 99% stained with anti-CD68 antibody (a human macrophage marker) and 1% stained with anti-GFAP and anti-galactocerebroside antibodies (astrocyte and oligodendrocyte markers, respectively). Enriched astrocyte cultures were prepared as previously described with minor modifications (Chao et al., 1996c). In brief, after removing microglia as described above, flasks were incubated with Ca2+- and Mg2+-free HBSS containing 0.125% trypsin for 20 min at 37C, which was followed by addition of 10% FBS-containing medium. After centrifugation, the cell suspension was seeded into new flasks with medium containing 10% FBS, and this culture medium was changed 24 hr later. This subculture procedure was repeated three times at a weekly interval. Finally, highly enriched (99% positive by anti-GFAP staining) astrocytes were seeded into 96-well plates at a density of 105 cells per well for NO and IL-1 production and into 12-well plates at 2 106 cells per well for RNA analysis. At the end of cell culture, in all experiments, 99% of cells remained GFAP-positive, and 1% stained positively with anti-CD68 antibodies. Glial cell cultures were treated with gp41 (20 m) for 8 hr before harvesting total RNA for evaluating iNOS, glyceraldehyde 3-phosphate dehydrogenase (GADPH), or IL-1 mRNA expression, for 48 hr for IL-1 bioassay (Chao et al., 1992a), and for 5 d for assaying nitrite levels. The optimal timing for harvesting supernatants or cells for mRNA expression and NO and IL-1 assays has been reported previously (Chao et al., 1996c). The concentration (20 m) of gp41 selected was based on a previous study demonstrating that lower concentrations of gp41 failed to trigger iNOS mRNA expression in rat Rabbit Polyclonal to ACK1 (phospho-Tyr284) glial cell cultures (Adamson et al., 1996). To evaluate the possibility that a soluble factor(s) was released from gp41-treated microglia, microglial cell cultures were first treated with gp41 for 48 hr, and supernatants were transferred to highly enriched astrocyte cultures in the absence or presence of 200 U/ml IFN- for 8 hr for assessing iNOS mRNA expression and for an additional 5 d for assaying nitrite levels. Glial cells were cultured in glass chambers for staining with the indicated reagents. Polymyxin B (20 g/ml) was used in one experiment to block endotoxin as a potential contaminant in gp41-induced cytokine expression. Polymyxin B had been shown previously to reduce 97% of lipopolysaccharide (LPS)-induced TNF- production by microglia (Peterson et al., 1995b). For studies of signaling transduction pathways, microglial cell cultures were incubated with gp41 in the absence or presence of the inhibitors H7 or G103 (30 m), as previously described (Chao et al.,.