Figure 2.

MMP7 and syndecan-1 are required for epithelial-mediated neutrophil activation. (A and B) Alveolar type II epithelial cells were isolated from WT, Mmp7^−/−, and Sdc1^−/− mice, grown to confluence, scratch wounded, incubated for 2 hours to respond to injury, then cocultured for 3 hours with 2 × 10⁶ normal human neutrophils. Myeloperoxidase (MPO) levels in the media samples were determined by immunoblot assay. Shown is a representative immunoblot for MPO. Values (25–100 μl, 50–200 μl) are the volumes of cell-free conditioned media spotted per dot. Neutrophils (2 × 10⁶; PMN alone) were stimulated for 3 hours with vehicle (Cnt), 1 μM N-formyl-L-methionyl-L-leucyl-L-phenylalanine (nFMLP [nFP]), or 1 μM phorbol-12-myristate-13-acetate (PMA). (B) MPO immunoblot signal was expressed in arbitrary units. Data are mean ± SE of cells isolated from 5 mice/genotype. ^#P < 0.01, relative to intact within the same genotype; *P < 0.05, relative to WT within same condition. (C) Data are the means of MPO release from neutrophils from duplicate experiments. (D) Confluent monolayers of alveolar type II epithelial cells were infected with 50 multiplicity of infection P. aeruginosa for 1 hour, washed with antibiotics, and cocultured for 3 hours with 2 × 10⁶ normal human neutrophils. Data are from one experiment. (E) Mmp7 mRNA levels in parental SW620 (620) and Mmp7 knockdown (620AS) human colon carcinoma cells were measured by quantitative RT-PCR and expressed as relative quantification (RQ). *P < 0.01. (F) Parental and Mmp7 knockdown human colon carcinoma cells were grown to confluence and cocultured with 2 × 10⁶ human neutrophils for 3 hours. MPO release into conditioned media was assessed by immuno-dot blot and expressed in arbitrary units. *P < 0.01. (G) Neutrophils (2 × 10⁶) were stimulated for 3 hours with vehicle (Cnt), 1 μM nFP, or 1 μM PMA.