Figure 4.

Zymosan depleted of its TLR stimulatory activity synergizes with a TLR2 agonist through activation of dectin-1. (a) RAW cells stably transfected with an NF-κB luciferase reporter (ELAM luciferase) were stimulated for 4 h with the indicated concentrations of zymosan (○) or depleted zymosan (▪), and luciferase activity was measured. (b) HEK 293 cells were transiently transfected with dectin-1 fused to green fluorescent protein at the receptor's intracellular amino terminus and dectin-1 localization was visualized before (left) and after (right) exposure to depleted zymosan. (c) Cells expressing V5-tagged dectin-1 were stimulated with 100 μg/ml zymosan, 100 μg/ml depleted zymosan, or PAM₃CSK₄ lipopeptide as indicated for 15 min in the presence or absence of 50 μg/ml laminarin. Total cell lysates were prepared and the protein was detected by immunoblot using an antibody for the tag (bottom). Tyrosine-phosphorylated proteins were immunoprecipitated from the lysates and recovery of dectin-1 was analyzed by immunoblot (top). (d) IL-12 p40 mRNA production was measured by quantitative real-time PCR in control RAW cells and cells overexpressing dectin-1 stimulated for 4 h with 100 ng/ml PAM₃CSK₄ lipopeptide or 100 μg/ml depleted zymosan in the presence or absence of 500 μg/ml laminarin.