Fig. 8. Macrophage Depletion using Clodronate.

Alveolar macrophages and circulating monocytes were depleted using IN and IP coldronate, respectively. Control mice received IN and IP liposome only. Forty-eight hours after treatment, mice were harvested for BALF, lung histology and circulating leukocyte cell counts and differential. Lungs were inflated with 10% formalin and embedded in paraffin. Sections were stained with MAC-2 to analyze pulmonary macrophage content. (A) BALF cell count and differential demonstrating a 10 fold reduction in alveolar macrophages with no significant difference in neutrophil recruitment in clodronate-treated mice. Immunostaining confirmed a significant reduction of pulmonary macrophages in the clodronate-treated group. (B) Flow cytometry of circulating leukocytes. Leukocytes were isolated from liposome-treated (left) and clodronate-treated (right) mice using gradient separation with Histopaque 1077. Cells were stained with FITC-conjugated anti-mouse CD11b and analyzed by flow cytometry. The dot plots represent the CD11b+ population of leukocytes, with CD11b staining intensity on the X axis. The CD11b+ cells are grouped into 3 subgroups, a-c, based on level of expression. The percentages represent number of CD11b+ cells in area/total CD11b+ cells. Clodronate-treated mice demonstrated depletion of a CD11b^high population of circulating monocytes (area c) from 21% to 8% of all CD11b+ cells (areas a-c). (C) Total circulating monocyte cell counts as determined by manual differential and cell counts from control and clodronate-treated wildtype mice. There was a significant reduction in the numbers of circulating monocytes in clodronate-treated mice (*p value <0.05).