FIG 6.

Antibody blocking of CD18 but not CD11b is sufficient to reduce bacterial internalization of P. aeruginosa in murine neutrophils. Murine neutrophils (WT or CD18 KO) were assayed by gentamicin protection assay for relative in vitro phagocytosis of PA14 WT, PA14 motABmotCD (inset), or PAO1 WT (MOI of 10) in the presence of 10 μg/ml of GAME-46 CD18-blocking antibody (A and B) and M1/70 CD11b-blocking antibody (C and D) with the respective isotype controls (as indicated). The neutrophils were pretreated with the antibodies for 15 min on ice. Phagocytic uptake levels were normalized as percentages of respective mean WT phagocytosis. (E) Relative association of GFP-expressing PAO1 WT (MOI of 25 at 45 min postinfection) to murine neutrophils (WT or CD18 KO) in the absence or presence of 10 μg/ml GAME-46 and 10 μM cytoD. The neutrophils were preincubated with GAME-46 and cytoD for 30 min at 37°C prior to infection. The data are normalized to the mean fluorescence intensity (MFI) of uninfected murine neutrophils analyzed by flow cytometry (set to 1,000). All data are analyzed using one-way ANOVA with Tukey’s post hoc analyses and are representative of at least two independent biological experiments (n ≥ 4). ***, P ≤ 0.0005; *, P ≤ 0.05; ns, not significant.