Figure 4.
Figure 4. Developing alveolar macrophages in LPL−/− pups do not upregulate PPAR-γ despite abundant GM-CSF. (A) Representative immunoblot of PPAR-γ from prealveolar macrophages and alveolar macrophages sorted from whole lung homogenates of WT and LPL−/− neonatal (PND3) pups. Both PPAR-γ1 and PPAR-γ2 were detected in prealveolar macrophages and alveolar macrophages, although only PPAR-γ1 is found in the activated peritoneal macrophages used as a positive control. Positive control contained lysate from thioglycollate-elicited peritoneal macrophages, and the negative control contained lysate from thioglycollate-elicited peritoneal macrophages from PPAR-γ-deficient animals. Immunoblot of actin used as loading control. Density of PPAR-γ band normalized first to actin, then to WT alveolar macrophage sample, set to “1.” (B) PPAR-γ levels (normalized) from 4 independent experiments. Bar shows median of 4 values with interquartile range. (C) GM-CSF concentrations, measured by enzyme-linked immunosorbent assay, from whole lung homogenates from WT (gray symbols) or LPL−/− (black symbols) neonatal pups. Data normalized to lung weight obtained prior to lysis. Each symbol represents data from 1 animal, line at median. Data from 3 independent experiments combined. (D) Flow cytometric analysis of CD131 on alveolar macrophages from BAL fluid from adult WT (filled gray histogram) and LPL−/− (solid line) mice. Cells that do not express CD131 shown as negative control (dotted gray line). Representative of at least 3 independent experiments. (E) Flow cytometric analysis of phospho-STAT5 in cells incubated for 15 minutes with (solid line) or without (filled gray histogram) GM-CSF. Prealveolar macrophages and alveolar macrophages from whole lung homogenates from WT and LPL−/− neonatal pups (PND1-2) and in alveolar macrophages from BAL fluid of adult WT and LPL−/− mice, defined by flow cytometric analysis as in Figure 3. Few fully mature alveolar macrophages were present in neonatal WT and LPL−/− pups. Percentage of cells positive for phospho-STAT5 given in each histogram. Representative of 2 independent experiments. (F) Intracellular flow cytometric analysis of PPAR-γ expression in cells from whole lung homogenates of WT and LPL−/− neonatal pups (PND1-3), with cell types defined as in supplemental Figure 3C. Median fluorescence intensity of each histogram is given. In lowest panel, cells were incubated overnight in vitro with GM-CSF (20 ng/mL); percentage and median fluorescence intensity of cells with upregulated PPAR- γ are given. Representative of 2 independent experiments. MW, molecular weight.

Developing alveolar macrophages in LPL−/− pups do not upregulate PPAR-γ despite abundant GM-CSF. (A) Representative immunoblot of PPAR-γ from prealveolar macrophages and alveolar macrophages sorted from whole lung homogenates of WT and LPL−/− neonatal (PND3) pups. Both PPAR-γ1 and PPAR-γ2 were detected in prealveolar macrophages and alveolar macrophages, although only PPAR-γ1 is found in the activated peritoneal macrophages used as a positive control. Positive control contained lysate from thioglycollate-elicited peritoneal macrophages, and the negative control contained lysate from thioglycollate-elicited peritoneal macrophages from PPAR-γ-deficient animals. Immunoblot of actin used as loading control. Density of PPAR-γ band normalized first to actin, then to WT alveolar macrophage sample, set to “1.” (B) PPAR-γ levels (normalized) from 4 independent experiments. Bar shows median of 4 values with interquartile range. (C) GM-CSF concentrations, measured by enzyme-linked immunosorbent assay, from whole lung homogenates from WT (gray symbols) or LPL−/− (black symbols) neonatal pups. Data normalized to lung weight obtained prior to lysis. Each symbol represents data from 1 animal, line at median. Data from 3 independent experiments combined. (D) Flow cytometric analysis of CD131 on alveolar macrophages from BAL fluid from adult WT (filled gray histogram) and LPL−/− (solid line) mice. Cells that do not express CD131 shown as negative control (dotted gray line). Representative of at least 3 independent experiments. (E) Flow cytometric analysis of phospho-STAT5 in cells incubated for 15 minutes with (solid line) or without (filled gray histogram) GM-CSF. Prealveolar macrophages and alveolar macrophages from whole lung homogenates from WT and LPL−/− neonatal pups (PND1-2) and in alveolar macrophages from BAL fluid of adult WT and LPL−/− mice, defined by flow cytometric analysis as in Figure 3. Few fully mature alveolar macrophages were present in neonatal WT and LPL−/− pups. Percentage of cells positive for phospho-STAT5 given in each histogram. Representative of 2 independent experiments. (F) Intracellular flow cytometric analysis of PPAR-γ expression in cells from whole lung homogenates of WT and LPL−/− neonatal pups (PND1-3), with cell types defined as in supplemental Figure 3C. Median fluorescence intensity of each histogram is given. In lowest panel, cells were incubated overnight in vitro with GM-CSF (20 ng/mL); percentage and median fluorescence intensity of cells with upregulated PPAR- γ are given. Representative of 2 independent experiments. MW, molecular weight.

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