FIGURE 7.

Phagocytic activity of macrophages from Sftpc^−/− mice. A, In vitro phagocytosis. Alveolar macrophages were collected from PND14 mice and incubated with fluorescent beads. The internalization was determined by FACS analysis. Peaks of descending magnitude indicate smaller populations of cells containing an increased number of beads. The phagocytic index of Sftpc^+/+ and Sftpc^−/− macrophages is shown (right) of the FACS profile (n = 3 mice). p = 0.04. B, In vivo phagocytosis. Phagocytosis by alveolar macrophages was determined by FACS analysis after instillation of P. aeruginosa that constitutively express bacterial GFP. The open peak corresponds to specific GFP fluorescence generated by ingestion of GFP expressing P. aeruginosa. The filled peak is nonspecific fluorescence produced by wild-type P. aeruginosa instilled into separate control mice. Phagocytosis of bacteria by Sftpc^−/− macrophages was reduced (n = 3 mice). Mean fluorescence intensity is summarized (right) for macrophage-associated fluorescence in cells. p = 0.01. C and D, Sftpc^−/− macrophages express markers of alternative activation. Arginase I gene (Arg I) expression in Sftpc^+/+ and Sftpc^−/− macrophages was assessed by RT-PCR of cDNA. Relative expression was normalized to β-actin expression (C). Gel electrophoresis identified a 40-kDa protein that was increased in macrophages and BALF from Sftpc^−/− mice (D). The band was collected for sequence analysis by mass spectroscopy. The sequence of peptide fragments that match the protein Ym1 from both the macrophages and the BALF samples is shown below the gel image.