Fig. 6

Profiling of cytokines in the supernatants from MC BBC2 and KO #7 cells cultured in vitro using LEGENDplex bead-based immunoassays over a 48-h period following stimulation with either LPS or IL-1β demonstrates specific and time dependent increases in IFN-γ only in the MC cells (A). Levels of IFN-α and IFN-β and GM-CSF do not show this specific activation. These data were confirmed at the mRNA expression level using RT-PCR (B, above) and protein level using ELISA (B, below) from independent treatments with either LPS or IL-1β (N = 3) compared individually with levels seen in the PBS treated cells. Analysis of the IFN-γ downstream targets CXCL9 and CXCL10 (C) shows increased levels only in the MC cells treated with IL-1β. In serum samples from mice engrafted with either MC or KO#7 cells (D), ELISA assays show significantly increased levels of IFN-γ in the MC cell engrafted mice compared with the KO cell engrafted group. Analysis of splenocytes from the same animals shows coincident increases in IFN-γ and CXCL9 mRNA expression in the MC cell engrafted mice only (E), which was confirmed at the protein level using western blotting (F; N = 4). ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001