Trace Levels of Staphylococcal Enterotoxin Bioactivity Are Concealed in a Mucosal Niche during Pulmonary Inflammation
Fig 1
BALF of mice treated with SEA contain a heat resistant proteinaceous pro-inflammatory factor.
(A) BALF obtained from mice 16 h after i.n. SEA (1 μg) diluted in BSS (BALF-SEA) or BSS alone (BALF-BSS) were tested in a bioassay as described in material and method section. Briefly, mouse splenocytes were pretreated with IL-12 (2 ng/ml) for 16–18 h, washed and rested for 5 h. Primed splenocytes (200,000/well) were incubated with BALF and controls. Culture supernatants were obtained after 16–18 h and assayed for IFN-γ by ELISA. For comparison, proinflammatory cytokines at a final concentration of 5 ng/ml, except IFN-α (1000 U/ml), and LPS (5 μg/ml) were added to the bioassay. Bar graphs show IFN-γ secretion measured in triplicate. Representative of 1 out 3 experiments is shown. (B) BALFs obtained 8 h and 18 h after i.n. SEA and BSS were compared in a bioassay. Bar graphs show IFN-γ secretion measured in triplicate. Representative of 1 out 4 experiments is shown. (C) BALFs and IL-33 (bioactive protein control) incubated with and without proteinase K at 37°C for 18 h, were either boiled for 10 min or left untreated and tested in a bioassay as described above. Bar graphs show IFN-γ secretion measured in triplicate. Representative of one out three experiments is shown. (D) Culture supernatants obtained with BALFs, cytokines and LPS applied to a bioassay as described in A were tested for IL-2. Bar graphs show IL-2 secretion measured in triplicate. Representative of 1 out 3 experiments is shown. For all panels statistical significance between groups was evaluated by two-tailed Student’s t tests. The error bars indicate the standard error of the mean between triplicates. * p<0.05, *** p<0.001.