Figure 4.

Effects of inhibition of angiopoietin-2 (Ang2) and/or tumor necrosis factor (TNF) on neutrophil influx and inflammatory cytokines. A–E: Confocal microscopic images of neutrophils (red, S100a9) and blood vessels [green, platelet endothelial cell adhesion molecule 1 (PECAM-1)] in tracheal whole mounts. Neutrophils are sparse in the pathogen-free trachea (A) but are abundant after Mycoplasma pulmonis infection for 7 days (B). Neutrophils are less numerous in mice treated with anti-Ang2 antibody (C) or anti-TNF antibody (D) and even less abundant in mice treated with both antibodies (E), but the number is still greater than in pathogen-free mice. F: Values for neutrophils stained for S100a9 were near 0 in the mucosa over cartilage rings in the pathogen-free group (0.02% ± 0.01%) but are numerous after infection (27% ± 4%). Neutrophil density reduces on treatment with anti-Ang2 antibody (19% ± 4%), anti-TNF antibody (17% ± 5%), or both antibodies (14% ± 4%), but is still greater than the pathogen-free baseline. G–I: Fold change in mRNA expression of S100a8 (G), Cxcl1 (H), and IL-1β (I) in the trachea of mice infected for 7 days with various concurrent treatments in relation to pathogen-free controls. Values for mice treated with anti-Ang2 antibody are approximately the same as the infected controls, but are significantly lower after treatment with anti-TNF antibody or both antibodies. Data are given as means ± SEM (F–I). N = 4 to 5 mice per group (F–I). ^∗P < 0.05 versus pathogen-free group (analysis of variance); ^†P < 0.05 versus infected group treated with phosphate-buffered saline vehicle. Scale bar = 200 μm (E, applies to A–E).