Mammary alveolar macrophages are essential for neutrophil recruitment elicited by LPS/TLR4 signaling in the mammary gland. Alveolar macrophages are demonstrated in the normal mammary gland of C57BL/6 mouse by fluorescence staining with DAPI, CMTMR and anti CD68 mAb (red on line) of cryosection (arrows in A). Mammary glands of lactating C3H/HeN mice were infused with either liposome-encapsulated PBS (C and D) or clodronate (E and F) followed by intramammary infusion of 10 μg LPS. H&E staining of formalin-fixed mammary tissues 24 h after LPS inoculation (C–F). Inserts of C and E are magnified in D and F. LPS infused into the alveolar macrophages-depleted gland failed to induce disease (E and F), only liposomes are visible in the alveolar spaces (arrows in E and F) while neutrophil recruitment into the alveolar space is clearly visible (large arrows in C and D) together with the control PBS-liposomes (small arrows in D). Neutrophil recruitment to the mammary gland was assessed by the MPO assay (B). Significant difference in relative (LPS to PBS-challenged gland) MPO activity was observed in the mammary glands of C3H/HeN mice infused with PBS-liposomes compared with clodronate-liposome infused mice 24 h after intramammary challenge with 10 μg LPS. Data are mean and S.E., n ≥ 6/group and p value compared PBS-liposome with clodronate-liposome infused mice using the Student t-test. Scale bars 20 μm (A), 200 μm (C and E), 50 μm (D and F). (A color version of this figure is available at www.vetres.org.)