FIG.1.

Microglial cells produce CXCL10 in response to stimulation with CMV. (A) Induction of CXCL10 protein. Microglial cells (2 × 10⁵) were exposed to CMV AD169 at an MOI of 5 TCID₅₀ (infected) or to virus-free HFF cell extracts (mock infected) or medium alone (uninfected) for 8, 24, 48, 72, and 96 h prior to measurement of supernatant CXCL10 levels by ELISA. Data are expressed as mean (± SEM) CXCL10 levels from pooled data obtained during three separate experiments with microglial cells isolated from three different brain specimens. *, P < 0.05; **, P < 0.01 (versus mock-infected control cells). (B) Kinetics of chemokine mRNA induction in response to CMV. Total RNA was extracted from CMV-exposed microglial cells at 3, 8, 24, and 48 h, and 4 μg was used in the multiprobe chemokine RPA in accordance with the manufacturer's (Pharmingen) instructions. Lane C, uninfected microglial cell RNA; lane P, probe alone. Ltn, lymphotactin. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (C) UV inactivation decreases CXCL10 induction in microglial cells. Microglial cells (2 × 10⁵) were stimulated with UV-inactivated CMV AD169 (equivalent to an MOI of 5 TCID₅₀). CXCL10 levels were measured by ELISA at 72 h p.i.. Data are presented as the mean ± the SEM of pooled samples from three separate experiments. ††, P < 0.01 versus infected cells. (D) Decreased CXCL10 mRNA expression in microglial cells by UV-inactivated CMV. Total RNA from microglial cells stimulated with replication-competent (V) or UV-inactivated (UV) CMV or from uninfected cells (C) was analyzed by RPA at 24 h p.i. (E) IFN-γ (200 U/ml)-exposed microglial cells (microglia) were used as a positive control. CXCL10 production was also examined following IFN-γ treatment of astrocytes (2 × 10⁵ astrocytes). The data shown are the mean ± the SEM of pooled data from three separate experiments with glial cells obtained from two different brain specimens. **, P < 0.01 versus untreated cells. (F) Microglial cells were treated with cycloheximide (10 μg/ml) 30 min before (pre) or 6 h after (post) the addition of CMV. Total RNA was extracted from cycloheximide-treated and control cultures at 18 h p.i. and analyzed for CXCL10 mRNA by RPA.