Fig. 2.

Temporal changes in cell composition of bronchoalveolar lavage fluid (BALF) and histological changes in the lungs following bleomycin injury. BALF was collected at 2, 4, 6, and 12 days following bleomycin injury and at 12 days from uninjured mice (A–D); for histology, lungs were collected at day 8 (E). A and D: total number of cells in BALF was counted with a disposable hemocytometer. Protein concentration (Conc) in BALF was determined by the BCA assay. Both total BALF cell number and protein concentration increased temporally. Both parameters were the highest by day 12 postinjury and were significantly higher than in the uninjured controls. Significant differences in total cells and protein concentration between groups were also observed at various time points as indicated in the figure. B and C: the cells were labeled with antibodies against macrophage (F4/80) and neutrophil (Ly6G) markers and analyzed by flow cytometry. Macrophages and neutrophils also increased temporally following bleomycin injury. However, on days 2 and 4, whereas total cells and macrophages were either similar to or lesser in number compared with uninjured mice, neutrophil numbers on those days were higher than the uninjured mice. E: representative images of Masson's trichrome-stained lung sections from bleomycin-injured and uninjured mice from day 8. Lower row represents magnification of area inside orange frame in upper row. Area of inflammation and fibrosis is clearly visible in bleomycin-injured lungs. Values are represented as arithmetic means ± SE (n = 3 mice per group). P values were calculated by 1-way ANOVA with a post hoc Bonferroni's multiple comparison analysis performed between all the groups. *P < 0.05, **P < 0.01, and ***P < 0.001.