Figure 10

Cell Adhesion to and Migration across HBME Monolayers and Phagocytosis of GFP-Expressing P. aeruginosa (A) HMBEs were cultured to postconfluence in 24-well plates. The HBME monolayers were pretreated for 4 h with LPS 200 ng/ml or medium alone and gently washed. Equivalent concentrations of calcein AM-labeled HL60 and dHL60 cells, after pretreatment for 2 h with IL-8 3 nM or medium alone, were washed and co-incubated for 0.5 h with HBME monolayers. After gentle washing to remove nonadherent cells, the attached cells were fluorometrically assayed (excitation 485 nm, emission 530 nm) in a fluorescence plate reader. (B) HBMEs were cultured to postconfluence on the filter supports of transwell chambers. Equivalent concentrations of viable calcein AM-labeled HL60 and dHL60 cells were introduced into the upper compartment whereas IL-8 or medium alone was placed in the lower compartment and the transwell chambers incubated for 2 h at 37 °C. After incubation for 2 h, each compartment of each chamber was sampled and labeled cells fluorometrically assayed. Vertical bars represent mean ± SE adhesion/migration. (C, D). The nondifferentiated and DMF-differentiated HL60 cells were mixed with PAK-GFP at a MOI of 25, incubated for 10 or 45 min, and fixed for flow cytometric analysis. The cells alone served as negative control (Ctrl). The histograms (C) and the percentage of GFP-positive cells (D) are shown. (E) Non-opsonized (BRC⁻/sera⁻) and BBRC⁻ -and BRC⁺ sera⁺-opsonized P. aeruginosa were mixed with either nondifferentiated or DMF-differentiated HL60 cells, incubated at 37 °C for 45 min, and the mixtures sampled for CFUs. *, significantly increased adhesion, migration, or opsonophagocytosis in dHL60 cells compared with that seen for HL60 cells at p < 0.05. The data generated in each panel represents experiments performed on ≥ 2 independent occasions.