Figure 4

Additive effects of different microbial agonists and lack of evidence of tolerance in reporter gene activation. Pooled stable transfectants of RAW264 cells, with either the ELAM or IL-12 promoter driving firefly luciferase, cotransfected with the IL-1β promoter driving renilla luciferase, were cultured overnight for 16–18 hrs with either no addition, LPS (100 ng/ml), PAM3-CSK lipopeptide (PAM, 100 ng/ml) or E. coli genomic DNA (bDNA, 10 μg/ml) as indicated on the vertical legend. The cells were washed by aspirating the medium, replacing it with warm medium, incubating for 5 minutes and replacing the medium a second time. The replacement medium contained no additional stimulus, or one of original stimuli as indicated on the legend to the X-axis, so that a 4 × 4 matrix of pretreatment and retreatment was established. After a further 8 hrs stimulation, the cells were harvested for determination of luciferase activity. Each datapoint is the average of duplicate wells. The experiments is representative of two with identical design. The pattern of restimulation has been confirmed for LPS, and for the ELAM/IL-1 line in separate experiments (not shown).