Figure 5.

Csf2^−/− mice lack preAMFs and mature AMFs throughout life. (A) Lung epithelial cells isolated at the indicated time points before and after birth were FACS sorted, and expression of GM-CSF mRNA was measured by RT-PCR. The expression levels were normalized by comparison with expression of the HPRT housekeeping gene. (B) GM-CSF levels in lung homogenates was measured by ELISA at the indicated time points before and after birth. (C) Csf2^−/− mice were sacrificed on their DOB. Lungs were homogenized and CD11b⁺F4/80⁺ myeloid cells (gated as indicated) were assessed for Ly-6C, CD64, CD11c, F4/80, and SiglecF expression. (D) GM-CSF-R (CD116) expression was evaluated on fetal MFs, fetal monocytes, preAMFs, and mature AMFs on the days mentioned using the gating strategy depicted in Fig. 3. (E) The presence of CD11c⁺SiglecF⁺ AMFs was evaluated in the BAL of WT and Csf2^−/− mice at the indicated time points after birth. (F and G) The presence of F4/80^hiCD11b^lo splenic MFs and liver MFs (Kupffer Cells) was evaluated in adult WT and Csf2^−/− mice. (H and I) BAL cells from adult WT and Csf2^−/− mice were subjected to a Percoll gradient to remove dead cells and protein debris. The resulting CD64⁺F4/80⁺ MFs were assessed for Ly-6C, CD11b, CD11c, and SiglecF expression (I). Data represent two (H and I) and three (A–G) independent experiments.