Figure 8: Role of CREB in MBP-primed Th2 cell contact-inhibited glial expression of iNOS and IL-1β
(A) Mouse BV-2 microglia were co-transfected with pCRE-Luc and pRL-TK. After 24 hr of transfection, cells were stimulated with Th1 and Th2 cells. After 1 hr, T cells were removed and adherent microglia received LPS (0.5 μg/ml) in serum-free media. After 5 hr, firefly and Renilla luciferase activities were assayed. Data are mean ± S.D. of three different experiments. ap<0.001 vs. control (no T cells), bp<0.01 vs. MBP-Th2, and cp<0.01 vs. MBP-Th2 treated cells. (B) Th2 cells were treated with 0.5 μg/ml of antibodies against αV (ΔαV), α4 (Δα4), β3, (Δβ3), that were previously transfected with pCRE-Luc and pRL-TK. ap<0.001 vs. control, bp<0.001 vs. LPS+MBP-Th2, and cp<0.001 vs. LPS+MBP-Th2 treated cells. Similarly BV-2 microglia was transfected with pCRE-Luc and pRL-TK. After 24 h of transfection, microglia were incubated with different concentrations of antibodies against either PDGFRβ (ΔPDGFRβ) or VEGF-R (ΔVEGFR) for 1 h followed by stimulation with Th2 cells (0.5:1 of T cell: glia). After 5 h, firefly and Renilla luciferase activities were assayed in microglia. Data are mean ± S.D. of three different experiments. (C) Primary microglia was incubated with 1 μM antisense (ASO) and scrambled (ScO) oligonucleotides against CREB. After 42 h of incubation, the expression of CREB mRNA was examined by RT-PCR. (D) Microglia preincubated with 1 μM ASO or ScO against CREB for 36 h were stimulated with Th2 cells and LPS as described above. After 5 h, microglia were analyzed for iNOS and IL-1β mRNAs by RT-PCR. (E) After 24 h, concentration of nitrite was analyzed in supernatants by ELISA. ap<0.001 vs. nitrite in Th2 -treated microglial cells (F) A schematic diagram of Th2 cell and microglial interaction leading to the activation of CREB and suppression of inflammation in microglial cells.