Figure 6: Effect of neutralizing antibodies against α4, αV, β1, and β3 integrins on MBP-primed Th2 cell-mediated inhibition of LPS-stimulated iNOS and IL-1β expression in mouse primary microglia.
Th2 cells were incubated with different concentrations of neutralizing antibodies against αV, α4, β3, and β1, and then added to mouse primary microglia at a ratio of 0.5:1 T cell: microglia. After 1 h of stimulation, T cells were removed followed by incubation of adherent microglia with LPS (0.5 µg/ml) in serumfree media. (A) After another 5 h of incubation, the expression of iNOS and IL- 1β was analyzed by RT-PCR in LPS-stimulated microglial cells incubated with either α4 or αV (1-2 µg/mL) neutralizing antibody-treated Th2 cells. (B) After 24 hrs, nitrite was measured in the respective supernatants by Griess method described under “Materials and Methods” section. ap<0.001 vs. nitrite in control glia and bp<0.001 vs. nitrite in only MBP-Th2 treated glia. Similarly, RT-PCR (C) analyses of IL-1β and iNOS were performed in microglial cells and nitrite (D) production was measured in the supernatants in LPS-stimulated microglia that received β1 or β3 (1-2 µg/mL) blocking antibody-treated Th2 cells. a <0.001 vs. control cells and bp<0.001 MBP-primed Th2 cells. Data are mean ± S.D. of three different experiments.