Comparison of LPS-induced lymphangiogenesis in HeN and HeJ mice and CD11b⁺ macrophage infiltration in the peritoneal side of diaphragm. (A) HeN and HeJ mice were treated intraperitoneally with the indicated doses of LPS for 7 days, and diaphragms were immunostained for LYVE-1 (brown) and visualized with DAB. Representative images of LYVE-1⁺ lymphatic vessels and lymphatic branching (black arrows) in the peritoneal side of diaphragm muscle. Right panels are higher magnifications of the black dotted rectangle and show lymphatic branching (n = 4). Scale bars indicate 300 μm. (B-E) HeN and HeJ mice were treated with LPS (0.5 mg/kg/day) intraperitoneally for 7 days. (B,C) Densities of LYVE-1⁺ diaphragmatic lymphatic vessels were measured in each given area (3.64 mm²); values were presented as a percentage per each area (n = 4). Numbers of LYVE-1⁺ lymphatic branching exceeding 50 μm in a given area (1 mm²) were counted and presented as the actual number per field (n = 4). Bars represent means plus or minus SD. *P < .05 versus HeN. (D) Diaphragms were double immunostained for LYVE-1 (green) and CD11b (red) and visualized with fluorescent dyes. Note the robust infiltration of CD11b⁺ macrophages in the diaphragmatic lymphatic vessels of HeN but not in HeJ mice (n = 3). Scale bars indicate 200 μm. (E) Whole diaphragms were harvested from HeN and HeJ mice, digested into single cells, and analyzed by flow cytometry. The number of CD11b⁺/F4/80⁺ macrophages that infiltrated into the diaphragm was presented as a percentage of the total cell number in the whole diaphragm. Data are presented as means plus or minus SD. Information on immunostaining and morphometric analysis is available in Document S1.