FIG. 6.

In vitro addition of anti-4-1BB MAb induces production of cytokines/chemokines in neutrophils, but not monocytes. (A) Neutrophils and monocytes were purified from the bone marrow of naïve mice. The cells were cultured in 96-well culture plates with combinations of rat IgG (5 μg/ml), 3E1 (5 μg/ml), HI-3E1 (5 μg/ml), and heat-killed L. monocytogenes (HKLM) (1 × 10⁴ bacteria/well). After 24 h, the concentrations of each cytokine/chemokine in the culture supernatants were measured with a CBA kit. (B) Total RNA was isolated from neutrophils cultured with control rat IgG or 3E1 for 12 h using TRIzol reagent. Cytokine and chemokine mRNA levels were quantified by an RNase protection assay as described in Materials and Methods. (C) Increase of neutrophil phagocytic activity by 4-1BB stimulation. Neutrophils purified from bone marrow were incubated with FITC-dextran (1 mg/ml) and rat IgG or 3EI (1 μg/ml) for the indicated times, washed, and analyzed by FACS for FITC-positive cells. The results are from one representative of three independent experiments and are expressed as means plus standard deviations. *, P < 0.05; **, P < 0.01.