LEC-specific LPS-TLR4 signaling is critical in LPS-induced lymphangiogenesis. N/N, N/J, J/N, and J/J mice were treated with intraperitoneal injection of LPS (0.5 mg/kg per day) for 7 days; the diaphragms were then harvested, whole-mounted, and immunostained for LYVE-1 (brown; scale bars indicate 300 μm) (A) or coimmunostained for LYVE-1 (green) and CD11b (red) (D). Scale bars indicate 200 μm. (A,D) Note that the LPS-induced increase of lymphatic densities and branchings and infiltration of CD11b⁺ cells adjacent to lymphatic vessels of the diaphragm in N/N mice are largely abrogated in N/J mice. (B,C) Densities of the LYVE-1⁺ diaphragmatic lymphatic vessels were measured in each given area (3.64 mm²), and the values are presented as the percentage of total area of the field. The number of LYVE-1⁺ lymphatic branching exceeding 50 μm in length in each given area (1 mm²) was counted, and the absolute numbers are presented. Bars represent means plus or minus SD (n = 4). *P < .05 versus N/N. (E) The CD11b⁺ cells in the diaphragm of HeN and HeJ mice after LPS stimulation (0.5 mg/kg per day) for 5 days were collected and enriched by MACS. The VEGF-C and VEGF-D mRNA levels in CD11b⁺ cells were analyzed by quantitative RT-PCR. mRNA levels for each gene were normalized to GAPDH, and the values are presented in arbitrary units (AU) compared with the value from HeN mice set as 1. Bars represent means plus or minus SD (n = 4). *P < .05 versus HeN.