Platelet interference with Gp96-induced DC maturation is dependent on neither cell-to-cell contact nor soluble platelet factors.
Peripheral blood–derived monocytes were cultured to obtain immature human DCs, as described for Figure 4. After 6 days, cells were harvested and 2 × 105 DCs were plated into a 96-well plate. Autologous platelets (1 × 105/μL) were placed in the chamber of a transwell insert. If indicated, 25 μg/mL Gp96 or 1 μg/mL LPS was added to the platelets. (A) Freshly prepared platelets or thrombin-activated platelets, which were fixed with PFA after activation, were used. After 20-hour transwell culture, TNF-α concentration in the supernatant was determined. Mean values of duplicates are shown. The experiment was repeated twice. (B) DCs were left untreated or were stimulated with Gp96 and LPS in the absence or presence of fresh platelets. After 48 hours, CD86 and CD83 expressions were analyzed by flow cytometry. Percentage increase of activated cell numbers compared to nonstimulated DCs in the absence of platelets is shown. Percentages of activated cells in control samples for CD83 and CD86 were 33% and 26%, respectively. Mean values of triplicates are shown with error bars representing SEM.
