(A) Experimental design for Kupffer cell depletion by clodronate liposomes and NP injection. Clodronate liposomes were injected intraperitoneally to mice 2 days before NP injection. Mice were sacrificed at 40 minutes, 1, and 2 weeks after NP injection for collection of liver tissues. (B) CD68-positive Kupffer cells (green) were completely depleted 2 days after clodronate liposome treatment. NPs (red) were retained in the liver even in the absence of Kupffer cells. Scale bar; 50μm. The percentages of NP-positive/CD68-positive cells in the total NP-positive cells were determined 40 minutes after NP injection. (C) Kupffer cells returned to the basal levels by 16 days after clodronate liposome treatment. Newly recruited CD68-positive Kupffer cells (green) were negative to NPs (red), but NPs still remained in CD68-negative cells even 2 weeks after their injection (arrow heads). Scale bar; 50μm. The ratios of CD68-positive Kupffer cells to those of the basal levels were determined 2, 9 and 16 days after clodronate liposome treatment. The number of NP-positive cells was counted at 200× magnification 40 minutes, 1 week and 2 weeks after NP injection. Data are shown as mean ± SEM. **p<0.01.