Monocyte trafficking into alveoli requires LPL. (A) Monocytes isolated from the bone marrow of WT (light gray circles) and LPL^−/− (black circles) mice were allowed to migrate across Transwell inserts for 90 minutes. The percentage of migrated cells was normalized to the maximum migration of WT monocytes stimulated with CCL2 (10 ng/mL) within each experiment. Averages from duplicate samples from 3 independent experiments shown; P < .01 by Student t test. (B) Representative flow cytometry of BAL fluid harvested 24 hours after intratracheal CCL2 challenge of adult WT and LPL^−/− mice. Neutrophils and alveolar macrophages excluded using Ly6G and SiglecF, respectively. Monocytes identified as CD45⁺CD11b⁺Ly6C⁺ cells. Total number of cells in BAL fluid shown in the upper right corner; the entire BAL sample was acquired to enumerate cells. (C) Quantification of total number of monocytes recovered from BAL fluid and percentages of monocytes in whole lung homogenates from adult WT (gray circles) or LPL^−/− (black circles) mice challenged with intratracheal injection of CCL2 (or PBS control). Each symbol represents data from 1 animal; data from 2 independent experiments. (D) Ratio of WT to LPL^−/− monocytes isolated from adult animals, mixed and cotransferred via retro-orbital injection into WT neonatal mice and recovered 1 day following CCL2 intranasal challenge. Input ratio is used as the control. Data combined from 14 recipient mice in 3 independent experiments. (E) Examples of images acquired via 2PM of cleared lungs from PND7 CD11c.YFP⁺-WT or CD11c.YFP⁺-LPL^−/− pups. PND7 mice were the smallest pups that could be consistently thoroughly perfused. CD11c⁺ prealveolar macrophages or alveolar macrophages were easily distinguished as round, YFP+ (green/yellow) cells (white arrows) from the thin and flat dendritic cells (yellow arrows). CD11c⁺ cells were also readily distinguished from many smaller, intensely autofluorescent bodies of unclear etiology located entirely within the lung parenchyma, which were found in lungs of both CD11c.YFP⁺-WT and CD11c.YFP⁺-LPL^−/− pups and may be artifact from the clearing process. Brightness and contrast were adjusted using ImageJ for display in print. Images representative of randomly selected fields from lungs of 9 CD11c.YFP⁺-WT and 9 CD11c.YFP⁺-LPL^−/− pups from 2 independent experiments. Scale bar represents 50 μM. (F) Percentage of round CD11c⁺ cells localized entirely within alveoli as determined by a blinded observer who scored Z-stacks from randomly selected fields from CD11c.YFP⁺-WT (gray circles) and CD11c.YFP⁺-LPL^−/− (black circles) pups. Data combined from 2 independent experiments.