Figure 5
Role of IL-1β and TNFα in macrophage-mediated NF-κB activation in RLE cells. A. Western blot analysis of IκBα degradation in RLE cells treated with 1 ng/ml IL-1β for 10 min or with 1 ng/ml TNFα for 5, 10 and 20 min, both pre-treated with neutralising antibodies. For neutralisation, the cytokine solution was incubated for 1 h with 0.25-2.5 μg/ml anti-IL-1β or 5 μg/ml anti-TNFα antibody. The asterisk indicates cells treated with IL-1β incubated at 37°C for 1 h, to investigate incubation effects. β-tubulin antibody was used as loading control. B. Expression of IκBα protein in RLE cells after treatment with supernatants of quartz-treated (40 μg/cm2, 24 h) NR8383 cells (Q-MS), that were pre-treated with neutralising antibodies (5 μg/ml anti-TNFα or 2.5 μg/ml anti-IL-1β). Supernatant of non-treated (medium, 24 h) macrophages (c-MS) was used as negative control for the TNFα neutralisation, β-tubulin served as loading control. C. and D. Effect of pre-treatment with both neutralising antibodies on NF-κB activation determined by Western blot (C) (phospho-IκBα, total IκBα) and (D) Immunocytochemistry (RelA). For Western blot experiments, RLE cells were treated for 5 to 20 min with native or cocktail-treated Q-MS. For immunocytochemical analysis, cells were treated with Q-MS or a cytokine mix (0.125 ng/ml IL-1β + 0.25 ng/ml TNFα) for 15 min. Macrophages were treated with 40 μg/cm2 DQ12 or medium for 24 h. Original magnification: 1000×. Pre-treatment conditions for both methods consisted of 1 h incubation with both 5 μg/ml anti-TNFα and 2.5 μg/ml anti-IL1β. β-tubulin was used as loading control.