β-1,3-glucan induces PLD activity in A549 cells.

<p>A549 cells were stimulated with the indicated concentrations of β-1,3-glucan for 30 min (A) or for the indicated duration of time with 50 µg/mL of β-1,3-glucan (B). Thereafter, the PLD activity was determined and differences in [³H] PtdEtOH formation between the control (indicated by “0”) and other groups were compared. In C and D, A549 cells were first incubated with HBSS (Control), HBSS containing 5 µg/mL of isotype control antibody and HBSS containing 5 µg/mL of anti-dectin-1 mAb GE2 (ab82888) for 30 min, respectively. Then, the cells were infected with swollen conidia of <i>A. fumigatus</i> 13073 at an MOI of 10 (C) or stimulated by HBSS and HBSS containing 50 µg/mL of β-1,3-glucan for 30 min, respectively (D). <i>A. fumigatus</i> internalization was analyzed by the nystatin protection assay (C) and the PLD activity (D) was measured. Differences in <i>A. fumigatus</i> internalization and [³H] PtdEtOH formation between the untreated cells (Control) and antibody-treated cells were compared. In E and F, A549 cells were infected with swollen conidia of <i>A. fumigatus</i> 13073 at an MOI of 10 or incubated with PBS (Control) for 30 min. Subsequently, the cells were stained with isotype (IC) antibody or the primary anti-dectin-1 mAb GE2 (ab82888) and analyzed by FACS Calibur flow cytometer. The geometric mean fluorescence intensity was determined by Cell Quest Pro software. Differences between uninfected cells (Control) and conidia-infected cells were compared. G. A549 cells were incubated with swollen conidia of <i>A. fumigatus</i> 13073 at an MOI of 10 or incubated with PBS (Control) for 30 min. Cells were analyzed for dectin-1 expression by immunoblotting using an anti-dectin-1 antibody (sc-26094). FACS profiles and immunoblots shown here are characteristic of 3 independent experiments. Data are represented as mean ± SE (n = 3–4). *P<0.05.