Granulysin activation of APCs is MyD88-dependent and requires TLR4. (A) Mouse macrophage cells from WT B6129 or B6 MyD88-deficient cells lines (MyD88 KO) were incubated at 106/mL in the presence of 10 μg/mL 15-kDa GNLY, 1 μg/mL Pam3, 1 μg/mL ultrapure LPS, or 5 μg/mL poly I:C for 48 hours, and the production of IL-6 in the culture supernatants was measured by enzyme-linked immunosorbent assay. The results of 1 experiment representative of 2 are presented as the average (mean ± SD) of triplicate wells. To analyze the role of TLR4 in GNLY-mediated activation, day 6 mouse BMDCs from WT (HeN) and TLR4 mutant (HeJ) mice were incubated at 106/mL in the presence of 10 μg/mL 15-kDa GNLY and 1 μg/mL ultrapure LPS for 48 hours. After treatment, cells were analyzed by FACS for induction of costimulatory molecules and production of proinflammtory cytokines in the culture supernatants by enzyme-linked immunosorbent assay. (B) Analysis of CD80 and CD86 surface markers compared between untreated and GNLY-treated cells. (C) Induction of proinflammatory cytokines by GNLY compared with untreated and LPS (positive control). One representative experiment of 4 is shown. *P < .01.
